Table of Contents
- P-01: The vector-pathogen anti-complement evolution dictates host skin immunomodulation for multimodal Lyme disease bacterial transmission
- P-02: New roles of intracellular C3 in triggering immune responses
- P-03: The complement system is activated and regulated differently during infection with the orthoflaviviruses, dengue and zika virus
- P-04: Complement evasion strategies of Candida glabrata clinical isolates and antibody independent binding of C1q to C. glabrata
- P-05: Complement activation and immune responses induced by Nuvaxovid and ChAdOx1 vaccines against Covid-19
- P-06: MicroRNA sequencing of plasma exosomes reveals complement dysregulation in COVID-19
- P-07: Molecular epidemiology and comparative virulence patterns of Acinetobacter baumannii isolates from war-related injuries in Ukraine
- P-08: A Novel Complement-Based Drug Candidate Against Fungal Infections With Promising Results in a Murine Model of Invasive Pulmonary Aspergillosis
- P-09: Evasion of Complement and Amyloid-Beta by the Neurotropic Pathogen Borrelia burgdorferi in Human 3D Cultures
- P-10: Major outer membrane protein P5 of non-typeable Haemophilus influenzae interacts with human vitronectin and contributes to serum resistance
- P-12: Complement expression determines disease severity in mouse models of SARS-CoV-2 and influenza pneumonia
- P-13: C5aR1 but not C5aR2 signaling promotes fungal clearance in the lung and host survival during systemic infection with Cryptococcus neoformans
- P-14: Epitope-Dependent Complement Activation and ADCC by Anti-NS1 Antibodies in Targeting Infected Cells: Implications for Dengue Vaccine Design
- P-15: Infection Risk in Pediatric Complement Inhibition: Insights from a Single-Center Experience
- P-16: Host Cell Origin Drives Divergent Complement Neutralization Mechanisms of Chandipura Virus
- P-17: Characterization of antibody Fc:Fc interactions by a VHH blocking technology
- P-18: Deciphering the lectin pathway among Systemic Lupus Erythematosus (SLE) patients from Western India
- P-19: Complement Factor I Functional Assay for Assessing CFI Variants
- P-20: Development and Evaluation of Alternative Pathway Factor B Neoantigen Assay Using Novel Monoclonal Antibodies
- P-21: Isoform-Specific Detection and Quantification of Complement C4A via Novel mAbs
- P-22: Profiling complement dysregulation in frontotemporal dementia using cerebrospinal fluid, plasma, and exosomes.
- P-23: Novel anti-clusterin monoclonals as tools for characterising clusterin expression in biofluids and tissues
- P-24: Development of a novel dual fluorescent ELISA (FELISA) for the simultaneous quantification of C3 and C3a in biological fluids.
- P-25: Cerebrospinal fluid complement system biomarkers in Alzheimer’s disease.
- P-26: Reconstitution of a fully functional recombinant membrane attack complex for molecular biology studies
- P-27: C3 and C3a as Indicators of Complement Dysregulation in Tauopathies and FTLD-Associated Disorders.
- P-28: A Novel Tool for Complement Activation Profiling in AAV Gene Therapy
- P-29: Advanced Assay Platform for Antibody Development: Precision Assays for ADCC, ADCP, and CDC
- P-30: Complement activation during the early postpartum period in healthy pregnant women.
- P-31: Novel antibodies and assays to ascertain the role of C4 protein variants in Schizophrenia risk
- P-32: Complement signaling as a T-cell checkpoint in the tumor microenvironment
- P-33: Distinct sites of complement activation and regulation in the tumor microenvironment
- P-34: Integration of Structural Modeling and CADD Scores Improve Variant Classification for CFH SCRs 5-18
- P-35: Comparison of complement activation products and eosinophils between bicarbonate-based and lactate-based peritoneal dialysis (PD) solutions in PD patients during introduction periods.
- P-36: Microglial Complement C5aR1 signaling drives inflammasome mediated neuropathology in Parkinson’s disease
- P-37: The Fc fragment of soluble IgMs binds C1q to activate the first step of the classical complement pathway, while inhibiting complement-dependent cytotoxicity
- P-38: A Novel Immunoassay Platform to Study the Dynamics of the Alternative Pathway
- P-39: Novel Quantitative ELISA Reveals Dynamics of Pro-Factor D Conversion During Alternative Complement Pathway Activation
- P-40: High-dose radiotherapy modulates the expression and secretion of complement (regulatory) proteins by tumor cells
- P-41: The Four Functional Segments of Factor H:Role in Physiological Target Recognition and Contribution to Disease
- P-42: MUCOSAL COMPLEMENT FACTOR B PRODUCTION BY INTESTINAL EPITHELIUM IS REQUIRED IN HOMEOSTASIS AND RESPONSE TO INJURY.
- P-43: Design and development of potent and selective C5aR2 agonists
- P-44: ROLE OF COMPLEMENT COMPONENTS 3A (C3A) AND 5A (C5A) IN THE ACUTE-TO-CHRONIC PAIN TRANSITION IN A MOUSE MODEL OF NEUROPATHIC PAIN.
- P-45: Ficolin-3 Recognizes Acylated and Carbamylated Lysine Residues and Activates Complement in a pH-Dependent Manner
- P-46: Update on protein array screening of the ficolin-3 interactome
- P-47: The Tumor- and Apoptosis-Associated Annexin A2 is a New Ligand for FHL-1 and Factor H-Related Proteins – Annexin-Bound FHR-5 Enhances Alternative Pathway Activation
- P-48: The human complement factor H-related protein FHR-2 enhances complement activation on host and bacterial ligands
- P-49: Complement regulation in genetically modified pig endothelial cells under xenogeneic and inflammatory conditions
- P-50: Live-cell single-molecule imaging of a new fluorescent C5a receptor antagonist reveals the heterogeneous spatiotemporal dynamics of C5aR1 at the plasma membrane
- P-51: Complement C3 inhibition mitigates complement activation in clinical platelet concentrates without preventing platelet storage lesions
- P-52: Differential Complement Responses in Radiation- vs. Chemotherapy-Induced Senescence
- P-53: Something about the at least 50 different complexes of the initiating molecules of the lectin pathway and the polyreactivity of the pattern recognition molecules.
- P-55: Molecular basis of naturally-encoded signaling-bias at the complement anaphylatoxin receptor, C5aR1
- P-56: Three cryo-EM structures of the complement C3d-complement receptor 3 complex reveal a novel layer of dynamics and the signaling mechanism underlying iC3b dependent phagocytosis
- P-57: The toxin CyaA from Bordetella pertussis as a potential modulator of Complement receptor 3 (CR3) function
- P-58: C5 inhibition by mAbs: There are many ways to skin a cat!
- P-59: A conserved molecular mechanism orchestrates diverse ligand recognition at the complement anaphylatoxin receptor C3aR
- P-60: A CD4+ T cell-intrinsic complement C5aR2-prostacyclin-IL-1R2 axis orchestrates Th1 contraction and is perturbed in Th1-driven disease states
- P-61: FHR2 Binds to HUVE Necrotic Cell and Renal Glomerular Basal Membrane, Inhibiting TCC Deposition and Exhibiting Cofactor Activity.
- P-62: Convergent patterns of protein binding promiscuity of complement proteins and immunoglobulins
- P-64: Characterization of disease-associated FHR-5 variants for their ligand binding and complement modulating capacity
- P-65: Targeted binding and activation of native C3 without proteolytic cleavage induced by flip/flopped cell membranes
- P-66: Structural basis of complement C3 recognition by both convertases
- P-67: Insights into potentiation of Factor H function by the Pneumococcal surface protein C (PspC): the structure of Factor H domains 8-14 in complex with PspC
- P-68: Structural insights into how the membrane attack complex is recognised by the diagnostic antibody aE11
- P-69: Utilising induced pluripotent stem cells (iPSCs) to model the outer retina for complement gene therapy delivery
- P-70: Moss-produced complement factor H (CPV-104) as a therapeutic regulator in complement-mediated diseases.
- P-71: Hypothalamic C3aR Activation by Astrocyte-Derived Complement C3a Drives Obesity and Cardiometabolic Dysfunction
- P-72: CSL040 ameliorates primary graft dysfunction in pre-clinical models of lung transplantation
- P-73: Pre-clinical characterization of CSL040, a soluble complement receptor 1 (CR1) fragment
- P-74: The Power of Fusion: Tuning Pharmacokinetic and Pharmacodynamic Properties of Complement-Modulating Peptides and Proteins Via Antibody Engineering
- P-75: Rapid Complement Inhibition with the C5 Inhibitor Crovalimab: Timing Analysis Using Animal Model and COMPOSER Trial Data
- P-76: Effects of Complement C3/C3b Inhibition on Control of Malignant Effusions and Neutrophil Phenotypes in Patients with Recurrent Epithelial Ovarian Cancer: Interim Analysis of a Phase 2 Clinical Trial
- P-77: Characterization and efficacy of a C2-blocking antibody in a rat kidney transplant model
- P-78: Exploring the Role of the Terminal Complement Pathway in the DSS-Induced Colitis Model of Inflammatory Bowel Disease
- P-79: Targeting C5a/C5aR1-mediated NETosis restores immunotherapy response in STK11-mutant lung cancer
- P-80: HDM-FH treatment during static cold storage protects against ischaemic injury in a simulated kidney transplant model.
- P-81: Brain-penetrant anti-C7 therapy reduces neurodegeneration and microgliosis in an Alzheimer’s mouse model
- P-82: Development of novel peptide ligands with improved selectivity and pharmacological properties for complement system receptors
- P-83: AAV-mHDM-FH: Safety and expression profile in two murine models of hyper-complement activation
- P-84: Targeting C5a Inflammation in ALS: Ex Vivo Findings from PMX205 Phase Ib Trial
- P-85: Inhibition of the complement component C5 and the Toll-like receptor molecule CD14 prevents systemic and local kidney inflammation in mice experiencing brain death.
- P-86: Compstatins abolish pathogenic fibroblast function associated with inflammatory maladaptation of tissues
- P-87: Mapping inhibitory epitopes on C7 with monoclonal antibodies
- P-88: The enigmatic role of C5aR2 in diabetic kidney disease
- P-89: Motor neuron disease C9orf72 dipeptides mediate neurotoxicity in human brain organoids through activation of C5aR1
- P-90: Exploring the rules for classical complement pathway activation by antibody pairs with co-dependent oligomerization
- P-91: Mechanism of action of bi-specific antibodies recruiting endogenous complement inhibitors for targeted local complement inhibition in the context of rheumatoid arthritis
- P-92: Regulation instead of inhibition: a triple fusion protein of complement regulators (TriFu) is superior in protecting animals in a rat xenotransfusion model when compared to C5-inhibition
- P-93: Complement Biomarker Responses to Factor B and C3 Inhibition in C3 Glomerulopathy
- P-94: AON-D21 – Complement C5a-Neutralizing Human/Mouse Cross-Reactive L-aptamer from Bench to Phase 2 Clinical Trial
- P-95: Eculizumab in severe HELLP syndrome – a case report
- P-96: Tumor-derived Factor B Drives Tumor Growth and anti-PD-1 resistance in STK11-mutant Lung Adenocarcinoma
- P-97: Analysis of Human Factor H-Related Gene and Protein Expression in Rheumatoid Arthritis Synovium Identifies a Novel Mechanism Promoting Dysregulated Complement Pathway Activation
- P-98: Renal Pathology as a Predictor of Complement Dysregulation in C3 Glomerulopathy
- P-99: Complement C3 and Complement Factor H affect glomerular IgA deposition in IgA Nephropathy
- P-100: Age-Dependent Plasma Factor D Levels and Their Clinical Implications in Complement-Mediated Kidney Diseases
- P-101: Mice lacking MASP- and/or MASP- are protected from the development of retinal degeneration in a murine model of NaIO3-induced dry age-related macular degeneration (AMD)
- P-102: The Complement C3a Receptor Regulates the Tumour Microenvironment
- P-103: Cell-intrinsic complement C3 and FB promote acute kidney injury
- P-104: Different patterns of complement activation markers are present in acute and chronic adult primary ITP patients
- P-105: Characterization of inherited C3 deficiency – a patient case
- P-106: Antibodies in the Healthy Population Influence Convertase Formation
- P-107: Baseline and Longitudinal Associations between serum C3/C4 levels, tissue complement biomarkers, and IgA Nephropathy in South Asians.
- P-108: The role of C3 in steatotic liver disease
- P-109: Comprehensive functional analyses of 110 complement Factor B variants provide novel diagnostic and structural insights
- P-110: A new screening approach to identify autoantibodies dysregulating the C3bBb convertase in C3 glomerulopathy and immunoglobulin-mediated membranoproliferative glomerulonephritis
- P-111: The Alzheimer’s disease-associated complement receptor 1 variant CR1*2 enhances glial phagocytosis of relevant targets and impacts neuroinflammation
- P-112: Doxycycline inhibits MMP- and inactivates microglia to reduce PNN breakdown and preserve motor neurons in SOD1(G93A) mice.
- P-113: Analysis of circulating immune complexes in serum of patients with ANCA-associated glomerulonephritis
- P-114: Beyond Systemic Complement Dysregulation: The Active Contribution of Renal Cells to Complement Dysregulation in C3 Glomerulopathy
- P-115: Complement is dysregulated in Huntington’s disease in mouse and man.
- P-116: A multi-omic pipeline identifies complement as a driver of age-dependent progression in a model of multiple sclerosis
- P-117: Modulation of Complement-Mediated Inflammation in the mdx Mouse Model of Duchenne Muscular Dystrophy by PK007
- P-118: Targeted Treatment With Pegcetacoplan for Adolescents With C3G or Primary (Idiopathic) IC-MPGN in the VALIANT Phase 3 Trial
- P-119: Assessing C3 Nephritic Factor Function Using Luminex-Based Formation and Stabilization Assays
- P-120: VALIANT: Randomized, multicenter, double-blind, placebo-controlled, phase 3 trial of pegcetacoplan for patients with native or post-transplant recurrent C3G or primary (idiopathic) IC-MPGN
- P-121: Metabolic changes in the retinal pigment epithelium of patients with complement factor H haploinsufficiency.
- P-122: Distinct associations of the lectin pathway proteins MASP-, MASP-, and MAP- with clinical and serological profiles in Systemic Lupus Erythematosus
- P-123: Charting the Path of Dendritic Cell Subsets in Allergic Asthma Using C5aR1 Fate Mapping Mice
- P-124: Features of Circulating Factor H Immune Complexes in Complement-Mediated Diseases
- P-125: Detecting FH–Anti-FH Immune Complexes in MGRS-C3G
- P-126: Complement C9-Mediated RBC Hemolysis Drives Microvascular Obstruction via Endothelial Necroptosis and Hemolyzed RBC Aggregation in COVID-19
- P-127: Senescent erythrocytes as a trigger of complement activation and implications for breakthrough haemolysis in Paroxysmal Nocturnal Haemoglobinuria patients
- P-128: Single-cell Transcriptomic Changes in Mouse Hearts during Collagen-Induced Arthritic Inflammation
- P-129: Deep neutrophil phenotyping unveils the impact of complement activation in sickle cell pathophysiology
- P-130: The conversion of native C3 to C3(H2O) is contact-driven facilitated by neutrophil extracellular traps
- P-131: The evaluation of clinical scoring system in complement mediated-TMA patients
- P-132: Genetic C4 deficiency in lower extremity peripheral arterial disease
- P-133: The C3-C3aR axis modulates trained immunity in alveolar macrophages
- P-134: NOVEL FUNCTIONS OF CD59 ISOFORMS IRIS-1 AND IRIS-2 IN INSULIN-PRODUCING PANCREATIC β-CELLS
- P-135: Complement C5aR1 Modulates Excitatory Synaptic Transmission in Cortical Pyramidal Neurons via a Calcium-Independent Mechanism
- P-136: Novel Metabolic Role of the Complement Activator MASP- in Regulating Pancreatic β-Cell Function
- P-137: Pregnancy complications in C5aR2-deficient mice are caused by an altered uterine immune cell network
- P-138: A ‘sweet spot’ for complement receptor CD46 activity: CYT-1 regulates glycolytic and moonlighting functions of glycolytic enzymes during human CD4+ T cell activation
- P-139: CD46 directly engages Sp1 to control gene transcription and HIV expression in human CD4+ T cells
- P-140: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction
- P-141: The role of intracellular and cell-autonomous C3 in NF-κB priming in human macrophages
- P-142: Exploring the role of intracellular C3 in activation of the NLRP3 inflammasome in human macrophages
- P-143: Neutrophil Plasticity in Epidermolysis Bullosa Acquisita: Bridging Innate and Adaptive Immunity via C5aR2 Signaling
- P-144: Complement Membrane Attack Complex Alters the Proliferation of Neural Progenitor Cells During Late Embryonic Murine Neurogenesis.
- P-145: Investigating complement receptor C3aR1 signalling in metabolic dysfunction in the R6/1 mouse model of Huntington’s Disease
- P-146: Peptide-mediated modulation of T cell-expressed complement-like CD109boosts T cell anti-tumor responses
- P-147: Intracellular C1r impacts tumor progression and immune microenvironment in renal cancer
- P-148: GC B cell autonomous complement activation rewires cell metabolism associated with delayed class switching and altered GC dynamics
- P-149: Factor H-Related Protein Dysregulation and Intracellular Localization in Osteoarthritis and Head & Neck Cancer
- P-150: HMGB1 cleavage by C1s modulates the pro-inflammatory signal induced by TLR2
- P-151: Inflammatory cytokines drive local C3 transcription to regulate metabolic reprogramming in kidney epithelial cells and subsequent fibrosis
- P-152: Spatial transcriptomics maps complementdriven celltocell interactions during acute kidney injury
- P-153: Targeting complement anaphylatoxin C3a receptor to break immunotherapy resistance in colorectal cancer
- P-154: Beta-Cell Intrinsic C3 Regulates Pancreatic Islet Responses to Metabolic Stress in vivo
- P-155: A Versatile Screening Platform to Guide Applications for the FH-Recruiting Peptide 5C6 on Non-Self Surfaces
- P-156: C5aR2 Fuels Autoimmune Skin Disease in Murine Epidermolysis Bullosa Acquisita
- P-157: Divergent Effects of Early Adult Microglial versus Global C1q Deletion on Amyloid Pathology and Synaptic Engulfment in an Alzheimer's Disease Mouse Model
- P-158: Opposing effects of animal models of Type 1 and type 2 diabetes on factor D expression and systemic alternative pathway activity
- P-159: Cell-autonomous complement C3 activity in prostate epithelial cells suppresses malignant transformation through tonic restrain of the MYC-tumorigenesis program
- P-160: Investigating the Role of Neuronally-expressed C5aR1 in Motor Neuron Disease
- P-161: A detrimental role for complement C5aR1 activation in Huntington’s disease
- P-162: Complement C3a receptor signalling mitigates motor neuron degeneration by modulating neurotoxic astrocytes
- P-163: CD93: A New Complement-Linked Target for Immunomodulation and wound healing in SCI
- P-164: Macrophage-Derived Complement Factor H Restrains Kidney Inflammation and Fibrosis in Immune Complex-Mediated Disease.
- P-165: Effects of Cannabinoid receptor-1 Antagonist MRI-1867 on C5ar1 Signaling in Attenuating Lung Remodeling in Mice Induced with Experimental Arthritis
- P-166: TiO2 nanoparticles act as an Alternative Pathway-specific C3 inhibitor
- P-167: CIMED: A High-Serum Concentration Diagnostic Platform for Complement Pathway Activity and Drug Response Monitoring in Patients
- P-168: Point-of-Care Assessment of sC5b-9: A Reliable Quantitative Tool for Rapid Detection of Complement Activation
- P-169: Evolutionary Insights into the Complement System: Functional Diversification and Ancestral Mechanisms in Bony Fish
- P-170: Classical and Lectin Pathway Activation in ANCA-Associated Vasculitis with Kidney Involvement: Prognostic Value and Comparison with Alternative Pathway Components
- P-171: Rescue Therapy with Pegcetacoplan in a Patient with C3 Glomerulopathy: A Case Report
Select an abstract from the Table of Contents above to view its content here.
P-01
The vector-pathogen anti-complement evolution dictates host skin immunomodulation for multimodal Lyme disease bacterial transmission
Carly Fernandes1,2, Ashley Marcinkiewicz1,2, Alan Wang2, Daniel Palmer2, Jill Malfetano2, Tristan Nowak2,3, Peter Kraiczy4, Gregers Andersen5, Sergios-Orestis Kolokotronis6, Yi-Pin Lin1,2
1Tufts University, North Grafton, USA. 2Wadsworth Center, NYSDOH, Albany, USA. 3SUNY Albany, Albany, USA. 4University Hospital of Frankfurt, Frankfurt, Germany. 5Aarhus University, Aarhus, Denmark. 6SUNY Downstate Health Sciences University, Brooklyn, USA
Background
Successful transmission of vector-borne pathogens hinges on molecular strategies of immune defense subversion at the skin interface localizing in the vector’s saliva. The causative agent of Lyme disease, Borrelia burgdorferi (Bb), invades reservoir hosts through blood feeding by Ixodes ticks. Tick-to-host transmission of Bb requires naïve larvae feeding simultaneously with physically close Bb-infected nymphs (cofeeding-mediated transmission) or subsequently to Bb-persistent hosts (dissemination-mediated transmission). Bb utilizes its outer surface protein CspZ to bind factor H (FH), thus evading the alternative complement pathway (AP). The tick vector secretes Salp20 in saliva that binds properdin, preventing AP activation. Given the temporal and spatial overlap of Salp20-mediated and CspZ-mediated complement evasion at the skin, we hypothesize that a synergistic interaction between these mechanisms is essential for efficient Bb transmission. We investigate if Salp20-properdin interactions facilitate CspZ-mediated AP evasion, thereby enhancing immunosuppression and establishing infection across multimodal transmission routes. Furthermore, we explore the evolutionary forces driving the development of Salp20-properdin interactions, seeking to understand the selective pressures that shaped this critical aspect of vector-pathogen synergy.
Methods and Results
To demonstrate the basis of Salp20-properdin interactions, we predicted the structure of the Salp20-properdin complex, revealing a loop in Salp20 that is critical for properdin binding. The role of this loop for AP inhibition was confirmed with functional assays. This synergy was attributed to collaborative AP inactivation and elevated Bb infection at bite sites. Transcriptomic analysis revealed that the CspZ-Salp20 synergy is crucial for the alleviation of Bb-triggered inflammation at tick bite sites. We further attributed our findings to the Salp20 loop and the CspZ FH-binding pocket, as highlighted by computational evidence for the footprints of positive Darwinian selection.
Conclusion
Our study reveals a synergistic mechanism by which Bb and its tick vector collaboratively modulate host skin immunity to facilitate multimodal transmission. We provide a novel framework for understanding the intricate interplay between vector and pathogen in establishing infection. This research not only elucidates the molecular underpinnings of Lyme disease transmission but establishes a model system for dissecting the broader mechanisms of pathogen transmission driven by vector-pathogen collaborations, offering potential avenues for targeted therapeutic interventions.
P-02
New roles of intracellular C3 in triggering immune responses
Katarzyna Kuska, Serena Bettoni, Frida Mohlin, Saleh Moradi, Maja Chrobak, Vaishnavi Dandavate, Kristian Riesbeck, Ben King, Anna M Blom
Lund University, Malmö, Sweden
Background
C3 is primarily recognized as a secreted protein that converges all complement activation pathways. Growing evidence suggests that C3 can participate in various processes beyond its traditionally appreciated bactericidal and opsonic activity. Many of those functions take place in the intracellular environment, where C3 can initiate signaling pathways or target pathogens for xenophagy. Previously, we demonstrated that intracellular, cytosolic C3 can opsonize Staphylococcus aureus and impact their virulence. Our current aim is to further investigate novel roles of cytosolic C3 in immune responses to infection.
Methods The human type II alveolar epithelial cell line A549, including wild-type and gene-edited variants deficient of C3 (C3 KO) or expressing only cytosolic C3 (ΔATG1) were subjected to transcriptomic analysis. Cells were either infected with bacteria, e.g., Moraxella catarrhalis, or treated with Toll-like receptor (TLR) agonists. Cytokine secretion was assessed with ELISA, while activation of signaling pathways was measured using dual-luciferase reporter assay. Signal transduction was investigated with cell fractionation and western blotting and gene expression was assessed using qPCR. Results
RNA-Seq analysis revealed potential differences in cytokine-cytokine receptor pathways when comparing C3 KO cells with either wild-type or ΔATG1 cells. Following bacterial infection and TLR agonist treatment, we observed reduced activation of NF-κB pathway and subsequent cytokine secretion in C3 deficient cells. In contrast, wild-type and ΔATG1 cells exhibited similar levels of NF-κB activation. Next, we investigated consecutive steps in the signal transduction cascade and detected similar patterns, where C3 deficient cells showed decreased protein phosphorylation and lower NF-κB translocation to the nucleus compared to wild-type cells. Furthermore, we found differences in mRNA expression array and later confirmed downregulation of TLR1 and TLR4 in C3 deficient cells. Interestingly, cytosolic C3 in ΔATG1 cells was able to rescue the wild-type phenotype restoring normal signal propagation and TLR expression. Conclusions Our findings suggest that C3 deficiency impairs signal transduction and NF-κB pathway activation when triggered by different stimuli, possibly through decreased expression of TLRs. The expression of cytosolic C3 can reverse this pattern. These results highlight a novel role of intracellular, cytosolic C3 in innate immune signaling in response to infection.
P-03
The complement system is activated and regulated differently during infection with the orthoflaviviruses, dengue and zika virus
Jill Carr1, Joshua Dubowsky1, Evangeline Cowell2
1Flinders University, Adelaide, Australia. 2Flinders, Adelaide, Australia
Background: The complement system is important for controlling viral infections but can also be part of a damaging host response. Here we have investigated changes in the complement system in the context of a dengue virus (DENV) infection - where viral replication and pathology is driven by peripheral responses in macrophages and endothelial cells to cause a vascular leak syndrome; and in zika virus (ZIKV) infection in the neonatal mouse brain that is associated with loss of neurons and developmental impact.
Methods: DENV or ZIKV infection, complement factor H (FH), FB and C3 were quantitated by RT-PCR, nanostring analysis and immunofluorescent staining. Infections were performed in vitro and in a neonatal mouse model of viral infection.
Results: In primary macrophages or endothelial cells in culture both DENV and ZIKV induced mRNA for FH, FB and C3. At the protein level, however, this corresponded to induction of FB and C3 and for ZIKV also FH and release from infected cells. During DENV infection FH protein was not induced and instead remained cell-associated and bound to surface heparin. DENV and ZIKV infection of the developing mouse brain similarly both induce C3 and FB, but components of the classical/lectin pathway, C2 and C4A, were uniquely induced by ZIKV only.
Conclusion: Although DENV and ZIKV are closely related viruses, these impact the complement system differently. During DENV infection, activation of the alternative pathway is associated with the sites of replication (macrophages) and pathology (endothelium). In the brain, we propose the ability of ZIKV, but not DENV, to activate the classical pathway will result in greater impact of ZIKV on other roles of the classical pathway component C1q in the developing brain, such as in the process of synaptic pruning, aligning with the higher propensity of ZIKV to impact neuronal development. Current work is aimed at assessing localization of C1q and C3 with sites of ZIKV pathology in the neonatal mouse brain.
References
Thank you to past and current collaborators Dr Sheila Cazebas-Falcon, Prof David Gordon, Prof Kim Hemsley, Prof Trent Woodruff. Part of this work has been published: Cowell et al., JNeurovirol, 2023, https://doi.org/10.1007/s13365-023-01123-5; Dubowsky et al. ExpImmunol, 2024, https://doi.org/10.37349/ei.2024.00147.
P-04
Complement evasion strategies of Candida glabrata clinical isolates and antibody independent binding of C1q to C. glabrata
Tahsin Ahamed Khan1, Umakhanth Venkatraman Girija1, Nisha Valand1, Randolph Arroo1, Sydney Javillo1, Krupa Valand1, Frances Alexander2, Stephen Taylor2, Steve Thomas2, Paul Bird3
1De Montfort University, Leicester, United Kingdom. 2UK Health Security Agency, Porton Down, United Kingdom. 3Leicester Royal Infirmary, Leicester, United Kingdom
Treating Candida species is made difficult by the emergence of antifungal resistance and incomplete knowledge of immune evasion mechanisms. Candida glabrata is listed in the WHO’s fungal priority pathogen list, which can cause invasive life threatening infection (World Health Organization, 2021). Additional research is required due to its clinical importance. The complement system connects the innate and adaptive immunity and is made up of proteins that work together to eliminate foreign invaders. Three pathways are involved in the activation of complement: classical, lectin, and alternative. It is important to understand how C. glabrata avoids complement activation to understand host-pathogen interactions and develop effective immunotherapy strategies.
To investigate complement evasion, crude overnight culture supernatants of C. glabrata isolates were tested for proteolytic activity against complement ligands (C1q, C3, C3b, C4, C4b, C5, MBL, L-ficolin, DC-SIGN, FH, and FI) and antibodies (IgA and IgG). Complement assays using ELISAs assessed the inhibition of all three complement pathways. The results showed that one clinical isolate cleaved C3 and C3b, key components of the complement pathways, inhibiting all three pathways by 40-50%. Pepstatin inhibited this proteolytic activity, indicating that aspartyl proteases were involved in the cleavage of C3 and C3b. Additionally, it was discovered that the classical pathway's complement C1q binds directly to C. glabrata (as well as other species including Candida albicans and Candida tropicalis) in a calcium-dependent, antibody-independent manner activating the complement. Higher concentrations of C1q were associated with increased C3b deposition, indicating that C1q can activate the complement system on C. glabrata without the need for antibodies.
In conclusion, several clinical isolates of C. glabrata cleaved C4 and IgG. Furthermore, C1q binds to C. glabrata in a calcium-dependent manner, triggering the classical complement pathway without antibodies. High concentrations of NaCl break this connection. These findings imply that secreted and membrane proteases could be potential targets for future therapeutic strategies in addition to antifungal agents. Antibody independent complement activation by C1q is a novel avenue for immunotherapeutic strategy against Candida infections.
References
World Health Organization, (2021). Global report on the epidemiology and burden of fungal infections. Available at: https://iris.who.int/bitstream/handle/10665/363682/9789240060241-eng.pdf?sequence=1 [Accessed 11 March 2025].
P-05
Complement activation and immune responses induced by Nuvaxovid and ChAdOx1 vaccines against Covid-19
Eija Nissilae1,2, Tommaso Poletto3, Elias Aho1,2, Gabriele Giudice3, Juha Kotimaa1,2, Karita Haapasalo1,4, Kurosh Kalantar5,1,2, Seppo Meri1,2
1Department of Bacteriology and Immunology, Medicum, University of Helsinki, Helsinki, Finland. 2Translational Immunology Research Program, University of Helsinki, Helsinki, Finland. 3Department of Biomedical Sciences, Humanitas University, Milan, Italy. 4Human Microbiome Research Program, University of Helsinki, Helsinki, Finland. 5Department of Medical Immunology, Shiraz University of Medical Sciences, Shiraz, Islamic Republic of Iran.
The Nuvaxovid (NVX-CoV2373) and ChAdOx1 vaccines have been used during the COVID-19 pandemic. To test the potential role of complement in their immunogenicity and side effects, we analyzed whether they induce complement activation in human serum. Nuvaxovid and ChAdOx1 vaccines were incubated in pooled and individual serum samples from healthy donors with different vaccination statuses. The formation of C5a, C3a and the terminal complement complex (sC5b-9) was quantified using enzyme immunoassays. Both Nuvaxovid and ChAdOx1 activated the complement system primarily through the classical pathway in a concentration-dependent manner. Both vaccines induced significant C5a formation, with higher levels observed through the classical than the alternative pathway. Nuvaxovid increased both C3a and sC5b-9 levels. There was significant variability in complement activation between individual serum samples. Strongest responses were observed post-vaccination. For the S-protein containing Nuvaxovid the complement activation product levels correlated with anti-S IgG levels and for the adeno-vectored ChAdOx1 with the adenoviral anti-hexon antibodies. The results have potential implications for the immunogenicity and safety profiles of these vaccines.
P-06
MicroRNA sequencing of plasma exosomes reveals complement dysregulation in COVID-19
Anthony Shadid1, Aleksey Domozhirov1, Lavanya Gunamalai1, Scott Collum1, Galina Florova2, Andrey Komissarov2, Michelle Felicella3, Marie-Francoise Doursout1, Harry Karmouty-Quintana1, Pooja Shivshankar1
1UTHealth, Houston, USA. 2UTHealth, Tyler, USA. 3UTMB, Galveston, USA
COVID-19 pathogenesis primarily impairs lung function through pneumonia, diffuse alveolar damage, and acute respiratory distress syndrome (ARDS), with severe cases of ARDS advancing to non-resolving pulmonary fibrosis. Mechanical ventilation can exacerbate lung injury and promote the release of profibrotic mediators, including microRNAs (miRNAs). We hypothesized that miRNAs shuttled in plasma exosomes may serve as superior biomarkers of COVID-19 pathogenesis during severe ARDS and the convalescent stage of infection. Plasma samples were deactivated at 56 °C for 45 minutes to eliminate microbial contaminants and stored at –80 °C. Exosomes were isolated via polyethylene glycol-based precipitation after thrombin pre-treatment to remove fibrin, and their presence was confirmed by transmission electron microscopy. Small RNA libraries were prepared from exosomal RNA using adaptor ligation and cDNA synthesis, followed by sequencing on an Illumina platform. Differential miRNA expression was analyzed using DESeq2 and edgeR, with known and novel miRNAs identified through miRDeep2 and miREvo pipelines. MicroRNA sequencing analysis of plasma exosomes revealed a unique repertoire of circulating miRNAs in patients with severe ARDS and during convalescence, compared to healthy controls. Strikingly, several critical miRNA targets were identified as complement immune factors, responsible for bridging host innate and adaptive immunity. Circulating miR-26b-5p, which targets the 3'-UTRs of complement components C1S, C3, and C5, was significantly upregulated in both critical ARDS and convalescence, indicating complement dysfunction. In contrast, miR-27b-3p which targets C1S and C5, and miR-16-2-3p which targets the complement anaphylatoxin receptor C5aR1, were significantly downregulated with disease severity and remained low during convalescence, suggesting sustained inflammatory C5aR1 signaling. Furthermore, miR-532-5p, which targets the terminal pathway regulator C7, was significantly downregulated, while miR-146a-5p, targeting soluble regulatory complement factor H (FH), was significantly upregulated during convalescence. These miRNA alterations correlated with complement component expression in lung tissues from patients with fulminant COVID-19. Finally, SARS-CoV-2 spike-overexpressing HUVECs cultured with C7- and FH-depleted serum exhibited elevated production of IL-6, IL-8, and CXCL10—key mediators of the cytokine storm—along with significantly increased C5aR1 expression. Together, these findings suggest that persistent complement dysregulation may contribute to vascular inflammation and post-acute COVID-19 lung disease.
References
Shivshankar, P., Mueller-Ortiz, S.L., Domozhirov, A.Y. et al. Complement activity and autophagy are dysregulated in the lungs of patients with nonresolvable COVID-19 requiring lung transplantation. Respir Res 26, 68 (2025). This study was supported by NIH award (R01AI158694-01) and NIH-clinical and translational science (CTSA) pilot award (5UL1TR003167-05) to PS.
P-07
Molecular epidemiology and comparative virulence patterns of Acinetobacter baumannii isolates from war-related injuries in Ukraine
Jale Boral1, Chaitanya Tellapragada2, Oskars Thofte1, Valdemar Öhnström1, Michal Magda1, Erika Matuschek3, Oleksandr Nazarchuk4, Christian Gieske2, Anna Blom1, Kristian Riesbeck1
1Lund University, Malmö, Sweden. 2Karolinska Institute, Stockholm, Sweden. 3EUCAST, Växjö, Sweden. 4National Pirogov Memorial Medical University, Vinnytsia, Ukraine
Background
Multidrug-resistant Acinetobacter baumannii remains as a global concern with its ability to adhere, disperse and sustain on areas involving suboptimal infection control measures, especially war zones. We aimed to evaluate the molecular epidemiology and virulence factors of A. baumannii isolates from conflict zone in Ukraine.
Methods
In 2022, 46 A. baumannii wound isolates were received from war-related injuries in Ukraine. Antimicrobial susceptibility testing and whole genome sequencing were performed to all isolates (n=46). The resistome, virulome, K- and O-types of isolates were detected using VFDB and Kaptive 2.0, respectively. Virulence traits of the isolates were tested in vitro and in vivo by phenotypic assays; capsule abundance, serum survival, biofilm formation, Galleria mellonella infection and mouse pneumonia.
Results
Five out of 46 (10.8%) isolates tested extensively drug resistant (XDR) and 84.78% of the isolates tested multidrug-resistant (MDR). Distance matrix analysis based on SNPs generated 5 main clusters of 46 isolates. Dominating sequence types of the isolates were ST2 (19%), ST19 (19%), ST400 (19%) and ST78 (19%). Comparative assessment of virulence profiling revealed that, KL235 and K91 are highly encapsulated K types and are found in ST2 and ST19 isolates. Virulome analysis revealed missing T66S encoding genes, Omp33-36 and biofilm production related genes (bap, blp1) in ST19 isolates. High level of encapsulation was observed in ST19 isolates followed by lower serum survival compared to the rest of the study cohort. ST19 isolates expressed a higher survival and virulence score at 24 h in mice and G. mellonella viability assay, respectively. However, prolonged infection up to 120 h in G. mellonella larvae and 72 h in mice showed that ST19 isolates have lower ability to kill larvae followed by clearance in lung and BAL fluid while ST2 isolates managed to persist the infection and lethality (p=0.0001).
Conclusion
Although A. baumannii is known for its strain specific behavioral pattern in terms of virulence, our study revealed that STs act parallel on genotypic and phenotypic basis. Instability and absence of TS66, OMP-3/36 and biofilm encoding genes and its effect on encapsulation should be studied further for possible antivirulent and immunization therapies.
References
Highly multidrug-resistant Gram-negative bacterial infections in war victims in Ukraine, 2022. Ljungquist O, Nazarchuk O, Kahlmeter G, Andrews V, Koithan T, Wasserstrom L, Dmytriiev D, Fomina N, Bebyk V, Matuschek E, RIESBECK K. Lancet Infect Dis. 2023 Jul;23(7):784-786. doi: 10.1016/S1473-3099(23)00291-8.
P-08
A Novel Complement-Based Drug Candidate Against Fungal Infections With Promising Results in a Murine Model of Invasive Pulmonary Aspergillosis
Verena Harpf1,2,3, Cornelia Speth3, Günter Rambach3, Verena Fleischer3, Mikkel-Ole Skjødt1, Reinhard Würzner3, Peter Garred2, Anne Rosbjerg2
1University of Copenhagen, Copenhagen, Denmark. 2Rigshospitalet, Copenhagen, Denmark. 3Medical University Innsbruck, Innsbruck, Austria
Background
Upon inhalation, the opportunistic fungal pathogen Aspergillus fumigatus can cause invasive pulmonary aspergillosis (IPA), a potentially life-threatening disease. There is an urgent need for novel antifungal therapeutics due to an emergence of antifungal resistant isolates, severe side effects of antifungals, and the increase of patients with risk factors such as immune deficiencies and therefore an increase of IPA cases. Our solution is a novel antibody-like antifungal drug candidate, which is comprised of a human IgG1 Fc region, a hinge region, and a peptide derived from MASP- instead of the Fab region. This peptide has been demonstrated to bind to Aspergillus fumigatus, activate complement, and trigger phagocytosis. The aim of this study was to investigate the peptibody in a murine model of IPA.
Methods
Cyclophosphamide-treated mice were intranasally challenged with a clinical isolate of Aspergillus fumigatus and either treated with three different peptibody dosages (low, medium, high) or “mock-treated” with a vehicle control (PBS) or Fc fragments at 1 h, 8 h, and 24 h post-infection. To investigate adverse effects of this novel antifungal drug candidate unifected animals were included, which were either given PBS or the high peptibody dosage. After 14 days of survival monitoring, organs and bronchoalveolar lavage (BAL) fluid were obtained, and the fungus burden was assessed.
Results
The intranasal challenge with Aspergillus fumigatus resulted in a lethal outcome for the animals receiving PBS. While the intranasal administration of Fc fragments did not improve the outcome, the low peptibody dosage rescued 30% of the animals. When administered the medium dosage, the survival increased to 80% and the outcome was further improved to 100% with the high peptibody dosage. Peptibody-treated animals also showed a lower fungal burden in their lung tissue and BAL fluid compare to “mock-treated” mice. No adverse effects by the peptibody were detected in uninfected animals.
Conclusion
The study concludes that peptibody plays a critical role in the fungal clearance and survival of the treated animals, highlighting the remarkable potential of this novel antifungal drug candidate as a salvage treatment for IPA in patients with compromised immune systems.
P-09
Evasion of Complement and Amyloid-Beta by the Neurotropic Pathogen Borrelia burgdorferi in Human 3D Cultures
Lilith Heiland1, Pavel Uvarov1, Alexander Moir2, Anna Maaser-Hecker2, Eeva Juselius1, Shahan Syed1, Antti Tuhkala1, Maija Lappalainen3,1, Jukka Hytönen4, Markku Varjosalo1, Doo Yeon Kim2, Taru Meri1, Deepak Kumar2, Rudolph Tanzi2, Karita Haapasalo1
1University of Helsinki, Helsinki, Finland. 2Massachusetts General Hospital and Harvard Medical School, Charlestown, USA. 3HUS Diagnostic Center, Helsinki, Finland. 4University of Turku and Turku University Hospital, Turku, Finland.
Background: Amyloid-beta is toxic to neurons and, in excess, can trigger Alzheimer's disease. Oligomers of the amyloid-beta peptide are known to activate the complement system. Previous findings suggest that amyloid-beta acts as an antimicrobial peptide and protects the brain from infectious agents. While evasion of the complement system is a common trait among pathogens, little is known about the role of amyloid-beta in host-pathogen interactions. The aim of this study was to determine whether, and how, amyloid-beta plays a role in the innate immune system, and whether a well-known neurotropic pathogen, Borrelia burgdorferi, has evolved strategies to evade amyloid-beta, a potential component of the innate immune system.
Methods: Human 3D neuronal tetracultures were used to model complement and amyloid-beta evasion by B. burgdorferi in a neuronal microenvironment. The effects of multiple interactions between amyloid-beta, bacteria, and complement molecules in infected 3D cultures were studied using ELISA, Western blot, transmission-electron microscopy, and immunofluorescence microscopy. In addition, neuroborreliosis patient cerebrospinal fluid (CSF) samples were studied to detect the presence of these innate immune molecules by mass spectrometry.
Results: We identified novel amyloid-beta-binding proteins with potential complement regulator binding domains from Borrelia spp. species. The association of amyloid-beta with the Borrelia surface reduced binding of Factor H (FH), increased complement-mediated attack against the pathogen and reduced microbial viability. However, B. burgdorferi exhibited resistance against complement and amyloid-beta. Increases in cytokine levels and reduction of soluble amyloid-beta in 3D cultures were detected in the presence of Borrelia spp. However, the cultures infected with B. burgdorferi showed no reduction in amyloid-beta levels. Furthermore, the neuroborreliosis CSF samples showed markers of complement activation and neuroinflammation including decreased CSF amyloid-beta levels, also observed in Alzheimer's disease patients.
Conclusions: These results indicate amyloid-beta’s target specificity in binding and oligomerizing with specific microbes and microbial molecules. The ability of some pathogens to evade amyloid-beta entrapment suggests that amyloid-beta plays a role in human innate immune defense. Whether this trait, exploited by B. burgdorferi, could be related to neuroinflammation and amyloid-beta plaque formation characteristic of Alzheimer’s disease warrants further investigation.
P-10
Major outer membrane protein P5 of non-typeable Haemophilus influenzae interacts with human vitronectin and contributes to serum resistance
Sandra Jonsson1, Martina Janoušková1, Vaishnavi Venkatesh Rao1, Juncal Garmendia2, Yu-Ching Su1, Kristian Riesbeck1
1Lund University, Malmö, Sweden. 2Consejo Superior de Investigaciones Científicas (IdAB-CSIC)-Gobierno de Navarra, Madrid, Spain
Background
Acquisition of complement regulators is a virulence strategy used by non-typeable Haemophilus influenzae (NTHi) to evade complement-mediated killing by the host. The major outer membrane protein of NTHi, P5, binds C4b-binding protein and factor H to promote bacterial serum resistance. Here we show that P5 can also bind vitronectin that inhibits the formation of the membrane attack complex at the terminal stage of the complement pathway.
Methods
Bacterial clinical isolates and isogenic mutants were analyzed. Complete open reading frame (ORF) of P5 was amplified from the genomic DNA of NTHi using primers with specific restriction sites. Surface expression of P5 in Escherichia coli was detected by flow cytometry using rabbit anti-P5_Loop33655 or anti-P5_Loop43655 polyclonal antibodies (pAb) and an FITC-conjugated goat anti-rabbit pAb. The cat gene was used to replace ompP5. NTHi P5 mutants were selected by chloramphenicol, and verified by PCR, flow cytometry and western blotting with anti-P5 specific pAbs. Bound purified vitronectin was detected with a mouse anti-human vitronectin monoclonal antibody and a FITC-conjugated goat anti-mouse pAb. A serum bactericidal assay (SBA) was performed and bacteria were in some experiments pre-incubated with purified human vitronectin. Peptides for loops 1 to 4 of P5 of each strain were synthesized and used in ELISA.
Results
Heterologous surface expression of P5 variants from NTHi strains 3655, KR271, KR317, and P652 promoted vitronectin binding to the P5-expressing E. coli. In contrast, deletion of P5 from the NTHi strains reduced vitronectin binding. Vitronectin acquisition conferred serum resistance to P5-expressing E. coli, but not to ΔompP5 mutants. Using site-directed mutagenesis, extracellular loops 2 and 4 of the P5 variants were identified as the binding sites for vitronectin, respectively.
Conclusion
Despite being highly variable, P5 is an outer membrane protein that functions as a receptor for vitronectin, promoting NTHi serum resistance.
References
Su YC, and Riesbeck K et al. Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host. Front Cell Infect Microbiol. 2023;13:1085908.
P-12
Complement expression determines disease severity in mouse models of SARS-CoV-2 and influenza pneumonia
Peter Szachowicz, Alexander Watson, Christine Wohlford-Lenane, Boopathi Sownthirarajan, Jeffery Dubert, Yuzhou Zhang, Balaji Manicassamy, Paul McCray, Richard Smith
University of Iowa, Iowa City, USA
Background: The role of complement in the host response to respiratory viral infections is debated, with evidence suggesting it can either promote an effective immune response, or induce excessive inflammation and acute lung injury. During the COVID-19 pandemic, complement was implicated in disease pathobiology and complement C5 inhibition was granted emergency use approval in severe cases. Currently, virulent strains of both SARS-CoV-2 and influenza still result in thousands of deaths annually, and new variants which could produce another pandemic event are an ever present threat. Thus, it is vitally important that we understand the mechanisms by which these respiratory viruses induce severe disease and identify new therapeutic options for the future.
Methods: We intranasally inoculated Balb/c WT, C3-/+, C3-/- mice with lethal doses of mouse-adapted SARS-CoV-2 (SARS2-N501YMA30) and PR8 influenza (5,000 PFU and 100 PFU, respectively) to determine whether varying degrees of complement expression altered the observed disease phenotype. Mice were monitored for weight loss and survival and euthanized on days 2 and 4 post-infection for sample collection.
Results: We found that both viruses induced a significant complement response in the lungs of wild-type mice by lung homogenate western blot for C3, C9, and FB. Interestingly, we found that mouse genotype directly influenced severity of illness, with homozygous mice having the best outcome and wild-type mice the worst, based on differences in weight-loss (p>0.05) and survival (p<0.05).
Conclusion: This data confirms that complement contributes to the pathobiology in mouse models of both SARS-CoV-2 and influenza pneumonia, suggesting that the mechanisms by which complement drives pathogenesis is conserved across various human pathologic respiratory viruses. This data will serve to support future research investigating the specific mechanisms by which complement influences the host response to these infections, and complement inhibition as a broad spectrum treatment option for severe viral pneumonia.
P-13
C5aR1 but not C5aR2 signaling promotes fungal clearance in the lung and host survival during systemic infection with Cryptococcus neoformans
Yiquan Wu, Meiqing Shi
University of Maryland, College Park, USA
Systemic infection, also known as disseminated infection, of fungal pathogens is a life-threatening condition. How the complement system responds to invasive fungal pathogens during systemic infection remains incompletely understood. Cryptococcus neoformans is an encapsulated fungal pathogen that causes disseminated infection, accounting for 112,000 deaths worldwide per year. Recent clinical studies have shown that complement-targeted monoclonal antibody therapy is linked to disseminated cryptococcosis. In this study, we examined the role of complement and its receptors in fungal clearance in the lung and host survival following systemic infection with C. neoformans. Complement C3-/- and C5aR1-/- but not C3aR-/- and C5aR2-/- mice infected intravenously with C. neoformans 52D died significantly earlier than their wild-type counterparts. The early death of infected C3-/- and C5aR1-/- mice was accompanied with enhanced lung fungal burdens. In contrast, the deficiency of C3aR and C5aR2 did not affect lung fungal burdens. Compared to infected wild-type mice, infected C3-/- and C5aR1-/- mice exhibited increased size and weight of lungs, as well as enhanced lung inflammation as assessed by histological studies. Flow cytometry analysis demonstrated that infected C3-/- and C5aR1-/- mice displayed higher numbers of total leukocytes, Ly6Chi inflammatory monocytes, interstitial macrophages, and total and activated CD4+ T cells and CD8+ T cells, with C3-/- mice also having higher neutrophil counts. Surprisingly, C3 and C5aR1 ablation dramatically reduced the frequency and number of alveolar macrophages. Compared to wild-type mice, C3-/- mice secreted higher amounts of IFN-γ, IL-1β, IL-4 and IL-5 in the infected lung. Higher levels of IL-4 and IL-5 was also detected in the infected lung of C5aR1-/- mice. Collectively, this work suggests that deficiency of C3 and C5aR1 but not C3aR and C5aR2 leads to death of mice during systemic infection with C. neoformans, associated with higher fungal burdens and enhanced inflammatory responses in the lung.
P-14
Epitope-Dependent Complement Activation and ADCC by Anti-NS1 Antibodies in Targeting Infected Cells: Implications for Dengue Vaccine Design
Somchai Thiemmeca1,2, Romchat Kraivong3,2, Nuntaya Punyadee1,2, Napon Nilchan3,2, Somchoke Traewachiwiphak3,2, Kessiri Kongmanus1,2, Prasit Luangaram3,2, Tanapan Prommool3,2, Ranyikar Poraha2, Preeyanuch Sayboonruan2, Pattarakul Pakchotanon3,2, Thaneeya Duangchinda3,2, Sansanee Noisakran3,2, Chunya Puttikhunt3,2, Panisadee Avirutnan1,2
1Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 2Siriraj Center of Research Excellence in Dengue and Emerging Pathogens, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. 3Molecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand
Dengue fever, caused by dengue virus (DENV), is an escalating global health threat inadequately addressed by current vaccines susceptible to ADE. The viral nonstructural protein 1 (NS1), involved in replication and pathogenesis (e.g., vascular leak), is a promising vaccine alternative. Anti-NS1 antibodies confer protection in vivo, likely via effector functions like antibody-dependent cellular cytotoxicity (ADCC) and complement activation (resulting in C3d/C5b-9 deposition), but the epitope-dependency of these mechanisms is unclear. We investigated how antibodies targeting distinct NS1 epitopes mediate these functions. Cell-surface NS1 binding, natural killer (NK) cell-mediated ADCC (CD107a/IFN-γ assays on infected B-cells), and complement activation (C3d/C5b-9 deposition on infected hepatocytes) were assessed. Antibodies recognizing hydrophilic surface regions effectively bound NS1. However, downstream functions diverged significantly by epitope: distal β-ladder binders primarily drove ADCC with minimal complement activation, while hydrophobic wing domain binders effectively activated complement, leading to C5b-9 deposition, but lacked ADCC activity. This demonstrates that distinct NS1 epitopes selectively trigger different antibody effector pathways. Rational NS1 vaccine design must therefore carefully consider epitope selection to optimize the desired balance of protective mechanisms like ADCC and complement activation.
References
This work was supported by Faculty of Medicine Siriraj Hospital, Mahidol University (R016536001), NSTDA (P-0-52570), and Siriraj Research Development Fund (R016637004, R016634008). P.A., N.P. and K.K. were supported by Faculty of Medicine Siriraj Hospital, Mahidol University through the Research Excellence Development (RED) program.
P-15
Infection Risk in Pediatric Complement Inhibition: Insights from a Single-Center Experience
Sara Graciaa1, Ronak Dave2, Logan Pickett1, Michael Briones1,2, Michelle Schoettler1,2, Satheesh Chonat1,2
1Children's Healthcare of Atlanta, Atlanta, USA. 2Emory University School of Medicine, Atlanta, USA
Background:
Complement inhibition (Ci) is an effective treatment for diseases characterized by complement dysregulation. These agents have revolutionized disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), by improving prognosis and quality of life. However, this approach is not without risks, as complement blockade increases susceptibility to infection. While this risk is mitigated with immunizations and antibiotics, patients remain vulnerable, and the true extent of infection risk is not well-documented in pediatrics.
Methods:
In this IRB-approved, retrospective study, we identified hematology patients treated with Ci at Children’s Healthcare of Atlanta from 2005 to 2025. Cultures and pathology reports were reviewed from 1st dose of Ci to 6 months following last dose.
Results
Long term infection risk was assessed in 71 patients over 161 patient-years. Median age at first dose was 15 years (range 4 months to 20 years) and 51% were male. Treatment indications included sickle cell hyperhemolysis (n=28), PNH (n=20), atypical hemolytic uremic syndrome (n=12), autoimmune hemolytic anemia (AIHA) (n=4), Shiga-toxin producing Escherichia coli-HUS (n=2), myasthenia gravis (n=2), thrombotic microangiopathy secondary to lupus (n=2), and SARS-CoV-2 (n=1).
One patient developed bacterial and fungal pneumonia. Six experienced bacteremia/ candidemia; identified organisms included Candida parapsilosis, Candida albicans, Pseudomonas aeruginosa, Acinetobacter, Staphylococcus cohnii, Streptococcal pneumoniae, Klebsiella pneumoniae, and Neisseria gonorrhoeae. Infection incidence at 6 months was 7.3% in those who received <5 Ci doses and 5.1% in those with >5 doses.
Ten deaths occurred, two of which were attributed to encapsulated bacterial infection. An AIHA patient on several immunomodulators and splenectomy died from necrotizing pancreatitis and Klebsiella pneumoniae bacteremia. This patient received eculizumab 40d before bacteremia. Additionally, a patient with sickle cell disease received one dose of eculizumab 4 days prior to dying from toxic-metabolic encephalopathy due to Streptococcal pneumonia sepsis and meningitis. Notably, infection was present prior to Ci.
Conclusion:
This comprehensive institutional review demonstrates the infection profile of Ci. While underlying diagnosis, splenectomy, immunosuppression and varying duration of Ci exposure could have contributed to reported infections, none of the reported deaths were directly attributable to Ci. Overall, with appropriate vaccination and antibiotic prophylaxis, Ci remains a well-tolerated and safe therapeutic option.
P-16
Host Cell Origin Drives Divergent Complement Neutralization Mechanisms of Chandipura Virus
JOHN JOHNSON, Umerali Kunnakkadan, Devika Rajani, Aranya Anunaya, Ashik Francis
Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India
Chandipura virus (CHPV), a neurotropic vesiculovirus of the Rhabdoviridae family, is implicated in pediatric encephalitis outbreaks across the Indian subcontinent. Although CHPV is highly susceptible to complement-mediated neutralization, its high case fatality rate suggests immune evasion strategies. We previously demonstrated that CHPV grown in Vero E6 cells activates the classical complement pathway in a C1q-dependent manner, leading to virus aggregation and neutralization. However, complement-virus interactions can vary with the host cell line, as shown for other RNA viruses like influenza A and vesicular stomatitis virus.
To investigate this, we cultured CHPV in BHK-21, HeLa, and Vero E6 cells and analyzed complement activation and neutralization outcomes. While all CHPV variants activated the classical pathway to similar extents, downstream mechanisms of neutralization differed. Using sucrose gradient ultracentrifugation, we found that BHK-21-derived CHPV underwent complement-mediated virolysis, evidenced by a shift of viral proteins to lighter fractions. Vero E6-derived CHPV was neutralized via aggregation, with viral proteins shifting to heavier fractions. In contrast, HeLa-derived CHPV showed no redistribution upon serum exposure, indicating resistance to complement-mediated neutralization.
To understand this resistance, we examined the expression of membrane-bound complement regulators (CD46, CD55, CD59) in host cells and their incorporation into CHPV particles. Viruses grown in HeLa and A549 cells incorporated these regulators expressed in these cells, as confirmed by qPCR, immunoblotting, flow cytometry, and electron microscopy. Functional assays showed that virion-associated RCAs retained their activity, inhibiting C3 and C5 convertase formation and membrane attack complex (MAC) deposition. In contrast, CHPV from RCA-deficient BHK-21 cells lacked this protection and remained highly susceptible to complement.
These findings highlight host cell origin as a key determinant of complement neutralization outcomes. While upstream complement activation is consistent, downstream effects—virolysis, aggregation, or evasion—depend on the host-derived molecular composition of the viral envelope. Understanding these mechanisms provides insight into CHPV pathogenesis and form theoretical basis for developing viral vectors for systemic delivery.
References
Acknowledgements: Karthika R, Priti K and Shivaja M for technical support and DST-SERB-India for funding.
P-17
Characterization of antibody Fc:Fc interactions by a VHH blocking technology
Els van der Meijden1, Inez Stoof1, Alexandra Terry1, Xascha van Engelen1, Alexander Davis2, Alexander Eletsky2, Xiaoguang Xue1, Riham Ghalaiyini1, Dennis Verzijl1, Boris Bleijlevens1, Adam Barb2, Rob de Jong1, Frank Beurskens1
1Genmab, Utrecht, Netherlands. 2University of Georgia, Athens, GA, USA
Human IgG antibodies can oligomerize upon binding to antigens on the cell surface. The HexaBody® technology enhances oligomer formation via a single-point mutation that strengthens Fc:Fc interactions between neighboring antibodies. These oligomers facilitate receptor clustering for outside-in signaling and improve binding to C1q, the first component of the complement system, leading to enhanced complement activation and complement-dependent cytotoxicity (CDC).
To validate and study the mechanism of action induced by the HexaBody format, we developed a VHH (HexBlock VHH clone 2E1) that disrupts antibody Fc:Fc interactions. The VHH was generated by immunizing llamas with an antibody variant capable of hexamer formation in solution, followed by phage display library selection.
Here, we characterize the biochemical and biophysical properties of HexBlock VHH clone 2E1– IgG complexes using cell surface target binding assays (FACS), biolayer interferometry (BLI), size-exclusion chromatography (HP-SEC), cryogenic electron microscopy (cryo-EM), and nuclear magnetic resonance spectroscopy (NMR). HexBlock VHH clone 2E1 bound to the CH2 domain of IgG antibodies and efficiently interfered with Fc:Fc interactions. In functional assays, this interference caused reduced receptor clustering and suppressed complement activity mediated by IgG1 or HexaBody molecules.
P-18
Deciphering the lectin pathway among Systemic Lupus Erythematosus (SLE) patients from Western India
Vandana Pradhan1, Kirti Rai1, Ridi Khatri1, Amrutha Jose1, Milind Nadkar2, Anjali Rajadhyaksha2, Lubka Roumenina3, Manisha Madkaikar1
1ICMR-National Institute of Immunohaematology, Mumbai, India. 2King Edward Memorial Hospital, Mumbai, India. 3Cordelier Research Centre, INSERM UMRS 1138, Paris, France
Background: The complement system, particularly the lectin pathway, plays an important role in removal of apoptotic cells and maintenance of tissue homeostasis. However, its role in pathogenesis of SLEis poorly known. The present study aimed to assess the association of serum levels of ficolins, MBL and other PRMs of the lectin pathway and their corresponding autoantibodies with various clinical manifestations and disease activity in SLE patients from Western India.
Methods: In this cross-sectional study a total of 282 clinically diagnosed SLE patients were included and classified into inactive (SLEDAI<7, n=138) and active (SLEDAI≥7, n=144) groups. Serum levels of ficolins, MBL, MASPs, Map-44, CL-L1 and their corresponding autoantibodies were quantified using ELISA. Group differences were analyzed using Mann-Whitney U tests, while associations/relationships were evaluated using chi-square tests and Spearman's correlations.
Results: Serum levels of ficolin-2 (p<0.001), MASP- (p=0.030) and MAp44 (p<0.001) were significantly elevated, while MBL (p<0.001) and MASP- (p<0.001) levels were significantly reduced in SLE patients in comparison to healthy controls (HCs). Active SLE patients had significantly higher ficolin-1 levels (p=0.009) and lower MASP- levels (p=0.022) in comparison to inactive SLE patients. Renal involvement was associated with elevated ficolin-1 (p=0.009) levels, while hematological manifestations were linked to reduced MASP- (p=0.018), MASP- (p=0.002), and MAp44 (p=0.002) levels. Mucocutaneous manifestations were associated with elevated MAp44 (p<0.001) and anti-ficolin-1 (p=0.038) autoantibodies. SLE patients further showed significantly elevated levels of anti-ficolin-1 (p=0.001), anti-ficolin-2 (p=0.001) and anti-ficolin-3 (p<0.001) autoantibodies compared to HCs. Anti-ficolin-2 autoantibodies had a negative correlation with ficolin-2 (r=-0.153, p=0.015) andC3 (r=-0.131; p=0.039) levels. Anti-MBL autoantibodies were found to be correlated with SLEDAI(r=0.169, p=0.007)and anti-dsDNA antibodies (r=0.178, p=0.005).
Conclusion: The present study provided novel insight into the dysregulation of lectin pathway components and their corresponding autoantibodies in Indian SLE patients. The observed dysregulation in ficolins, MASPs, MAp44, CL-L1 levels, and their corresponding autoantibodies, as well as their association with clinical manifestations, disease activity, and serological parameters, suggest their potential as immunological indicators in SLE.
References
We are thankful to the Director General, ICMR-New Delhi, India and ICMR North East Task force (ICMR-NTF) for the financial support (Project no. NTF/NER/NER Cell/RBMCH-2020).
P-19
Complement Factor I Functional Assay for Assessing CFI Variants
Renee Goodfellow, Nicole Meyer, Nicolo Ghiringhelli Borsa, Amanda Taylor, Carla Nester, Richard Smith, Yuzhou Zhang
Molecular Otolaryngology and Renal Research Laboratories -University of Iowa, Iowa City, USA
Background
Complement Factor I (FI) is a serine protease essential for regulating complement activity across all pathways. With the assistance of cofactors—factor H (FH), membrane cofactor protein (MCP/CD46), soluble complement receptor 1 (sCR1), or C4 binding protein (C4BP)—FI inactivates C3b and C4b through proteolytic cleavage. Rare variants (RVs) in CFI can cause FI deficiency via two primary mechanisms: reduced protein expression (haploinsufficiency, type I) or impaired functional activity (type II). Differentiating type II defects is crucial, as these variants may influence therapeutic decisions in complement-mediated diseases.
Methods
To evaluate functional defects independent of FI expression levels, we developed a standardized cell-based assay. C3b-coated sheep erythrocytes were incubated with patient serum or plasma (diluted 1:32), supplemented with purified cofactors (FH, MCP, or sCR1), and incubated at 37°C for 15 minutes. Residual C3b was quantified using FB and FD with diluted rat serum EDTA as a source of C5-C9, enabling membrane attack complex-mediated hemolysis. Controls included sera/plasma from healthy donors (negative control) and a previously known loss-of-function CFI variant (c.1006C>G, p.Arg336Gly).
Results
Genetic analysis identified 59 patients with 42 distinct CFI missense RVs, including 10 novel variants. Of these, 20 RVs showed reduced plasma FI expression (type I), while 22 had normal FI levels. Among variants with normal expression, our functional assay identified five loss-of-function variants clustered in two regions of the serine protease domain.
Conclusion
Our validated assay reliably identifies type II (functional) CFI variants, emphasizing the importance of integrating genetic and functional analyses for accurate classification and informed clinical decision-making in patients with complement-mediated diseases.
References
Acknowledgements. Supported in part by National Institutes of Health R01 DK110023.
P-20
Development and Evaluation of Alternative Pathway Factor B Neoantigen Assay Using Novel Monoclonal Antibodies
Naia Vad Stampe, Laura Pérez-Alós, Beatrice Linnea Christina Fageräng, Karsten Skjødt, Peter Garred
Laboratory of Molecular Medicine, Copenhagen, Denmark
Background and Aim:
Factor B plays a critical role in the alternative pathway (AP) of complement activation, where its cleavage by Factor D generates fragments Ba and Bb. Fragment Bb forms active convertases, amplifying complement activity, while released Ba serves as a biomarker of AP activation. Elevated AP activation significantly contributes to complement-mediated diseases, such as IgA nephropathy and paroxysmal nocturnal hemoglobinuria. Currently, invasive kidney biopsies remain the primary diagnostic approach due to the absence of reliable non-invasive biomarkers. Therefore, developing accurate, minimally invasive assays to measure Factor Ba in plasma and urine is clinically essential.
Methods:
Monoclonal antibodies (mAbs) targeting Factor Ba neoantigens were produced using hybridoma technology. Selected clones, identified via direct ELISA, underwent purification through Protein G affinity chromatography, with some antibodies biotinylated. The specificity and affinity of these mAbs were rigorously evaluated using Western Blot and Sandwich ELISA (S-ELISA).
Results:
We identified unique monoclonal antibodies that specifically recognize Factor Ba fragments in zymosan-activated serum, which are absent in control samples. Using these antibodies, we developed a sensitive, quantitative S-ELISA demonstrating time-dependent increases in Factor Ba upon E. coli-induced activation in plasma.
Conclusion:
These novel mAbs significantly advance non-invasive monitoring of AP activation, improving diagnostic and therapeutic evaluations of complement-related diseases through plasma and urine Factor Ba measurements.
P-21
Isoform-Specific Detection and Quantification of Complement C4A via Novel mAbs
Bettina Eide Holm1, Mikkel-Ole Skjoedt2, Karsten Skjoedt1, Peter Garred1
1Laboratory of Molecular Medicine, Copenhagen, Denmark. 2Novo Nordisk, Copenhagen, Denmark
Background:
The complement component C4, a critical mediator of the classical and lectin pathways, plays dual roles in innate immunity and neurodevelopment. Humans express two nearly identical C4 isoforms, C4A and C4B. Despite sharing more than 99% sequence homology, these isoforms exhibit distinct functional properties: the preferred targets of C4A are amino groups on proteins. In contrast, C4B preferentially targets hydroxyl groups on carbohydrates and glycoproteins. Notably, C4A has emerged as a key regulator of synaptic pruning. Approximately 1–3% of Caucasian individuals exhibit homozygous deficiencies of C4A or C4B. Copy number variations (CNVs) in C4A and C4B are further associated with autoimmune, infectious, and neuropsychiatric disorders. Additionally, C4A and C4B define the Rodgers/Chido blood group antigens, yet clinical reagents to distinguish these isoforms remain scarce. To address this gap, we aimed to develop a quantitative sandwich ELISA (s-ELISA) to measure C4A in plasma.
Methods:
Monoclonal antibodies (mAbs) were generated using hybridoma technology with C4A-specific peptides as immunogens. Candidate clones were screened for C4A peptide recognition and further validated using ELISA against purified C4, and plasma from individuals deficient in C4A (C4A0), C4B (C4B0), and healthy controls. Specificity was further confirmed by Western blot under reducing and non-reducing SDS-PAGE conditions.
Results:
A panel of mAbs demonstrated high specificity for C4A peptides without cross-reactivity to C4B. The selected clones reacted with control and C4B0 plasma but not with C4A0 plasma and showed strong reactivity to purified C4 in ELISA setups. Moreover, after SDS-PAGE, Western Blot confirmed the ELISA results, showing reactivity under non-reduced and reduced conditions matching the molecular weight of the C4 alpha chain. Based on these mAbs, a selective, quantitative C4A s-ELISA was successfully established.
Conclusion:
The development of these novel C4A-specific monoclonal antibodies represents a critical advancement for complement research, enabling accurate isoform-specific quantification and functional analysis. This assay lays the groundwork for improved biomarker profiling in diseases linked to C4A variation, including autoimmune and neuropsychiatric disorders. Parallel efforts to generate C4B-specific mAbs are underway.
P-22
Profiling complement dysregulation in frontotemporal dementia using cerebrospinal fluid, plasma, and exosomes.
Lewis M Watkins1,2, Laura E Nicholls1, Kok Yung Lee1, Robert AJ Byrne1, Rebekah S Cooke1, B Paul Morgan1, Aitana Sogorb-Esteve2, Wioleta M Zelek1
1Cardiff University, Cardiff, United Kingdom. 2University College London, London, United Kingdom
Background: Frontotemporal dementia (FTD) is the commonest early-presenting dementia, typified by synaptic dysfunction, neuronal loss, and inflammation. Several genetic mutations are linked to FTD; however, the exact causes are not fully understood. Limited evidence suggests complement involvement in genetic and sporadic forms of FTD, yet reliable complement biomarkers are lacking. Here we investigated whether measurement of complement markers in plasma, cerebrospinal fluid (CSF), and plasma/CSF-derived extracellular vesicles (EVs) could provide a biomarker for early diagnosis in FTD.
Methods: Two distinct cohorts were utilized in this study: 1) Plasma was obtained from sporadic FTD cases (spFTD, n=20) and healthy controls (HC, n=20). EVs were isolated plasma using Exo-spinTM kits. EVs were validated by measuring particle size using nanoparticle tracking analysis and electron microscopy, confirmed by EV marker staining for CD9/CD63/Alix/Flotillin1/CD46/CD55/CD59. 2) Plasma and CSF were obtained from presymptomatic and symptomatic FTD mutation carriers (C9orf72 (n=42); GRN (n=23); MAPT (n=28)) and non-carrier controls (n=59). Complement activation products (Ba, iC3b, and TCC), regulators (FH, properdin, sCR1, and clusterin), and components (C1q, C3, FB, C4, C5, C7, and C9) were measured by ELISA in plasma, CSF, and EVs.
Results: In plasma, most complement analytes were not significantly different between spFTD and HC; the exceptions were C4 and FB, significantly decreased in spFTD vs HC (p=0.0192 and p=0.0181, respectively), suggesting complement consumption in classical and alternative pathways. In EVs, levels of components (C4 p=0.0029; FB, p=0.0007; C5, p=0.0243; C7, p=0.0211; and C9, p=0.0065) and regulators (FH, p=0.0362; properdin, p=0.0069; and clusterin, p=0.0001) were significantly reduced in spFTD compared to HC, suggesting complement dysregulation or consumption. EV western blots were immunopositive for complement markers sCR1/FH/CD46/CD55/CD59/clusterin. In CSF, complement activation markers (iC3b, p=0.0325), components (C3, p=0.0021; C4, p=0.0036; FB, p=0.0013; C5, p=0.0018; C7, p=<0.0001; and C9, p=0.0001), and regulators (FH, p=0.0022; properdin, p=0.0053; and clusterin, p=0.0117, respectively) were significantly increased in symptomatic vs presymptomatic FTD, supporting increased dysregulation.
Conclusions: We demonstrate complement dysregulation in sporadic and genetic FTD cases using plasma, CSF, and EVs. Measurement of complement biomarkers, particularly in CSF and on EVs shows promise for early disease detection and monitoring of progression.
P-23
Novel anti-clusterin monoclonals as tools for characterising clusterin expression in biofluids and tissues
Robert AJ Byrne, Aurora Veteleanu, Hannah L Cramp, Olivia G Bacon, Matthew Bright, Rebekah S Cooke, Nikoleta Daskoulidou, Kok Yung Lee, Wioleta M Zelek, B Paul Morgan
UK Dementia Research Institute, Cardiff, United Kingdom
Background: The clusterin gene (CLU) is a top GWAS risk locus for Alzheimer’s disease (AD) while biomarker studies consistently report elevated plasma clusterin levels in AD, suggesting an important role in the disease. Clusterin is a multifunctional protein, a negative regulator of the complement terminal pathway but also involved in lipid and cholesterol transport. Although a handful of commercial ELISA kits are available to quantify clusterin in human biofluids, there is a lack of validated reagents for isolation, detection, and characterization of clusterin in tissues and fluids, required for exploring roles in AD. To address this, we generated a panel of anti-clusterin monoclonal antibodies (mAbs) and applied them to relevant biofluids and tissues.
Methods: Clusterin was isolated from human plasma and used to immunize mice. Clusterin-positive hybridomas were taken to monoclonality; eight mAbs (clones: 1A4, 1D5, 2D5, 3C9, 4C7, 5D11, 6C12, 6D1) were selected, purified by protein G affinity chromatography and validated in ELISAs, western blot (WB), immunostaining, surface plasmon resonance (SPR), and functional (classical haemolytic and reactive lysis) assays.
Results: All selected mAbs bound strongly to native clusterin in ELISA; mAbs 2D5 and 4C7 were specific for clusterin in plasma WB and were used to develop a sensitive (detection limit 8 ng/ml) and specific sandwich ELISA to measure clusterin in plasma, serum, cerebrospinal fluid (CSF), and cell lysates, as already described (PMIDs: 36149090, 37480051, 38359836). Assay reproducibility was excellent (intra-assay CV=3.93%; inter-assay CV=8.5%). Immunoaffinity purification of clusterin from serum using immobilized mAb 2D5 yielded pure, functional clusterin in a single step. None of the anti-clusterin mAbs prevented complement inhibition by clusterin in haemolysis assays. The mAbs were used to detect and quantify clusterin in control and AD brain tissue, astrocytoma lines, and induced pluripotent stem cell (iPSC)-derived astrocytes. One mAb (4C7) specifically detected the known intracellular isoforms of clusterin.
Conclusions: We have developed novel mAbs against human clusterin that enable accurate quantification of clusterin in human biofluids and tissues, and visualization of clusterin in AD and control brain tissue and iPSC-derived astrocytes. These mAbs provide essential tools for elucidating the role of clusterin in AD.
P-24
Development of a novel dual fluorescent ELISA (FELISA) for the simultaneous quantification of C3 and C3a in biological fluids.
Gareth D. Fenn, Wioleta M. Zelek, B. Paul Morgan
UK Dementia Research Institute, Cardiff University, Cardiff, United Kingdom
Background: Enzyme linked immunosorbent assay (ELISA), enable rapid quantification of specific proteins within biological samples. ‘Sandwich' ELISAs utilize a pair of antibodies in combination to detect specific proteins. A ‘capture antibody' is used to bind the target protein in the sample and, after washing, a second‚ ‘detection antibody', specific for a different epitope in the target protein is applied. The detection antibody can be directly HRP-tagged or detected with an anti-IgG-HRP. Bound HRP is measured by adding an appropriate chromogenic substrate. In a fluorescence ELISA (FELISA) the detection antibody is fluorophore conjugated, providing greater dynamic range and sensitivity, thus beneficial for high-throughput applications. FELISA offer a further advantage, facilitating the detection of more than one analyte simultaneously by using different detection antibodies labelled with different fluorophores. Here we describe a FELISA that simultaneously measures intact C3 and the C3a fragment in biological samples.
Materials and Methods: Black 96 well MaxiSorp plates were coated with a monoclonal anti-C3/C3a antibody, blocked, washed and sample or standards (C3a or intact C3) added in duplicate. After incubation and washing, monoclonal anti-C3a labelled with Alexafluor-488 and monoclonal anti-C3 (β-chain) labelled with Alexafluor-647 were added simultaneously, plates incubated, washed and fluorescence measured in a BMG Clariostar platereader. Concentrations of intact C3 and C3a concentrations in samples were automatically read from the relevant standard curve and C3a:C3 ratios automatically calculated.
Results: For both analytes, the FELISA showed improved sensitivity and increased working range compared to separate in-house ELISAs. Values obtained for C3a and intact C3 in plasma and serum samples using the FELISA closely matched those from the ELISA. Time taken to perform the assay was reduced from 8 to 3 hours. Elevated C3a levels and C3a/C3 ratios were apparent in serum compared to plasma in matched samples. The increased sensitivity enabled quantification of C3a and C3a/C3 ratios in cerebrospinal fluid.
Conclusion: We report a dual fluorescent ELISA (FELISA) enabling the simultaneous quantification of C3 and C3a and the C3a/C3 ratio in biological samples. This assay is readily adaptable to measurement of other C3 fragments or multiplexing for measurement of other complement components and activation products.
P-25
Cerebrospinal fluid complement system biomarkers in Alzheimer’s disease.
Aurora Veteleanu1, Vinira Wijesuriya1, Aitana Sogorb-Esteve2, Henrik Zetterberg2, Wioleta Zelek1, Paul Morgan1
1Cardiff University, Cardiff, United Kingdom. 2University College London, London, United Kingdom
Background: Complement is implicated in Alzheimer’s Disease (AD) as a neuroinflammatory driver, with complement activation evident in plasma. Evidence of complement involvement from cerebrospinal fluid (CSF) measures, which may more accurately reflect changes in brain, is currently lacking. Few studies have measured CSF complement levels, and none have comprehensively explored CSF complement proteins as AD biomarkers. To address this, we applied our complement analyte panel to CSF samples from two sources.
Objective: CSF samples were obtained from two independent cohorts, one comprising very early onset AD (mean age 48.5 years; Gothenburg) and the other later onset (mean age 63 years; UCL), both with age-matched cognitively healthy controls. Neurodegeneration markers, complement proteins and total protein were measured and disease-related changes identified.
Materials and methods: Complement proteins (C1q, C1s, C3, C4, C9), regulators (Factor I (FI), Factor H (FH) , FH-related proteins 4 (FHR4), Properdin, Clusterin, C1 Inhibitor (C1Inh) and activation products (iC3b and TCC) were measured and quantified in CSF samples using our established ELISA set. CSF neurodegeneration markers amyloid beta (Aβ), phosphorylated tau (p-Tau), total tau (t-Tau) were measured in the Gothenburg set using the Innotest ELISA. Total protein was measured using the BCA assay and results were expressed either uncorrected or relative to total protein (μg/mg).
Results: CSF t-Tau and p-Tau were elevated and Aβ reduced in AD versus control CSF samples in both cohorts. All fourteen of the complement proteins were quantifiable in CSF using the ELISA set. AD CSF had increased levels of complement proteins (C1q, C3,C4) and regulators (Clusterin, Factor H) across both cohorts, although these differences were lost when corrected for total CSF protein. Complement activation products (iC3b, TCC) were not significantly different from controls in either cohort.
Conclusions: We show that all tested complement analytes are present in and can be reliably measured in CSF in health and disease. Elevated levels in AD CSF compared to controls were lost when corrected for total protein, implying blood-brain barrier leak. Complement activation products were not elevated in CSF in the two cohorts, implying absence of significant complement activation in CSF in these early AD cohorts.
P-26
Reconstitution of a fully functional recombinant membrane attack complex for molecular biology studies
Bill Ho, Suzanne Wang, Charles Bayly-Jones, Bradley Spicer, Michelle Dunstone
Monash Biomedicine Discovery Institute, Clayton, Australia
The membrane attack complex is one of the main complement effectors and is known to be associated with a plethora of inflammatory diseases. Currently, the tools used to study the significance of the MAC are limited to either serum or by adding serum-purified components C5b6, C7, C8 and C9. These systems are limited to native forms and may present confounding factors by other complement components, contaminant proteins and potential loss of protein activity.
Here, we have developed a completely sufficient recombinant MAC system that exhibits all the known characteristics of serum-derived proteins with respect to both function and binding interactions. We can control for MAC initiation in vitro with a stable and soluble C5 convertase, which activates C5 to form C5b6 in the presence of C6. This C5b6 complex can form MAC pores in the presence of C7, C8 and C9 on both erythrocytes and artificial membranes, such as liposomes.
Overall, the recombinant MAC can be used as an in vitro tool to investigate the molecular mechanism of the MAC or in animal models to investigate the role of the MAC in inflammation. Furthermore, the system enables mutagenesis studies to investigate the portions of the pore that are responsible for inflammation.
P-27
C3 and C3a as Indicators of Complement Dysregulation in Tauopathies and FTLD-Associated Disorders.
Laura Nicholls1, Jacqui Nimmo1, Rebekah S. Cooke1, Taine J. Baggott1, Matthew Bright1, Robert A.J. Byrne1, Gareth D. Fenn1, Samuel Keat1, Weronika J. Forys2,3, John T. O'Brien4, James B. Rowe2,5, B. Paul Morgan1, Maura Malpetti2,3, Wioleta M. Zelek1
1UK DRI at University of Cardiff, Cardiff, United Kingdom. 2Department of Clinical Neurosciences, Cambridge University, Cambridge, United Kingdom. 3UK DRI at University of Cambridge, Cambridge, United Kingdom. 4Department of Psychiatry, Cambridge University, Cambridge, United Kingdom. 5Medical Research Council Cognition and Brain Sciences Unit, Cambridge, United Kingdom
Background: Dementia encompasses a range of neurodegenerative diseases (NDDs) presenting with progressive decline in memory, cognition, language and ability to complete daily tasks. Whilst Alzheimer’s disease (AD) is the most common form of dementia, less prevalent subtypes include frontotemporal lobar degenerative associated disorders (FTLD) frontotemporal dementia (FTD), corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP). FTLD are NDDs characterized by progressive atrophy and neuronal loss in brain frontal and temporal lobes, predominately affecting behavior, language and cognition. Of these, FTLD, CBS and PSP are primary tauopathies, pathologically classified by predominant accumulation of aggregates of tau, a microtubule-associated protein responsible for mediating intracellular transport and structure. Some FTD subtypes, such as Pick’s disease, are also defined by tau pathology. Although complement dysregulation is broadly accepted as a feature of AD, this has not been convincingly shown in tauopathies and FTLD. Here we measured a panel of complement biomarkers in serum from FTLD and tauopathy cases and compared these with non-demented matched controls (HC).
Materials and Methods: In-house ELISAs were used to quantify twelve complement analytes, comprising activation products (C3a, iC3b, TCC), regulators (Factor H (FH), Properdin, Clusterin) and components (C1q, C3, FB, C5, C7, C9) in serum samples. Samples were matched for age and sex from FTLDs: CBS (n=16), FTD (n=57), PSP (n=42), healthy controls (HC; n=41).
Results: Compared to HC, C3, C3a and properdin were elevated in FTLD (p=0.0002, p=0.0187, p=0.0451 respectively). Other complement analytes were not significantly different between FTLD and HC samples. Subgroup analysis demonstrated significantly higher C3 and C3a compared to HC in FTD (p<0.00001 and p=0.0455 respectively) and PSP (p=0.0051 and p=0.0179 respectively) but not CBS, while properdin was significantly increased only in FTD cases (p=0.0097).
Conclusions: The data demonstrates a degree of complement dysregulation in tauopathies and FTLD across the different disease subtypes with C3, C3a and properdin levels significantly different to matched controls. These findings suggest dysregulation in the alternative pathway and lead us to suggest that complement dysregulation represents a potential therapeutic target for FTLD and tauopathies.
P-28
A Novel Tool for Complement Activation Profiling in AAV Gene Therapy
Klaudia Kulak, Viktoria Kozma, Cecilia Klint, Camilla Nilsson, Selvi Celik, Hannes Brehm, Samuel Butler, Jordi Rodo
SVAR, Malmö, Sweden
Gene therapy utilizing adeno-associated virus (AAV) vectors demonstrates excellent safety profiles and remarkable clinical efficacy in treating diverse diseases. Despite its relative safety, evidence from clinical trials has shown that AAV can trigger severe life-threatening inflammatory responses, especially when administered intravenously at high doses. Many of these adverse reactions are associated with the secondary activation of the complement system, leading to complications such as acute kidney injury due to atypical hemolytic uremic syndrome–like (aHUS-like) complement activation, thrombotic microangiopathy (TMA), thrombocytopenia, and immune-mediated myocardial inflammation. Though AAV-mediated complement activation is a significant safety concern, we still lack understanding of the activation mechanism and a predictive method to prevent or mitigate activation in administered patients. In this context, our research has been focused on the development and evaluation of an ELISA-based AAV-mediated Complement Activation assay. The technique combines a serotype specific AAV-coated plate with a tailored buffer composition, selectively allowing evaluation of the distinct classical, lectin, or alternative complement system pathways. The assay detects de novo-generated terminal complement complex (TCC) deposition, making it a robust tool for testing and predicting complement reactions to AAV in analyzed sera.
Utilizing this assay, we demonstrated a clear dose-response relationship between complement activation and different anti-AAV antibody sources, including both IgG and IgM. Experiments using C3- and Factor H-depleted sera confirmed the specificity of complement responses and highlighted a potential role for the alternative pathway in AAV-mediated complement activation. Moreover, the assay effectively captured individual complement system characteristics in response to AAV, independent of baseline complement activity within the sample, demonstrating that it measures the formation of newly generated complement components specifically induced by AAV. Human sera from 100 healthy donors were screened for complement activation in response to AAV2 and AAV9, revealing variability in individual responses. Importantly, the AAV-COMPL assay demonstrated high intra-assay and inter-assay precision for both AAV2 and AAV9.
Given the complexity of serum interactions with anti-AAV antibodies and the complement system, our assay holds promise in shedding light on pivotal immune interactions and offers valuable insights to enhance the safety and efficacy of AAV-based gene therapies.
P-29
Advanced Assay Platform for Antibody Development: Precision Assays for ADCC, ADCP, and CDC
Christoffe Lallemand1, Caroline Grygar1, Ulrich Mayer2, Michael Schwenkert2, Lone Frier Bovin2
1Svar, Paris, France. 2Svar, Malmö, Sweden
Early in therapeutic antibody development, it is crucial to characterize their mode of action (MoA). The Fc regions of antibodies significantly influence their therapeutic characteristics, including half-life and cytotoxic effects like ADCC, ADCP, and CDC. Traditional ADCC and ADCP bioassays rely on human primary cells, which suffer from donor variability, limited expansion capacity, and labor-intensive culturing. To streamline this evaluation, we have developed advanced assays to assess ADCC, ADCP, and also CDC activities. Our Fc Effector assay platform uses reporter gene effector cells in combination with engineered target cells. Furthermore, to assess CDC, we have engineered target cells based on a B-cell line expressing no or only low levels of membrane-bound complement inhibitors (CD46, CD55 and CD59). The readout is based on Svar luciferase (SL) constitutively expressed by the target cells, which has some unique features like a bright signal and enhanced stability allowing its accumulation in cell culture medium upon killing without loss of enzymatic activity. This cell line can be rapidly customized to express a target of interest and allows to measure cell killing via CDC but also by primary T- and NK cells or CAR-T cells.
We showcase the use of these target cells to assess ADCC and ADCP with the above-described surrogate reporter gene systems and also complement-dependent cytotoxicity induced by rituximab, daratumumab, alemtuzumab and other drug antibodies.
P-30
Complement activation during the early postpartum period in healthy pregnant women.
Norimitsu Inoue1, Hiroshi Tsujimoto1, Katsuki Ohtani2, Takahiro Yamashita3, Tomoko Adachi3, Nobutaka Wakamiya4
1Department of Molecular Genetics, Wakayama Medical University, Wakayama, Japan. 2Department of Clinical Nutrition, Rakuno Gakuen University, Ebetsu, Japan. 3Aiiku Maternal and Child Health Center, Aiiku Hospital, Tokyo, Japan. 4Department of Medicine and Physiology, Rakuno Gakuen University, Ebetsu, Japan.
Background: Pregnant women are known to have a unique immune system to tolerate their immunologically semi-allogeneic fetus. However, it is not yet clear how the complement system fluctuates throughout the gestational period. In this study, we prospectively collected blood from healthy pregnant women and measured their plasma complement levels over time, from the first trimester to one month after delivery.
Methods: We enrolled 115 healthy pregnant women without any complication managed at Aiiku Hospital. Peripheral blood samples were collected at 7 points: the first, second, and third trimester, the first stage of labor, postpartum (1 to 3 days after delivery), one week and one month after delivery. Plasma C3, C4, CFH, CFI, Ba, sC5b-9, C5a and CH50 levels and C1 inhibitor activity levels were measured. The differences between the mean levels at each time point and the complement levels in healthy non-pregnant women held by the Japanese Society for Complement Research were compared.
Results: After excluding cases with miscarriages and complicated pregnancies, plasma complement levels were analyzed in 36 healthy pregnant women for whom plasma samples were obtained at all seven time points. C3 was higher than that of non-pregnant women throughout the entire gestational period. sC5b-9 remained relatively high from early pregnancy and increased markedly in the first week after delivery. C4, CH50, and Ba were similar to those of non-pregnant women until antepartum, but increased after delivery, reaching their highest levels at 1 week postpartum. CFH and CFI were also higher from the second trimester to antepartum, but CFH temporarily decreased after delivery and increased again at 1 week postpartum. C1-INH decreased from the second trimester and recovered after delivery. All other parameters except C3 recovered to non-pregnant levels at 1 month postpartum.
Conclusion: Pregnant women have an increased production of complement components, with a decrease in CFH and an increase in C4, Ba and sC5b-9 after delivery. It has been reported that some complement diseases tend to be induced during pregnancy and atypical hemolytic uremic syndrome can develop after delivery. Our results suggest that excessive activation of the complement system after delivery may induce these maternal complications.
P-31
Novel antibodies and assays to ascertain the role of C4 protein variants in Schizophrenia risk
Kirsten L Baillie1,2,3,4, Robert A.J Byrne3,4, Erik J.M Toonen5, Laura J Westacott2, B. Paul Morgan3,4, Wioleta M Zelek3,4
1Hodge Centre for Translational Neuroscience, Cardiff, United Kingdom. 2Division of Psychological Medicine and Clinical Neurosciences, Cardiff, United Kingdom. 3Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom. 4UK Dementia Research Institute at Cardiff University, Cardiff, United Kingdom. 5Hycult Biotech, Uden, Netherlands
Schizophrenia is a chronic psychiatric disorder affecting over 21 million people worldwide and conferring huge economic/social costs. Schizophrenia is highly heritable (~80%)1; GWAS identified strong genetic association in the major histocompatibility complex, later localized to the C4 gene2. C4 is duplicated; C4A and C4B encode C4 proteins that differ by four amino acids in a six amino acid stretch in C4d, conferring distinct chemical reactivities. Schizophrenia risk correlated with C4A copy number2. The capacity to measure C4A and C4B in fluids and tissues would aid diagnosis and management but antibodies that distinguish the isoforms are unavailable.
Over 100 donors were screened by PCR to identify individuals lacking either C4A or C4B, one C4AQ0 donor was identified and a C4BQ0 donor was gifted by Prof Garred (University of Copenhagen). C4 protein was purified by classical methods and functionality confirmed by addback to methylamine-treated human sera.
Novel monoclonal mouse antibodies were generated by hybridoma technology utilizing recombinant C4d as the immunogen. Two mAbs, E8 and D5H3, showed preferential binding to C4B. Sandwich ELISAs using E8 or D5H3 as capture and polyclonal anti-C4 detect failed to detect C4 in C4BQ0 plasma with normal levels detected in C4AQ0 plasma, implying specificity for C4B. Furthermore, serum depleted on an E8/D5H3 column retained haemolytic activity and residual C4 in westerns and ELISAs utilizing anti-C4 pAbs. We have obtained the necessary standard by elution of C4B from an E8/D5H3 column and quantified plasma C4B using the specific ELISA in a healthy cohort. Work is ongoing to produce C4A-specific mAbs and a C4A ELISA.
We have generated C4B-specific mAbs, purified the necessary proteins and identified C4-isotype donors for mAb characterization and C4 isotype quantification. The completed assays will enable analysis of C4 isoform protein levels in schizophrenia and other contexts.
PMID:10813800
PMID:26814963
References
PMID:10813800, PMID:26814963
P-32
Complement signaling as a T-cell checkpoint in the tumor microenvironment
Thejaswini Giridharan1, Sora Suzuki2, ANM Nazmul Khan3, Tiffany Emmons4, Michael Yaffe4, Xiaokang Lun4, Brian Joughin4, Martin Kolev5, Pascal Deschatelets5, Claudia Kemper6, AJR McGray2, Emese Zsiros2, Brahm Segal3
1University of Michigan, Ann Arbor, USA. 2Roswell Park Comprehensive Cancer Center, Buffalo, USA. 3Moffitt Cancer Center & Research Institute, Tampa, USA. 4Massachusetts Institute of Technology, Cambridge, USA. 5Apellis Pharmaceuticals, Waltham, USA. 6National Institute of Health, Bethesda, USA
Background: Durable anti-tumor immunity requires the recruitment, infiltration, and activation of antigen-directed T-cells within the tumor. Cancer immunotherapy is largely based on the expansion and persistent activation of T-cells within the tumor microenvironment (TME) and overcoming barriers to anti-tumor immunity. Using malignant effusions (MEs) as an authentic component of the TME, we observed that neutrophils acquire a complement-dependent T-cell suppressor phenotype characterized by inhibition of signaling and metabolic pathways necessary for T-cell activation and proliferation. These results contributed to the rationale for the first clinical trial of C3 and C3b inhibition in cancer (NCT04919629). An important question related to the clinical trial, and more broadly to our understanding of complement’s effects on tumor immunity, is the direct effect of complement signaling on T cells in the TME independent of neutrophil suppressor function.
Methods and Results: Ascites fluid supernatants (ASC) from patients with newly diagnosed ovarian cancer inhibited CD3/CD28-stimulated T-cell cytokine production (IL-2, interferon-γ, and IL-10) and metabolism (but not proliferation) in the absence of neutrophils. ASC-induced suppression of stimulated T-cell cytokine responses was dependent on complement activation. A similar effect occurred in MEs from other solid tumors, demonstrating generalizability. C3 activation inhibitors restored stimulated T-cell cytokine responses and metabolic function. Factor B and factor D inhibition each rescued cytokine responses, demonstrating that suppression was alternative pathway dependent. C3b/iC3b deposition on T-cells was increased with ASC exposure but reduced by C3/C3b inhibition or by addition of recombinant complement receptor-1 (rCR-1). Moreover, rCR-1 fully abrogated the suppressive effect of ASC on T-cells while rCD46 had no significant effect. rCR-1 may function both as a decoy for membrane-bound CR-1 signaling on T-cells and a complement activation inhibitor through its direct interactions with C3b and C4b. Phospho-CyTOF analysis showed that complement signaling in the TME inhibited stimulated phosphorylation of multiple protein targets required for T-cell activation.
Conclusion: These results show a novel complement-driven T-cell checkpoint in the TME and support a model in which critical T-cell effector functions and metabolism are impaired by neutrophil and complement signaling cues in the TME that cumulatively result in T-cell non-responsiveness.
References
Acknowledgements: R01CA267690 (to Emese Zsiros and Brahm Segal) and Apellis Pharmaceuticals
P-33
Distinct sites of complement activation and regulation in the tumor microenvironment
Joanne van Keulen1,2,3, Julie Alles1, Esther de Boer1,2, Gerard van Mierlo1, Liudmila Kodach4, Taco Kuijpers1,5, Richard Pouw1,2,6, Jan Paul de Boer3
1Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands. 2Amsterdam institute for Immunology and Infectious Diseases, Inflammatory diseases, Amsterdam, the Netherlands, Amsterdam, Netherlands. 3Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam, the Netherlands, Amsterdam, Netherlands. 4Department of Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands, Amsterdam, Netherlands. 5Department of Pediatric Immunology, Rheumatology, and Infectious Diseases, Emma Children’s Hospital, Amsterdam University Medical Centers, Amsterdam, the Netherlands, Amsterdam, Netherlands. 6Sanquin Diagnostic Services, Amsterdam, the Netherlands, Amsterdam, Netherlands
The role of the complement system in the tumor microenvironment (TME) remains inconclusive. Complement activation could result in antitumor responses through complement-dependent cytotoxicity of tumor-targeting antibodies. However, complement may also promote tumor development by supporting chronic inflammation and hampering antitumor immune responses. To elucidate these opposing effects it is important to determine local complement activation and regulation. This was conducted by analyzing the presence of membrane-bound complement regulatory proteins (mCRP) and distinguishing the C3 activated fragments (C3b) from the C3b regulated fragments (iC3b, C3dg) in human colorectal cancer (CRC) and head and neck cancer (HNC).
To distinguish C3 activated or regulated fragments, in house anti-C3 antibodies were characterized. Subsequently, CRC and HNC human tissue was stained with the specific anti-C3 antibodies and stained for mCRP CD46, CD55 and CD59. In addition, the presence of various (immune) cell types was determined. In CRC, we observed that C3 deposition was located in vimentin positive areas, indicating that mesenchymal tissue activated complement instead of epithelial tumor tissue. For HNC, we observed C3 deposition in mesenchymal tissue as well. However, C3 deposition was adjacent to dividing cells, which was not detected in CRC. Moreover, we identified distinct regions of C3 activation and C3 regulation in CRC and HNC. Regarding the presence of mCRPs, in CRC we showed expression of CD46 only on tumor cells and CD59 mainly on endothelial cells. In HNC, we could not detect the presence of mCRPs.
To conclude, we are able to distinguish and quantify sites of activated and regulated C3 in the TME of CRC and HNC. Furthermore, CRC showed different characteristics compared to HNC. The high abundance of CD46 on tumor cells in CRC, together with the poor penetration of complement within the tumor, suggests a crucial role for CD46 in supporting CRC. This is contrasted by HNC, where CD46 expression is virtually absent, highlighting again the ambiguous involvement of complement in tumor immunology. More research is needed to connect the effect of complement activation and regulation to disease progression, immune cell infiltration and responsiveness to therapies.
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Integration of Structural Modeling and CADD Scores Improve Variant Classification for CFH SCRs 5-18
Cobey Donelson1, Hector Martín Merinero1, Seth Welsh1, Santiago Rodríguez de Cordoba2, Richard Smith1, Yuzhou Zhang1
1University of Iowa, Iowa City, USA. 2Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
Background:
Complement Factor H (FH) is a soluble glycoprotein consisting of 20 short consensus repeats (SCRs) organized into three functional regions: N-terminal SCRs 1-4 accelerate the decay of the C3-convertase of the alternative pathway (AP) and act as a cofactor for Factor I-mediated inactivation of C3b; C-terminal SCRs 19-20 recognize cell surfaces by binding sialic acids and deposited C3b; and mid-region SCRs 5-18, the function of which remain poorly characterized. Here, we used a structural prediction algorithm in conjunction with Combined Annotation Dependent Depletion (CADD) scores to provide additional evidence for variant classification in SCRs5-18.
Methods:
As no experimentally determined structure is available for FH SCRs 5-18, we generated a model using AlphaFold3 (AF3) and optimized it with ForceFieldX (FFX) to reduce steric clashes and improve side-chain conformations. We next experimentally validated known pathogenic and benign variants as training sets to determine the impact of missense mutations on protein stability as predicted by ddGun. We used classification thresholds for likely pathogenic (CADD score > 20 and |ddGun| > 1.0 kcal/mol) and likely benign variants (CADD score < 10 and |ddGun| < 0.5 kcal/mol) based on previous studies (1, 2). These thresholds were subsequently applied to evaluate 994 VUSs identified in gnomAD.
Results:
Our pathogenic training set consisted of 9 variants while the benign had 41 variants. Our combined approach demonstrated a positive predictive value (PPV) of 89% with an error rate of 11%, and a negative predictive value (NPV) of 59% with an error rate of 41% for the test sets. When our methods were applied to the 994 VUSs, we identified 77 (8%) variants supporting a likely pathogenic classification; 8 replace highly conserved residues and 16 are within five positions of such residues. Additionally, we identified 340 variants (34%) with evidence supporting their reclassification as likely benign.
Conclusion:
Our study demonstrates the feasibility of combining structural modeling and stability predictions for variant interpretation in CFH SCRs 5-18. Future research should focus on expanding the training set and experimental validations. These findings highlight the importance of domain-specific approaches in evaluating CFH variants to improve diagnosis for complement-mediated diseases.
References
(1)Martin Merinero, et al.(2021). "Functional characterization of 105 factor H variants associated with aHUS: lessons for variant classification." Blood 138(22): 2185-2201. (2)Tollefson, M. R., et al. (2023). "Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome." Hum Genet 142(6): 819-834.
P-35
Comparison of complement activation products and eosinophils between bicarbonate-based and lactate-based peritoneal dialysis (PD) solutions in PD patients during introduction periods.
Masafumi Suzuki, Hangsoo Kim, Sosuke Fukui, Satoshi Ototake, Yasuhiro Suzuki, Masashi Mizuno
Nagoya University Graduate School of Medicine, Nagoya, Japan
Background
We previously reported temporary increase of eosinophils in peritoneal dialysis (PD) fluid (PDF) accompanied with C3a elevation in pH-adjusted PDF with lactate buffer (L-PDF) overt half of patients during PD initiation period. Recently, pH-adjusted PDF with bicarbonate-based buffer was introduced in Japan. In the present study, we investigated the effects of the different buffers for increase of eosinophils and the complement activation products in PDF during the initiation timing of PD.
Methods
We compared effects of B-PDF (group B) with L-PDF (group L); 28 patients on PD in group B who started PD in the University hospital and 28 patients from 2014 to 2016. Number of eosinophils, levels of the complement activation products, levels of IL-5 and IL-6 in PDF during overnight dwell were investigated on days 1, 2 and 4 after staring PD. As a reference, we used relevant data in 28 PD patients on PD with L-PDF (group L) in the previous report (ref .1) to compare each factor between groups B and L. We also analyzed between groups B and L in patients who started PD less than 30 days after PD catheter insertion (groups EB and EL, respectively).
Results
Number of eosinophils significantly correlated with C3a in PDF during the initiation periods. Increase of eosinophils was observed under usage of B-PDF as same as L-PDF after starting PD. When comparing between groups B and L, levels of IL-5 in PDF was significantly decreased in group B, but not eosinophils, IL-6, C3a, C5a, and sC5b-9. In contrast, number of eosinophils, levels of IL-5, C3a and sC5b-9 but not C5a, in PDF of group EB were significantly less than group EL on days 1, 2 and/or 4 after starting PD.
Conclusion
From our results, increase of eosinophils in PDF was frequently observed during initiation of PD even if buffer in PDF changed from lactate to bicarbonate. Compared with L-PDF, B-PDF might contribute to improve response of eosinophilia and the C system when PD started less than 30 days after PD catheter insertion.
References
Shigemoto E, et al. Perit Dial Int. 2019.
P-36
Microglial Complement C5aR1 signaling drives inflammasome mediated neuropathology in Parkinson’s disease
Eduardo Albornoz1, Richard Gordon2, Ibrahim Javed3, Gabriela Bodea4, Vinod Kumar1, Cedric Cui1, Julio Aguado5, Karine Mardon6, Rajiv Bhalla6, Gary Cowin6, Trent Woodruff1,4
1School of Biomedical Sciences, The University of Queensland, Brisbane, Australia. 2Queensland University of Technology, Brisbane, Australia. 3University of South Australia, Adelaide, Australia. 4Queensland Brain Institute, The University of Queensland, Brisbane, Australia. 5University of Colorado Anschutz medical campus, Denver, USA. 6Centre for advanced imaging, The University of Queensland, Brisbane, Australia
Parkinson's disease (PD) is one of the fastest-growing neurological disorders, characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein (Syn) aggregates. Immune-mediated neuroinflammation driven by microglia contributes to PD pathology. We previously showed that inhibiting the microglial NLRP3 inflammasomes prevents Syn pathology in mice, but the role of the complement system remains unexplored. Using transcriptomic and proteomic data, we identified widespread upregulation of complement in PD patient brains at sites of dopaminergic neuron loss and in peripheral blood. We demonstrated that fibrillar Syn activates complement in human plasma, with proteomic studies supporting C5a as a key factor upregulated in PD patient serum, along with markedly increased C5aR1 expression in human and mouse microglia.
In three distinct preclinical PD models, complement and microglial C5aR1 were upregulated following dopaminergic degeneration in the nigrostriatal pathway. Genetic deletion of key complement effectors at the C3, C5, and MAC levels in a neurotoxin-based PD model highlighted a critical role for C5aR1 in neurodegeneration. To test the therapeutic potential of this pathway, we first examined the efficacy of the C5aR1 inhibitor, PMX205, in reducing neuropathology and motor dysfunction in the 6-OHDA model. We correlated behaviour, microglial activation, and dopamine spatial distribution using mass spectrometry imaging (MALDI-MSI), showing neuroprotection in 6-OHDA-injected human C5aR1 knock-in mice and in wild-type mice orally dosed with PMX205. Next, we tested PMX205 in a 12-month Syn fibril model of PD. In parallel, we utilized F18-DPA-714 PET/CT imaging to visualize microglial activation in living mice. We identified that prophylactic (0-12 months) and therapeutic (4-12 months) oral administration of PMX205 ameliorated motor deficits, dopaminergic neurodegeneration, and F18-DPA-714 signals in this model.
Mechanistically, we show that Syn triggered cell intrinsic microglial C5a-C5aR1 engagement, leading to NLRP3 inflammasome activation via potassium efflux. This process was impaired in the absence or inhibition of C5aR1 in human and mouse microglia, suggesting that persistent complement activation by fibrillar Syn exacerbates disease pathology. Our findings indicate that targeting complement and C5aR1 with pharmacological inhibitors could mitigate microglial-driven neuroinflammation, offering a potential strategy to slow or halt disease progression in PD.
P-37
The Fc fragment of soluble IgMs binds C1q to activate the first step of the classical complement pathway, while inhibiting complement-dependent cytotoxicity
Andrea J. Pinto1, Chantal Dumestre-Pérard2, Anne Chouquet1, Isabelle Bally1, Véronique Rosse1, Nicole Thielens1, Wai-Li Ling1, Renate Kunert3, Christine Gaboriaud1, Jean-Baptiste Reiser1
1Institut de Biologie Structurale, UMR 5075, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, France. 2Laboratoire d’Immunologie, Pôle de Biologie, CHU Grenoble Alpes, Grenoble, France. 3Department of Biotechnology, BOKU University, Vienna, Austria
Background
Soluble type-M immunoglobulins (IgMs) are among the most potent activators of the classical pathway of the complement, playing a crucial role in complement-dependent cytotoxicity (CDC). Like other immunoglobulin classes, they comprise a heavy and a light chain but uniquely form highly oligomerized structures - either pentamers ((H2L2)5J)), which include the additional Joining (J) chain, or hexamer ((H2L2)6). Their distinct assemblies are made possible by the fragment crystallizable (Fc) region, or Fc-core, which interacts with cellular or soluble immune receptors to activate their effector functions, notably C1q and the complement cascade. These properties make IgM Fc-core inspiring candidates for novel therapies targeting autoimmune and inflammatory diseases. However, challenges in recombinant production and characterization of engineered molecules, as well as uncertainties regarding their functional properties, necessitate a deeper understanding of their mechanisms of action.
Methods
We performed biochemical and functional in vitro characterization of IgM Fc-core fragments in their pentameric and hexameric forms. Using biophysical methods, in-house enzyme-linked immunosorbent assay (ELISA) and hemolytic assay for detection of complement activity, we assessed their structural integrity, C1q-binding capacity, and functional activities.
Results
Biophysical analysis confirmed the essential role of the J chain in promoting homogenous pentamer formation, whereas its absence led to heterogeneous oligomers. By combining size-exclusion chromatography with mass photometry, we enriched samples containing IgM hexamers or pentamers without the J chain. Biolayer interferometry demonstrated that both IgM-Fc forms bind C1q, while ELISA showed their ability to induce in vitro C4b deposition on solid phase. Additionally, our data confirmed the higher efficacy of IgM hexamers compared to pentamers in activating the first component of the classical pathway. Importantly, hemolytic assays revealed the ability of IgM-Fc constructs to inhibit Ig-induced complement-dependent cytotoxicity, likely due to the absence of antigen-binding Fab regions.
Conclusions
Taken together, these findings suggest a possible mechanism by which IgM cores can sequester C1 complex in plasma, leading to consumption of the initial complement component C4 and preventing CDC. Our study provides important insights for the development of IgM-based anti-inflammatory molecules specifically targeting complement activation.
P-38
A Novel Immunoassay Platform to Study the Dynamics of the Alternative Pathway
Boglár Gál1, Beatrice Fageräng1, Laura Pérez-Alós1, Anne Rosbjerg1, Tom Eirik Mollnes2,3, Peter Garred1
1Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. 2Department of Immunology, Rikshospitalet University Hospital, Oslo, Norway. 3Research Laboratory, Nordland Hospital Bodø, Bodø, Norway
Background:
The complement system is a key component of innate immunity, activated by pathogen- or danger-associated molecular patterns. The alternative pathway (AP) acts as an amplification loop for the other complement pathways and plays a central role in immune regulation and inflammation. Due to its critical function, components of the AP are attractive therapeutic targets. This study aimed to develop immunoassays for core AP proteins, with a focus on the C3 convertase (C3bP), to enhance mechanistic understanding and support therapeutic development.
Methods:
Monoclonal antibodies were generated via hybridoma technology. A novel antibody clone (11-13-05) targeting both native and complexed Factor P, was employed for assay development. The C3bP complex was measured using 11-13-05 as a capture antibody and polyclonal anti-C3c for detection, with zymosan-activated serum (ZAS) as standard. Total C3 was quantified using a dual anti-C3c/C3 antibody setup and purified C3 as standard. C3 activation products were detected using an in-house neoepitope-specific monoclonal antibody (bH6). Assays were validated in various sample types including serum, ZAS, EDTA plasma, and E. coli-activated lepirudin plasma. Gel filtration chromatography was used to assess complex formation, while immunoprecipitation followed by SDS-PAGE and Western blotting verified protein interactions.
Results:
All target components were consistently detected and stable across sample types. A time-dependent increase in C3bP was observed in E. coli-activated plasma, confirming progressive AP activation. The bH6 antibody did not bind to C3bP, suggesting specificity for fluid-phase active C3. Gel filtration revealed C3, Factor P, and limited Factor B co-eluting, but most Factor B appeared unbound. Western blot and ELISA confirmed this, with Factor B primarily found in the soluble fraction, not stably associated with the convertase complex.
Conclusion:
The developed immunoassay platform effectively quantifies key AP proteins and activation products while providing insight into convertase dynamics. These findings indicate that Factor P and Factor B may not form stable associations within the C3bP complex, suggesting new avenues for studying AP regulation and therapeutics.
P-39
Novel Quantitative ELISA Reveals Dynamics of Pro-Factor D Conversion During Alternative Complement Pathway Activation
Tereza Alica Plchová1, Anne Rosbjerg1, Beatrice Fageräng1, Sophie Peen1, Mikkel-Ole Skjoedt2, Peter Garred1
1Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. 2Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
Background:
Factor D (FD) is a serine protease essential for activating the alternative complement pathway (AP). Synthesized as an inactive zymogen (pro-FD), it must be cleaved to form active FD, enabling the formation of the AP C3 convertase. The presence of pro-FD in circulation has recently been discovered, but knowledge about its specific amount and conversion rate in circulation remains limited. Thus, we aimed to develop an ELISA capable of distinguishing pro-FD from total FD and study the conversion of pro-FD to FD during complement activation.
Methods:
A pro-FD-specific monoclonal antibody (mAb) was generated in mice immunized with a synthetic peptide unique to the pro-form of FD. Its specificity was confirmed using recombinant pro-FD and recombinant mature FD. This mAb and a general FD mAb were used to construct a pro-FD-specific sandwich ELISA. EDTA plasma from healthy donors was analyzed to determine baseline pro-FD: total FD ratios. Human serum was stimulated with either zymosan (alternative pathway activator) or mannan (lectin pathway activator) and incubated at 37 °C to assess conversion dynamics. Samples were collected at 0, 0.5, 4, and 24 hours.
Results:
The developed assays reveal that pro-FD accounts for ~33% of total FD in the circulation (approx. 0.5 µg/mL). Over a 24-hour timeline, a steady decrease in pro-FD was found in serum, while no change in total FD was found, indicating a steady conversion of pro-FD into FD. Setting the pro-FD baseline of normal human serum to 100% (0 hours), levels decreased to 54% at 4 hours, and 7% at 24 hours in zymosan-activated serum. Mannan stimulation resulted in a more moderate decline, with pro-FD levels falling to 61% at 4 hours and 24% at 24 hours.
Conclusion:
We have developed a novel, specific ELISA for pro-FD quantification and, for the first time, can show the relationship between pro-FD to FD conversion during complement activation. A consistently lower quantity of pro-FD was seen in the zymosan compared with mannan-activated serum, revealing differences in the efficiency of activating the AP loop. Its application may reveal new insights into dysregulated complement activation and identify potential therapeutic targets.
P-40
High-dose radiotherapy modulates the expression and secretion of complement (regulatory) proteins by tumor cells
Magnus Trottnow1, Teresa Irianto1,2, Lisa Deloch1,2, Rainer Fietkau3,2, Udo S. Gaipl1,2, Michael Rückert1,2
1Translational Radiobiology, Department of Radiation Oncology, Universitätsklinikum Erlangen, Erlangen, Germany. 2Comprehensive Cancer Center Erlangen-EMN, Erlangen, Germany. 3Department of Radiation Oncology, Universitätsklinikum Erlangen, Erlangen, Germany
Background
The complement system plays a dual role in cancer by facilitating tumor cell recognition and leukocyte activation, leading to tumor cell elimination, while also potentially creating an immunosuppressive environment through key proteins such as C3, C3a, and its receptor C3aR. Despite ionizing radiation’s known immunomodulatory effects, its influence on tumor-driven complement regulation remains unclear. This study aimed to investigate how ionizing radiation shapes the regulation of the complement system in tumor cells.
Methods
Three tumor cell lines (MN/MCA1 sarcoma, B16 melanoma, and 4T1 breast cancer) were irradiated with doses ranging from 0.01 to 10 Gy. We analyzed the expression and secretion of complement components and regulatory proteins - including C3, C1-INH, CFH, CD35, CD55, and CD59a - using flow cytometry, ELISA, and qPCR. To assess the activation of the complement cascade, cells were cultured in the presence of mouse serum. The binding of complement fragments (C3b/iC3b/C3c) on the tumor cell surface 2 h after irradiation was then analyzed by flow cytometry.
Results
Only 4T1 cells released significant amounts of C3 and CFH when irradiated with higher doses of 2-10 Gy. However, a significant upregulation of the surface expression of the complement regulatory protein CD35 was observed on all three tumor cell lines after a high radiation dose of 10 Gy at 24 hours. The upregulation of CD59a on B16 and MN/MCA1 and the upregulation of CD55 on 4T1 were also significant 24 hours after 10 Gy irradiation. These results were supported by qPCR data. Complement binding assay showed no direct effect of radiotherapy on C3b binding to any of the three tumor cell lines.
Conclusion
These data indicate that high doses of ionizing radiation induce cell type-specific modulations of the complement system by tumor cells. The radiation-induced upregulation of complement regulatory proteins and the selective secretion of complement components by 4T1 cells suggest that tumor cells may adopt mechanisms to protect themselves from complement-mediated lysis. Although radiotherapy did not immediately alter the binding of activated complement fragments, there may be differences at later time points when tumor cells begin to die, which will be investigated next.
References
Acknowledgment: This work was funded by the German BMBF (Federal Ministry of Education and Research; 02NUK091B, ENDORSE) and the Interdisciplinary Center for Clinical Research (IZKF) at the Universitätsklinikum Erlangen (MD-Thesis Scholarship)
P-41
The Four Functional Segments of Factor H:Role in Physiological Target Recognition and Contribution to Disease
Peter Zipfel1, Karin Heidenreich2, Margarida Mesquita2
1Leibniz Institute for Natural Product Research, Jena, Germany. 2eleva GmbH, Freiburg, Germany
Factor H controls proximal complement activation, and its dysfunction leads to diseases that often manifest in the kidney. Structural and functional analyses have identified four distinct functional segments: an N-terminal regulatory unit, a cell binding unit, a segment with combined low-affinity C3b and Heparin sites, and a C-terminal recognition or sensor unit with overlapping C3b/C3d and Heparin sites. Three of these segments are associated with diseases. The regulatory segment is implicated in C3 glomerulopathy (C3G) and ANCA-anti neutrophil cytoplasmic antibodies - associated vasculitis (AAV). The second segment includes the Y402H-polymorphism of age relate macular degeneration (AMD), which is associated with cancer, and is also targeted by pathogens. The C-terminal sensor segment is involved in atypical hemolytic uremic syndrome (aHUS), in FHR1:FHR3 deficient and autoantibody positive HUS (DEAP-HUS), and is also exploited by pathogens. Factor H functions are modulated by two plasma proteins: Factor H like protein 1 (FHL1) and Factor H related protein 1 (FHR1), both of which share structural segments with Factor H. This interplay is critical for fine-tuning local complement activity. In addition, new noncanonical functions of Factor H and alternative sites of action, including intracellular roles, are being elaborated. A deeper understanding Factor H’s physiological role, as well as the impact of its absence, mutations, or autoantibody targeting, provides valuable insights into disease mechanisms and opens opportunities for therapeutic intervention by using full length Factor H, its fragments, or complement-modulatory compounds.
P-42
MUCOSAL COMPLEMENT FACTOR B PRODUCTION BY INTESTINAL EPITHELIUM IS REQUIRED IN HOMEOSTASIS AND RESPONSE TO INJURY.
Devesha Kulkarni1, Aayusha Thapa1, Aasritha Nallapu1, Khushi Talati2, Shyam More3, Kathrin Michelsen3, Hrishikesh Kulkarni1
1University of California Los Angeles, Los Angeles, USA. 2Washington University School of Medicine, St. Louis, USA. 3Cedar Sinai Medical Center, Los Angeles, USA
Background:
The complement system is an evolutionarily conserved arm of immunity. Key components of this system such as C3 and Factor B are activated in inflammatory bowel disease (IBD), and a coding variant in Factor B (rs4151651) is associated with severe IBD complications. Although these proteins are primarily derived from the liver and operative in the circulation, we and others have shown that they are active in the gut. Yet, major knowledge gaps remain regarding the production, activation and regulation of Factor B in IBD in order to develop tailored therapies for modulating complement activity in the gut.
Methods and Results:
Spatial transcriptomic analyses of human colonic tissue revealed that Factor B is primarily expressed by the epithelium (Visium). Using novel transgenic mouse models and an organoid system, we demonstrate that gut-derived Factor B is sufficient to maintain epithelial cell homeostasis under steady state and facilitates epithelial cell proliferation and differentiation. Additionally, we report that local Factor B production protects against chemical-induced colitis independent of circulating Factor B. However, although Factor B has key protective effects in gut mucosal immunity, multiple studies have demonstrated elevated Factor B production in inflamed tissues of patients with ulcerative colitis. Single-cell analyses of publicly available datasets from patients with ulcerative colitis reveal an increase in activators of the complement cascade, notably Factor B, and a concomitantly reduced expression of certain complement regulatory genes such as Factor H, in the epithelium.
Conclusion:
These observations indicate that local complement Factor B production by intestinal cells is required for gut homeostasis and response to injury, and creates the precedent for its regulation, rather than inhibition, in IBD.
P-43
Design and development of potent and selective C5aR2 agonists
Joshua Dent, Sam Gibson, Richard Clark
University of Queensland, Brisbane, Australia
Background:
The anaphylatoxin C5a plays a key role in Complement-mediated inflammation, inducing pro-inflammatory effects such as chemotaxis, leukocyte degranulation and cytokine/chemokine production through interaction with its endogenous receptor, C5aR1. In comparison to C5aR1, the understanding of the role of the second C5a receptor, C5aR2, is limited. Previous studies have relied predominantly on knockout mouse models to elucidate the role of C5aR2, however the impact of the receptor on trafficking and expression of C5aR1 and/or C3aR is still not fully understood. Ideally, these studies would be complemented with pharmacological experiments to further elucidate the immunomodulatory role of C5aR2 in both healthy and pathological conditions. In order to perform these studies, selective ligands for the receptor are needed. Previous studies have uncovered selective partial agonists, however selective full agonists have, to-date, remained elusive.
Methods:
Using FMOC-based solid phase peptide synthesis, a series of peptide analogues of C5aR1 agonist peptides were constructed and the ability of these analogues to modulate C5aR2-mediated β-arrestin 2 recruitment was assessed in HEK-293 cells transiently expressing C5aR2. Analogues that exhibited ≥100% efficacy of 100 nM C5a were then counter-screened for activity at C3aR and C5aR1 in ligand-induced ERK 1/2 phosphorylation and β-arrestin 2 recruitment assays.
Results:
This study identified 11 small peptide ligands that act as full agonists of C5aR2-mediated β-arrestin 2 recruitment. 8 of these analogues exhibited functional selectivity for the receptor, with no induction of C3aR/C5aR1 ERK1/2 activation or C5aR1-mediated β-arrestin 2 recruitment up to concentration of 100 µM.
Conclusions:
This study has identified the first reported full agonists of C5aR2-mediated β-arrestin 2 recruitment. These functionally selective ligands represent valuable tool compounds for modulation of C5a activity and further probing the role of C5aR2 within the complement-mediated immune response.
References
https://doi.org/10.1016/j.biocel.2009.04.005 https://doi.org/10.1146/annurev.immunol.23.021704.115835 https://doi.org/10.4049/jimmunol.1900371 https://doi.org/10.1038/icb.2016.43
P-44
ROLE OF COMPLEMENT COMPONENTS 3A (C3A) AND 5A (C5A) IN THE ACUTE-TO-CHRONIC PAIN TRANSITION IN A MOUSE MODEL OF NEUROPATHIC PAIN.
Marina Vygonskaya1, Maree Smith2, Trent Woodruff2, Felicity Han1
1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia. 2School of Biomedical Sciences, Faculty of Health, Medicine and Behavioural Sciences, The University of Queensland, Brisbane, Australia
Background:
C3a and C5a are associated with neuropathic pain (NP) behaviour in rodent models[1]. Additionally,C5a receptors C5aR1 and C5aR2 are expressed by immune (macrophages, T-cells), and glial (microglia, astrocytes) cells that are implicated in the development of neuroinflammation that contributes to the pathobiology of chronic pain [1]. However, the specific time course for activation of the complement signaling system in the development of NP remain unclear.
Methods:
NP was induced in wild type (WT) male and female mice, using the chronic constriction injury (CCI) model. The development of a mechanical allodynia is in the ipsilateral hind paws of CCI-mice was documented using Von Frey filaments. Plasma, liver, and lumbar spinal cord (SC) samples were collected on days 0, 4, 7, 14 and 28 post-CCI induction. The experiment involved measuring plasma concentrations of C5a and C3a using ELISA, quantifying C5 liver expression levels by qPCR, assessing C5aR1 expression in lumbar SC tissues sections using immunohistochemical methods.
Results:
There was a significant increase in the plasma concentration of C5a for all timepoints in female CCI-mice compared with the corresponding concentrations in control mice. In the male mice, C5a concentrations did not differ between the CCI and sham-group at any of the time points assessed. Liver C5 expression levels were aligned with the ELISA results, showing increased C5 mRNA in females from day 4, whereas expression levels remained low in the liver of male mice. Interestingly, the C3a plasma concentrations remained stable across all timepoints. C5aR1 immunofluorescence (IF) intensity inspections of the lumbar SC was highest on day 4 after CCI, which then decreased below the corresponding IF levels by day 14. No significant sex differences were observed in C5aR1 IF levels in sections of lumbar SC from CCI-mice.
Conclusions:
Our data suggest a role for C5a signaling via C5aR1 in the pathobiology of NP in female but not male mice in peripheral components of the somatosensory nervous system. In female CCI-mice, NP may involve peripheral cells, as previous studies have shown T-cell involvement [2], which could explain the increase in peripheral C5a. No sex differences were observed in central mechanisms.
References
1. Vygonskaya M, Wu Y, Price TJ, Chen Z, Smith MT, Klyne DM, Han FY. Therole and treatment potential of the complement pathway in chronic pain. The Journal of Pain. 2024 Oct 1:104689. 2. Sorge RE, Totsch SK. Sex differences in pain. Journal of neuroscience research. 2017 Jun;95(6):1271-81.
P-45
Ficolin-3 Recognizes Acylated and Carbamylated Lysine Residues and Activates Complement in a pH-Dependent Manner
Linnea Lindelöf1, Roland Ebert1, David Eikrem1, Anna Berling2, Daniella Hamnqvist2, Hanna Tegel2, Solbritt Rantapää-Dahlqvist3, Peter Garred4, Oskar Eriksson1
1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. 2Department of Protein Science, School of Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden. 3Department of Public Health and Clinical Medicine/Rheumatology, Umeå University, Umeå, Sweden. 4Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, University Hospital of Copenhagen, Copenhagen, Denmark
Background Ficolin-3 is a multimeric plasma protein and the main activator of the lectin pathway of complement in humans. In vitro, it binds strongly to proteins containing lysines modified by acetylation. Still, ficolin-3’s biological relevance remains incompletely defined.
Methods To elaborate on the physiological and pathophysiological relevance further, we systematically assessed the binding of both recombinant and native serum-derived ficolin-3 to post-translational protein modifications (PTMs) structurally related to acetyl-lysine, including various lysine acylations, carbamylation, and citrullination. Binding was evaluated using a custom ficolin-3 ELISA and validated via quartz crystal microbalance with dissipation monitoring (QCM-D) under physiological (pH 7.4) and mildly acidic (pH 6.0) conditions mimicking inflammation.
Results Ficolin-3 binding was restricted to shorter lysine acylations and discriminated between acetyl, propionyl, and buturyl adducts. Based on conservation with known acetyl-lysine binding proteins and site-directed mutagenesis, we identified a conserved asparagine anchor residue in the ficolin-3 binding pocket that was critical for binding to acetylated proteins, supporting that ficolin-3 has evolved to recognize acetylated lysine residues. We also screened for binding to immunogenic PTMs with structural similarities to acetyl-lysine. Recombinant ficolin-3 bound to carbamylated but not citrullinated proteins. Further studies also revealed a pH-dependent shift in ligand recognition properties. Native serum-derived ficolin-3 showed negligible binding and complement activation on carbamylated proteins at neutral pH, but the binding was enhanced at slightly acidic conditions. During these conditions, carbamylated proteins activated complement via the lectin pathway, as demonstrated by C4 deposition. Sera from SLE patients with high ficolin-3 activity displayed enhanced complement activation on carbamylated proteins during mildly acidic conditions.
Conclusion Our findings show that ficolin-3 recognizes lysine-PTMs with a pH-sensitive ligand specificity and subsequent complement-activating potential. This suggests a novel pH-dependent mechanism for ficolin-3 by which PTMs may activate the lectin pathway in tissues during inflammatory and autoimmune processes.
P-46
Update on protein array screening of the ficolin-3 interactome
Roland Ebert, Linnea Lindelöf, Oskar Eriksson
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
Methods
The Ficolin-3 interactome was explored using HuProtTM Human Proteome Microarrays v4.0, coated with over 20,000 recombinant human (rh) proteins (expressed in S. cerevisiae). Arrays were incubated under simulated physiological conditions with rhficolin-3 (expressed in CHO cells). Bound ficolin-3 was detected by a biotinylated goat anti-ficolin-3 antibody followed by streptavidin-coupled DyLight680. For normalization, binding to human serum albumin (HSA) was used as baseline.
Results
Among the top ficolin-3 interaction partners were several proteins related to cell signaling or immune response cascades. Examples from signal transduction are Crk and the Crk-like (Crkl) proteins, which both function as intracellular adapter proteins. Binding was also observed to galectin-1, a protein with lectin properties that is reported to have an immune-modulatory function. Therefore, it could be imaginable that a direct interaction of galectin-1 with ficolin-3 might modulate lectin pathway-mediated complement activation.
As mentioned, many of the identified hits are intracellular proteins while ficolin-3 circulates in the bloodstream. This spatial separation questions how realistic an interaction of those proteins might be. Considering events of cell disruption like necrosis an interaction of then exposed proteins with circulating ficolin-3 seems reasonable. This hypothesis is supported by reports of ficolin-3 interaction with apoptotic and necrotic cells. [2]
Interestingly, with the human N-acetylneuraminate synthase we identified a potential binding partner of which bacterial orthologues exist. Therefore, it might be possible to draw information about potential natural targets from the human interaction partner of ficolin-3.
Conclusion
With this systematic characterization of human interaction partners of ficolin-3 we hope to be able to identify previously unknown molecular crosstalk and natural targets of the lectin pathway.
References
[1] Garred P, et al. A journey through the lectin pathway of complement-MBL and beyond. Immunol Rev. 2016 Nov;274(1):74-97. [2] Honoré C, et al. The innate immune component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells. Arthritis Rheum. 2007 May;56(5):1598-607.
P-47
The Tumor- and Apoptosis-Associated Annexin A2 is a New Ligand for FHL-1 and Factor H-Related Proteins – Annexin-Bound FHR-5 Enhances Alternative Pathway Activation
Dávid Szakács1, Dániel Bencze1, Eszter Boros2, Patrik Horváth2, László Nyitray2, Barbara Uzonyi1,3, Mihály Józsi1,3
1Department of Immunology, ELTE Eötvös Loránd University, Budapest, Hungary. 2Department of Biochemistry, ELTE Eötvös Loránd University, Budapest, Hungary. 3HUN-REN-ELTE Complement Research Group, Hungarian Research Network, Budapest, Hungary
Annexin A2 (ANXA2) is a membrane-binding protein with diverse intra- and extracellular roles and involvement in certain tumorous and immune processes. Elevated ANXA2 cell-surface levels are characteristic of apoptotic cells where it serves as a ligand for factor H (FH)1. In mice, ANXA2 enhanced complement activation both in vitro and in vivo by blocking complement regulatory functions of FH, suggesting a mechanism by which ANXA2 contributes to complement-mediated diseases2. However, interactions between ANXA2 and other FH family proteins have not yet been studied, and little is known about the implications of ANXA2 as a ligand for FH or the FH-related proteins (FHRs).
We purified FH from normal human serum (NHS) and produced recombinant FH fragments, FH-like protein 1 (FHL-1), FHRs, full-length and N-terminally truncated ANXA2 to map the respective binding sites on FH and ANXA2, and to test whether the FHRs bind ANXA2 using ELISA. We studied the functional effects of these interactions using ANXA2 as a surface-bound ligand in cofactor activity and alternative pathway (AP) activation assays.
Our results indicate that the N-terminal peptide of ANXA2 plays no significant role in binding to FH family proteins. Previously, the binding site for ANXA2 on FH was mapped to complement control protein domains (CCP) 681. In our assays, all proteins containing FH CCP 19-20 or highly similar domains (FHR-1A, FHR-1B, FH fragments CCP 15-20 and 19-20) bound to ANXA2, suggesting the existence of a second binding site in the C-terminal region of FH. Additionally, FHL-1 and FHRs except FHR-2 bound ANXA2. We demonstrated that FH and FHL-1 retain their cofactor activity when bound to ANXA2. Accordingly, immobilized ANXA2 does not activate the AP in NHS. On the other hand, in NHS supplemented with FHR-5, we observed significant AP activation, detected as factor B, properdin and C5 deposition.
In conclusion, ANXA2 is a common self-ligand for FH family proteins that does not inhibit cofactor activity of FH or FHL-1. Moreover, FHR-5 can enhance AP activity on ANXA2-covered surfaces. Taken together, our findings suggest that ANXA2 plays an important role in the balanced regulation of local complement activation on certain self-surfaces.
References
1. Leffler J. et al. J Biol Chem. (2010) 285:3766–3776. 2. Renner B et al. J Immunol. (2016) 196:1355–1365. Supported by grants from the National Research, Development and Innovation Office (PharmaLab National Laboratory, RRF-2.3.1-21-2022-00015, OTKA K146911), the Hungarian Research Network (0106307), the European Union (nr. 899163 SciFiMed project).
P-48
The human complement factor H-related protein FHR-2 enhances complement activation on host and bacterial ligands
Andrea E. Schneider1, Marcell Cserhalmi1, Domonkos Czárán1, Csenge Réka Papp1, Dániel Bencze1, Dávid Szakács1, Barbara Uzonyi1, Mihály Józsi1,2
1Department of Immunology, ELTE Eötvös Loránd University, Budapest, Hungary. 2HUN-REN-ELTE Complement Research Group, Hungarian Research Network, Budapest, Hungary
Regulation of the alternative complement pathway (AP) and that of the amplification loop is important to limit physiological complement activation to the necessary extent on target surfaces, such as microbes or dead cells. Conversely, AP dysregulation contributes to the pathogenesis of various (auto)inflammatory diseases. Factor H family proteins are central to the proper regulation of the cascade as testified by their disease-associations. In contrast to the complement inhibitor factor H (FH), the function of the FH-related (FHR) proteins is less defined. Recent studies support the role of FHRs in enhancing complement activation by competing with FH for binding to certain ligands and surfaces and by promoting C3 convertase assembly.1 FHR-2 was described to inhibit the AP C3 convertase and the terminal pathway2, but its role in complement is poorly characterized. Therefore, our aim was to further investigate FHR-2 for ligand interactions and function.
FHR-2 was expressed in insect cells and outer surface protein E (OspE) of Borrelia burgdorferi and that of B. garinii were expressed in E. coli. Binding of C-reactive protein (CRP) and pentraxin 3 (PTX3), assembly of the C3bBb AP convertase, and complement activation were measured by ELISA. Hemolysis assays were performed using sheep and rabbit erythrocytes; in certain assays, lysis was induced by adding the anti-factor H monoclonal antibody OX24.
FHR-2 supported the assembly of the AP C3 convertase by binding C3b. The FHR-2 bound convertase was active as it could generate C3a from C3. This activity was confirmed by detecting C3 activation on immobilized FHR-2 in human serum, and deposition of Bb. FHR-2 bound to PTX3 and both to pentameric native CRP and monomeric CRP. FHR-2 enhanced AP activation on immobilized pentraxins when added to serum. FHR-2 bound to OspE of Borrelia spp., inhibited FH binding and cofactor activity, and enhanced complement activation as indicated by increased C3, factor B, properdin and C5 deposition. FHR-2 slightly inhibited OX24-induced hemolysis of sheep erythrocytes at high concentrations.
Altogether, our results identify FHR-2 as a soluble pattern recognition molecule of the innate immune system that enhances complement activation on selected host and non-host ligands.
References
1. Sándor N et al. Front Immunol. 2024;15:1135490. doi:10.3389/fimmu.2024.1135490 2. Eberhardt HU et al. PLoS One. 2013;8:e78617. doi:10.1371/journal.pone.0078617. Supported by grants from the National Research, Development and Innovation Office (OTKA K146911 and PharmaLab National Laboratory RRF-2.3.1-21-2022-00015), the Hungarian Research Network (HUN-REN, 0106307), and the European Union (nr. 899163 SciFiMed project).
P-49
Complement regulation in genetically modified pig endothelial cells under xenogeneic and inflammatory conditions
Mitra Gultom1, Nina Thomi1, Alain Despont1, Jane Shaw1, Elisabeth Kemter2, Eckhard Wolf2, Robert Rieben1
1University of Bern, Bern, Switzerland. 2Ludwig Maximilian University of Munich, Munich, Germany
Introduction: Multigene modifications in donor pigs have significantly advanced xenotransplantation by overcoming interspecies innate immune barriers. A prominent model includes α1,3-galactosyltransferase-knockout pigs expressing human CD46 and thrombomodulin (3GM), which have demonstrated promising life-supporting graft function in preclinical settings. As the initial interface between graft and host, endothelial cells (ECs) are critical regulators of complement activation, coagulation, and inflammation. Here, we investigate how these genetic modifications modulate complement responses in pig ECs during xenogeneic and inflammatory conditions.
Methods: Primary ECs from 3GM pigs were cultured under physiological shear stress and perfused with normal human serum (NHS) or recalcified citrated human plasma to assess complement and coagulation activation, respectively. Inflammatory conditions were simulated using recombinant human TNF-α (rhTNF-α) perfusion. Antibody (IgG, IgM) and complement deposition (C1q, C3b/c, and C5b-9) and coagulation markers (clot formation time) were assessed, and bulk RNA-seq was performed to investigate transcriptomic changes.
Results: 3GM ECs showed significantly reduced antibody and complement deposition following NHS perfusion, as well as better anticoagulant properties upon citrated human plasma exposure, indicating effective regulation of xenogeneic complement and coagulation pathways. However, this protection was reduced under rhTNF-α-induced inflammation conditions. Interestingly, the increased complement deposition under inflammatory conditions was not attributed to increased deposition of IgG, IgM, or C1q, although differences in endothelial glycocalyx dynamics were observed. Transcriptomic analysis revealed that 3GM ECs exhibit reduced expression of key proinflammatory, coagulation, and complement-related genes (e.g., VCAM1, CCL2, C1QBP, SERPINE1) compared to wild-type (WT) ECs. In addition, upon xenogeneic activation, 3GM ECs displayed minimal transcriptomic changes and distinct responses to that of WT ECs. In contrast, rhTNF-α-induced inflammation led to more aligned gene expression profiles between 3GM and WT ECs, where upregulation of immune and defense response pathways was observed, potentially contributing to increased complement and coagulation dysregulation.
Conclusion: Our findings highlight that 3GM pig ECs were able to effectively prevent complement activation in xenogeneic settings. However, this protection is compromised under inflammatory conditions. These results suggest that incorporating additional anti-inflammatory transgenes may be necessary to maintain immune quiescence and improve graft survival in clinical xenotransplantation.
P-50
Live-cell single-molecule imaging of a new fluorescent C5a receptor antagonist reveals the heterogeneous spatiotemporal dynamics of C5aR1 at the plasma membrane
Jinda Sun1, Sandra Parker1, Jonathan Lee1, Declan Gorman1, Xaria Li1, Eduardo Albornoz Balmaceda1, Mark Bellingham1, Richard Clark1, Trent Woodruff1,2, Pranesh Padmanabhan1,3,4
1School of Biomedical Sciences, The University of Queensland, Brisbane, Australia. 2Queensland Brain Institute, The University of Queensland, Brisbane, Australia. 3Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Australia. 4NHMRC Centre of Research Excellence in Mechanisms in NeuroDegeneration – Alzheimer's Disease, Brisbane, Australia
The anaphylatoxin C5a and its corresponding G protein-coupled receptor C5aR1 play a pivotal role in immune signaling and inflammation. However, validated imaging tools for reliably visualizing C5aR1 expression patterns and its cell surface dynamics at the nanoscale level are still lacking. To address this, we developed a molecular probe termed PMX403 by attaching fluorescein to PMX53, a potent and insurmountable C5aR1 antagonist. We first confirmed that fluorescein conjugation did not affect receptor binding, selectivity, or antagonistic function of PMX403 by using Chinese hamster ovary cells stably overexpressing human C5aR1 (CHO-C5aR1) and human primary monocyte-derived macrophages. We next employed confocal microscopy to demonstrate that PMX403 enabled precise detection of cell surface C5aR1 in CHO-C5aR1 cells and in human primary monocyte-derived microglia. Since PMX403 is non-toxic in living cells and significantly smaller than typical super-resolution imaging probes, such as nanobodies, we assessed its compatibility for single-molecule imaging of C5aR1 dynamics at the plasma membrane. Using total internal reflection fluorescence microscopy, we detected and tracked a high density of individual PMX403 molecules with nanometre- and millisecond-level resolution near the plasma membrane of live CHO-C5aR1 cells. In contrast, the detection density of PMX403 was significantly lower in both native CHO-K1 cells and CHO cells stably overexpressing C3aR, demonstrating high specificity of PMX403 for single-molecule detection of C5aR1 at the plasma membrane. We also detected and tracked the dynamics of PMX403 at the plasma membrane of phorbol myristate acetate (PMA)-treated, lipopolysaccharide (LPS)-stimulated U937 monocytic cells that natively express C5aR1, but not in those preincubated with the parental ligand PMX53, indicating that PMX403 can reliably quantify the dynamics of endogenous C5aR1. Finally, we found that PMX403 exhibited multiple mobility states and transitioned between these states, highlighting spatiotemporal heterogeneity in the dynamics and organization of C5aR1 receptors at the plasma membrane. Overall, our study using confocal and super-resolution microscopy demonstrates that PMX403 is a valuable tool for investigating the localization and dynamics of C5aR1 in living cells.
P-51
Complement C3 inhibition mitigates complement activation in clinical platelet concentrates without preventing platelet storage lesions
Linnea Andersson1, Per Sandgren2, Dick J. Sjöström1, Camilla Mohlin1, Kim Hägerström3, Ivar Tjernberg3, Tom Eirik Mollnes4, Per H. Nilsson1
1Linnaeus University, Kalmar, Sweden. 2Karolinska Institutet, Huddinge, Sweden. 3Region Kalmar County, Kalmar, Sweden. 4Oslo University Hospital and University of Oslo, Oslo, Norway
Background: The storage of platelets in concentrates requires an environment that supports their metabolic needs and maintains their function without activating the plasma components stored alongside the platelets. Over time, stored platelets develop storage lesions—progressive functional deterioration caused by morphological changes, altered surface markers, metabolic shifts, and the release of proteins, RNA, and other modifiers. We recently demonstrated increased complement activation during platelet storage, as evidenced by elevated levels of C1rs/C1-INH, MASP-/C1-INH complexes, C4c, C3bc, and sC5b-9 (1). This activation correlated with heightened platelet activation and diminished responsiveness, particularly to TXA2R stimulation.
Methods: This study explored whether complement C3 inhibition could reduce complement activation and platelet storage lesions in clinical platelet concentrates. Eight concentrates were prepared in platelet additive solution-E (PAS-E) with ~35% plasma and stored at 22°C for seven days. Each unit was split: one half was supplemented with the C3 inhibitor compstatin Cp40, the other serving as the control. Complement activation, platelet function, and metabolic parameters were assessed every two days.
Results: Cp40 significantly reduced C3bc and sC5b-9 levels, but not C4c, indicating inhibition of complement activation at the level of C3. Importantly, C3 inhibition did not alter platelet-specific markers or metabolic stability. CD62P expression and NAP- release increased during storage, while CD63, PF4, and TSP- levels remained stable. Platelet responsiveness to PAR-1 (TRAP-) and TXA2R (U46619) stimulation declined over time, as indicated by reduced CD62P and CD63 expression and diminished soluble factor release. No drop in platelet count was observed, and metabolic markers remained within their critical limits.
Conclusion: In summary, complement activation occurs during the storage of platelet concentrates in PAS-E/plasma, but C3 inhibition with Cp40, while effective in reducing complement activation, does not influence platelet activation or metabolic markers. These findings suggest there is no direct causal link between complement activation and the development of storage lesions in clinical platelet concentrates.
References
1. Andersson LI, Sjostrom DJ, Quach HQ, Hagerstrom K, Hurler L, Kajdacsi E, et al. Storage of Transfusion Platelet Concentrates Is Associated with Complement Activation and Reduced Ability of Platelets to Respond to Protease-Activated Receptor-1 and Thromboxane A2 Receptor. Int J Mol Sci. 2024;25(2).
P-52
Differential Complement Responses in Radiation- vs. Chemotherapy-Induced Senescence
Anas Abu-Humaidan1, Sabal Al Hadidi2, Fatima Ahmad3, Sofian Al Shboul4, Nidaa Ababneh2, Tareq Saleh4
1School of Medicine, The University of Jordan, Amman, Jordan. 2Cell Therapy Center, The University of Jordan, Amman, Jordan. 3School of Science, The University of Jordan, Amman, Jordan. 4Faculty of Medicine, The Hashemite University, Zarqa, Jordan
Background: Sub-lethal chemotherapy or radiation can drive cancer cells into senescence—a state marked by irreversible growth arrest, apoptosis resistance, and a pro-inflammatory secretory profile, potentially undermining treatment success. This study explored how senescent cancer cells interact with the complement system.
Methods: Senescence was confirmed via β-galactosidase activity, p21Cip1 upregulation, and lamin B1 loss. Complement activation and component expression were analyzed using qPCR, ELISA, and immunofluorescence.
Results: Senescent cancer cells exhibited enhanced complement activation compared to non-senescent cells, regardless of whether senescence was induced by radiation or chemotherapy, as evident by increased C5b-9 formation. Both inducers led to increased expression of C3 and CD59. Notably, complement Factor B was upregulated only in radiation-induced senescence. Experiments with C1q-depleted/ reconstituted serum indicated that complement activation was largely dependent on the presence of C1q.
Conclusion: The findings emphasize the involvement of complement in senescence, through complement activation and the upregulation of complement components following two different senescence inducers, and suggest that the senescence inducer may influence specific complement responses.
References
https://pmc.ncbi.nlm.nih.gov/articles/PMC10522834/#s6
P-53
Something about the at least 50 different complexes of the initiating molecules of the lectin pathway and the polyreactivity of the pattern recognition molecules.
Steffen Thiel, Annette Hansen, Kirstine Hymøller
Aarhus University, Aarhus, Denmark
We have a tendency to simplify the depiction of the complement system to ease the discussions of this phenomenal arrangement of recognition molecules and enzyme cascades, and enzyme inhibitors. As an illustration of the complexity, we have studied the pattern recognition molecules (PRMs) of the lectin pathway of the complement system. The mannan-binding lectin (MBL, mannose-binding lectin), H-ficolin (ficolin-3), L-ficolin (ficolin-2) and M-ficolin (ficolin-1) are polydisperse oligomers organized from homotrimeric subunits, whereas collectin-LK (CL-L1/CL-K1 or collectin-10/collectin-11) is organized from heterotrimeric subunits. These oligomers of lectin pathway PRMs are found in complexes with dimers of the MBL-associated serine proteases (MASPs) MASP-, MASP-, MASP-, and the two MBL-associated proteins (MAp), MAp44 and MAp19. For MBL, we find approximately 50% tetramers and 30 % trimers and 20% “more than tetramers”. In human serum, we find only little MASPs bound to the trimers. Assuming the 3 MASPs and 2 MAps are found in complexes with the tetramer, pentamer and hexamer MBL, we have 15 different unique complexes. We also studied the other lectin pathway PRMs, which are also found in serum in various oligomeric states, with associated MASPs and MAps. This results in a total of at least 50 different unique complexes of molecules in the initiating molecules of the lectin pathway.
The five different PRMs have a preference for binding to diverse structures found on microbial entities. We compared the ability of MBL, H-ficolin, L-ficolin and M-ficolin to bind to physiologically relevant carbohydrate structures in an array representing 120 different lipopolysaccharide (LPS) structures. All four PRMs preferentially bind to the O-antigens of LPS rather than the lipid A or the core oligosaccharide, contradicting previous suggestions. Each PRM displayed distinct binding patterns to different LPS structures, although some overlaps were observed. The interactions were confirmed with whole bacteria, where the PRM-LPS interactions led to complement activation. This expands and clarifies the spectrum of bacterial glycoconjugates that interact with the PRMs.
The truth about the initiating molecules of the lectin pathway is that we have a large array of unique complexes, and each of the PRMS has unique as well as overlapping specificities – fascinating. P-54
Transcriptional crosstalk regulates complement C3 expression during CD4⁺ Tcell activation
Jorge-Luis Trujillo-Ochoa1, Daniel Chauss1, Luopin Wang2, Didier Portolla3, Claudia Kemper4, Majid Kazemian2
1National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH), Bethesda, USA. 2Purdue University, West Lafayette, USA. 3University of Virginia, Charlottsville, USA. 4National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH), Bethesda, USA
Background Intracellular production of complement component 3 (C3) has emerged as indispensable for Tcell survival, proliferation, and the metabolic reprogramming that fuels Th1 differentiation. Cytokineresponsive transcriptional networks govern CD4⁺ Tcell fate and effector function and are vital for dictating effective versus aberrant responses. Yet how cytokine-activated transcription factors (TFs) are integrated in the context of other broadly modulatory TFs and cooperate at chromatin to govern C3 and other metabolismlinked genes remains incompletely understood.
Methods Here, we studied a cytokineresponsive transcription factor (TFA) and a broadly modulatory transcription factor (TFB). We combined bulk RNAseq from wildtype and TFAdeficient CD4⁺ T cells with TFspecific ChIPseq and H3K27ac HiChIP to map binding and enhancerpromoter contacts. Functional relevance was tested using TF overexpression, competition assays, CRISPR-Cas9 editing of ciselements, pharmacological blockade of cytokine signaling, and C3 reporter mice.
Results We found pronounced enrichment of cytokine-responsive and complement gene signatures in TFB-deficient CD4+ T cells. There was ~40% overlap between TF-A and TF-B-bound loci genome-wide and ~70 % of differentially expressed genes displayed overlapping TFA/TFB binding, indicating extensive genomic cooccupancy. TF-A and TF-B single and coexpression experiments, followed by RNAseq, in TFAdeficient T cells resolved distinct clusters where the factors acted synergistically or antagonistically. This was epitomized at the C3 locus, where TF-A/B co-occupancy was essential for expression, evidenced by lost C3 transcription in TF-B–/– cells, inhibition of cytokine signalling or deletion of bound loci. HiChIP showed activationdependent looping between these elements and the C3 promoter.
Conclusion Coordinated action of a cytokineresponsive factor (TFA) and a broadly modulatory factor (TFB) is essential for complement C3 induction during CD4⁺ Tcell activation. This mechanistic framework links cytokine signalling to intracellular complement regulation and offers a tractable axis for modulating Tcelldriven inflammation.
References
Liszewski, Kolev et al. Immunity 2013; 39(6): 1143-1157.
Kolev et al. Immunity 2020; 52(3): 513-527.
References
This has been submitted by the PI on behalf of an ECR
P-55
Molecular basis of naturally-encoded signaling-bias at the complement anaphylatoxin receptor, C5aR1
Annu Dalal1, Sudha Mishra1, Manish K Yadav1, Ravi Yadav2, Tomasz M. Stępniewski3, Asuka Inoue4, Jana Selent5, Ramanuj Banerjee1, Cornelius Gati6, Arun K. Shukla1
1Indian Institute of Technology, Kanpur, India. 2University of Southern California, Los Angeles, USA. 3Pompeu Fabra University, Barcelona, Spain. 4Tohoku University, Sendai, Japan. 5Pompeu Fabra University (UPF), Barcelona, Spain. 6University of Southern California,, Los Angeles, USA
Complement anaphylatoxin receptors, namely C3aR and C5aR1, are prototypical G-protein-coupled receptors (GPCRs) that play key roles in our innate immune response and represent important therapeutic targets in autoimmune and inflammatory disorders. While the complement anaphylatoxins, C3a and C5a, are well-characterized as receptor agonists, their naturally-derived d-Arg versions i.e., C3a-d-Arg and C5a-d-Arg, remain relatively less explored in terms of receptor activation and signaling. In this backdrop, we have carried out a comprehensive pharmacological profiling of the d-Arg version of C3a and C5a on the C3aR and C5aR1, respectively using cellular and biochemical assays. While C3a-d-Arg is incapable of activating C3aR as previously reported, we discover that C5a-d-Arg acts as a robust G-protein-biased agonist at the C5aR1 as it displays significant reduction in β-arrestin recruitment but maintains full efficacy for the G-protein activation. Taking a lead from this intriguing observation, we have determined the cryo-EM structures of C5a-d-Arg-bound C5aR1, which reveals distinct ligand interaction and receptor conformation compared to C5a-bound C5aR1 structure. In particular, the structural snapshot reveals a distinct positioning of TM7 and Helix 8 in the C5a-d-Arg-bound receptor, which is further corroborated with structure-guided molecular dynamics simulation and site-directed mutagenesis experiments. Interestingly, we also discover that C5a-d-Arg fails to promote efficient GRK recruitment and receptor phosphorylation, which in turn results in attenuated β-arrestin interaction with the receptor leading to G-protein signaling-bias. Taken together, our study elucidates a naturally-encoded signaling-bias mechanism at C5aR1 that is likely to have significant implications for the cellular and physiological responses regulated by the C5a-C5aR1 axis, and it also opens up novel avenues for the design of better therapeutics targeting this receptor.
P-56
Three cryo-EM structures of the complement C3d-complement receptor 3 complex reveal a novel layer of dynamics and the signaling mechanism underlying iC3b dependent phagocytosis
Gregers Andersen, Marlene Fruergaard, Josefine Lorentzen
Aarhus University, Aarhus, Denmark
Background
Complement receptor 3 (CR3) also known as integrin αMβ2, Mac-1, and CD11b/CD18 is highly expressed on macrophages, monocytes, dendritic cells, microglia, and neutrophils. The main function of CR3 is phagocytosis of iC3b opsonized cells. The iC3b ligand may be further degraded to C3d(g) and C3d, which has lower CR3 affinity but still supports phagocytosis. Genetic knock-out of CR3 in animal models of Alzheimer’s and Huntington’s disease has proven efficient in preventing complement mediated phagocytosis of synapses
Methods
Through nanobody selection and rational ligand design, we determined three cryo-EM structures of the CR3/αMβ2 receptor headpiece in complex with the complement C3d ligand at 2.9 Å resolution.
Results
Our structures reveal for the first time that the ligand binding αI domain have two preferred and opposite orientations relative to the CR3 β2 subunit. The structure of a C3d bound receptor αM variant associated with systemic lupus erythematosus reveals a reorganized main chain at the mutated residue that we suggest leads to altered dynamic properties of the ligand binding domain. Binding of C3d induces an ordered conformation of the αM internal ligand region that is tightly packed between the αM β-propeller and the β2 βI-domain. Recognition of the internal ligand induces an open βI conformation that is practically identical to that known from ligand bound αI-less integrins proving that ligand binding and signaling are coupled by a universal mechanism covering all integrins. Integration of our results with existing data allows us to propose a model for C3d/iC3b-bound CR3 in the phagocytotic cup.
Conclusions
Our structures of the αMβ2HP-C3d complex offer a detailed glimpse into the conformational spectrum of ligand bound CR3 that may apply also to the cell-bound ligand bound conformation. Recent discoveries in relation to the role of CR3 in neurodegenerative disease witness on the therapeutic potential for a CR3 antagonist. Our structures offer a groundbreaking framework for future rational design of CR3 function modulating molecules.
P-57
The toxin CyaA from Bordetella pertussis as a potential modulator of Complement receptor 3 (CR3) function
Marie Jachmann, Jannik Sichau, Sümeyye Erdemci Evin, Holger Barth, Christoph Q. Schmidt
Ulm University, Medical Center, Ulm, Germany
Background: C3-activation products (C3b, iC3b, C3dg) are one major class of opsonins crucial for opsonophagocytosis, which is vital for cellular maintenance and immune surveillance. Complement receptor 3 (CR3; CD11b/CD18, αMβ2) is a key receptor on phagocytes enabling C3-opsonin decorated material to be engulfed efficiently. When engaging certain ligands, CR3 switches from the inactive, bent into an extended, high affinity conformation. Most ligands bind CR3 via its conserved I-domain harboured in the CD11b chain. However, the CyaA toxin from Bordetella pertussis is unique as it binds CR3 outside of its I-domain and prefers the inactive conformation, displaying a non-canonical binding mode1. Interfering with CR3 binding may modulate the inflammatory signature of phagocytes. We hypothesise that CyaA interferes with the recognition of C3-opsonins and consequently alters the activation state of the phagocyte.
Methods: Recombinant protein expression was achieved using bacterial, yeast and mammalian cells. Combinations of conservative and tag-based purification strategies were employed for protein purification. Ligand receptor interactions were characterised by surface plasmon resonance (SPR) and spectral shift technology.
Results: We expressed the major ligand binding domain αM-I, the complete 6 domain spanning CR3 headpiece and the C-terminal receptor binding part of the CyaA toxin at high purity. Functionality of the purified αM-I domain and the CR3 headpiece was validated by determining binding affinities to C3-opsonins, which are in accordance with published data. The CR3 headpiece showed a 3- to 10-fold higher affinity than αM-I for binding to iC3b: KD Headpiece= 149 nM and KD αM-I = 484 nM for iC3b immobilised on an SPR chip; KD Headpiece = 94.6 nM and KD αM-I = 1.01 µM for interaction with iC3b in the fluid phase. These data support the hypothesis of additional binding sites for iC3b being present outside of the I-domain. The CyaA toxin binding domain bound to the CR3 headpiece (KD = 240 nM) and may proof a valuable tool for receptor competition.
Conclusion: The assembly of a recombinantly expressed molecular toolkit for CR3 protein interaction and the successful validation of C3-opsonin and CyaA toxin binding provides a basis for CR3 modulation on the phagocytic cell level.
References
Goldsmith, J. A., DiVenere, A. M., Maynard, J. A. & McLellan, J. S. Structural basis for non-canonical integrin engagement by Bordetella adenylate cyclase toxin. Cell Rep. 40, 111196 (2022).
P-58
C5 inhibition by mAbs: There are many ways to skin a cat!
Rebekah S Cooke1, Kok Yung Lee1, Georgina E Menzies2, B Paul Morgan1, Wioleta M Zelek1
1UK Dementia Research Institute, School of medicine, Cardiff University, Cardiff, United Kingdom. 2Molecular Biosciences Division, School of Biosciences, Cardiff University, Cardiff, United Kingdom
Background: Complement dysregulation drives inflammation in diverse diseases; inhibition of C5 has proven a robust therapeutic target. C5 cleavage releases C5a and seeds formation of the membrane attack complex. We generated a panel of C5 function-blocking monoclonal antibodies (mAbs) and compared them to the gold standard C5-blocking mAb therapeutics, eculizumab and crovalimab.
Methods: Complement inhibitory activity was tested using classical pathway haemolysis assays. C5 binding and binding competition between mAbs was tested by ELISA. Surface plasmon resonance epitope binning was performed to confirm competing pairs and identify distinct blocking epitopes. C5 cleavage inhibition was tested using both CVF and native C5 convertase in haemolysis assays; C5a generation was detected using westerns and/or ELISA.
Results: All novel mAbs bound C5 in direct ELISA and strongly inhibited haemolysis with comparable efficacy to the gold standard C5-blocking mAbs. In westerns, mAbs 4G4, 7D4, 2B11, and eculizumab bound the α-chain; 10B6, and crovalimab bound the β-chain. Competition binding assays identified four distinct function-blocking epitopes on C5: 7D4 and eculizumab competed for the same epitope (epitope 1) while 10B6 and crovalimab competed for another (epitope 2). 2B11 and 4G2 were not competitive with epitope 1/2 binders or each other, marking two distinct blocking epitopes (epitopes 3 and 4). Impact on C5 cleavage and C5a release differed for CVF and native C5 convertases; epitope 1 binders (7D4 and eculizumab) blocked C5 cleavage and C5a release in the CVF assay, no other mAbs prevented C5a release. In the native C5 convertase assay, all mAbs, except for the epitope 3 binder 2B11, blocked C5 cleavage and C5a release.
Conclusions: We identified four distinct function-blocking epitopes on C5 utilised by commercial and novel C5-blocking mAbs, epitope 1 targeted by eculizumab and 7D4, epitope 2 targeted by crovalimab and 10B6, and epitopes 3 and 4 targeted by 2B11 and 4G2 respectively. Although all efficiently blocked haemolysis, we reveal differences in mechanism, notably whether C5 cleavage is blocked in different assays. These data provide novel insights into C5 inhibition mechanisms, showing that there are (at least) four blocking epitopes on C5 and (at least) two distinct mechanisms of inhibition.
P-59
A conserved molecular mechanism orchestrates diverse ligand recognition at the complement anaphylatoxin receptor C3aR
Sudha Mishra1, Annu Dalal1, Manish Kumar Yadav1, Ravi Yadav2,3, Manisankar Ganguly1, John Lee4, Ramanuj Banerjee1, Trent Woodruff4, Cornelius Gati2,3,5, Arun Kumar Shukla1
1Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, Kanpur, India. 2Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA. 3The Bridge Institute, Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA. 4School of Biomedical Sciences, Faculty of Health, Medicine, and Behavioural Sciences, The University of Queensland, Brisbane, Australia. 5Department of Chemistry, University of Southern California, Los Angeles, CA, USA
The complement anaphylatoxin receptor C3aR is a prototypical G protein-coupled receptor (GPCR) that plays a crucial role in orchestrating various immune response mechanisms, including inflammation, chemotaxis, metabolic responses, and allergic reactions. Aberrant activation and signaling of C3aR have been implicated in various pathophysiological conditions, including the Alzheimer’s disease, asthma, and metabolic disorders, and therefore, it represents an attractive therapeutic target. Here, we have comprehensively characterized the pharmacology and functional responses of a broad set of C3aR ligands using cellular and structural approaches. We discover a previously unanticipated level of species-specific differences in ligand-receptor interactions and resulting downstream transducer-coupling, especially for the VGF-derived neuropeptide TLQP-1. Moreover, we also uncover that small molecule ligands such as SB290157 and JR14a, previously characterized as C3aR antagonists, are indeed robust agonists of the receptor. Finally, the cryo-EM structures of C3aR reveal that diverse agonists engage a highly conserved five-point switch in the C3aR spanning TM2, TM3, ECL2, TM6, and TM7 to orchestrated receptor recognition and activation. Taken together, our findings provide important insights into C3aR activation, species-specific pharmacology, and biased signaling and set a strong foundation for rational design of next-generation C3aR-targeted therapeutics with improved specificity and efficacy.
P-60
A CD4+ T cell-intrinsic complement C5aR2-prostacyclin-IL-1R2 axis orchestrates Th1 contraction and is perturbed in Th1-driven disease states
Jubayer Rahman1, Jack Bibby1, Parul Singh1, Nicolas Merle1, Erin West1, Andrea Bohrer1, Katrin Mayer-Barber1, Chengyu Liu1, Lauren Brinster1, Behdad Afzali1, Ana Briones2, Sara Alehashemi1, Farzana Bhuyan1, Yingbi Zhou3, Murray Clarke4, Bin Liu3, Raphaela Goldbach-Mansky1, C Henrique Serezani5, Claudia Kemper1
1NIH, Bethesda, USA. 2Departmento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain. 3Shantou University, Shantou, China. 4Cambridge University, Cambridge, United Kingdom. 5Vanderbilt University, Nashville, USA
Timely contraction of successful Th1 responses, characterized by downregulation of IFNg production and co-expression of the anti-inflammatory cytokine IL-10, is essential for limiting Th1-driven tissue pathologies and preventing autoimmune disease. T cell-intrinsic complement components are central orchestrators of Th1 induction and contraction. Specifically, CD4+ T cell-intrinsic C5 activation and stimulation of intracellular C5aR1 triggers NLRP3 inflammasome-dependent secretion of mature IL-1b by Th1 cells, which augments IFNg production and restrains IL-10 co-expression. The cell-surface expressed alternative C5aR2, engaged by T cell-secreted C5a/C5a-desArg, fosters IL-10 co-secretion and Th1 contraction through an unknown mechanism. Uncontrolled IL-1b production by CD4+ T cell cells has been pinpointed as driver of hyper-active Th1 response in patients with cryopyrin-associated periodic syndromes (CAPS), of sustained tissue inflammation in rheumatoid arthritis (RA), and of CD4+ T cell depletion during human HIV-1 infection. However, the mechanism by which CD4+ T cell-autonomous IL-1b production is regulated remains elusive. Here, we define a CD4+ T cell-autonomous pathway in which complement C5 orchestrates a shift from C5aR1-controlled prostaglandin E2 (PGE2) dominance to enhanced prostacyclin (PGI2) production via activation of C5aR2. This pivot in lipid mediators induces autocrine signaling through the PGI2 receptor and expression of the IL-1 decoy receptor IL-1R2, which sequesters Th1-driving intrinsic IL- 1b, facilitating the transition into Th1 contraction. Disruption of this C5aR2-PGI2-R axis is a hallmark of pathologically persistent Th1 activity in inflammatory conditions including CAPS, Crohn’s disease, and RA. Furthermore, rebalancing this axis through selective PGE2 synthase inhibition effectively rectifies the hyperactive Th1 phenotype in vitro in CAPS. These results reveal that complement C5 and its receptors are unexpected key controllers of prostanoid metabolism and of IL-1b auto-regulation and that the cell-autonomous C5-prostaglandin axis represents a novel, and potentially druggable, checkpoint for the cessation of Th1 effector responses.
P-61
FHR2 Binds to HUVE Necrotic Cell and Renal Glomerular Basal Membrane, Inhibiting TCC Deposition and Exhibiting Cofactor Activity.
Emma Diletta Stea1, Loreto Gesualdo2, Teresa Rampino1,3, Marilena Gregorini1,3, Mariantonietta Grignano1, Thorsten Wiech4, Giuseppe Castellano5, Christine Skerka6, Franz Peter Zipfel6
1Unit of Nephrology, Dialysis and Transplantation, Fondazione IRCCS Policlinico San Matteo., PAVIA, Italy. 2Nephrology, Dialysis and Transplantation Unit, Department of Precision and Regenerative Medicine and Ionian Area (DiMePRe-J), University of Bari Aldo Moro, Bari, Italy. 3Department of Internal Medicine and Therapeutics, University of Pavia, PAVIA, Italy. 4Section of Nephropathology, Institute of Pathology, University Hospital Hamburg-Eppendorf., Hamburg, Germany. 5Unit of Nephrology, Dialysis and Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico di Milano, Milano, Italy. 6Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany
Background: The FHR2 is the smallest protein in the Factor H-related protein family and an alternative complement pathway inhibitor. Mutations in the FHR2 gene have been linked to atypical Hemolytic Uremic Syndrome (aHUS) and C3 glomerulonephritis (C3GN). Despite its importance in disease pathology, the role of FHR2 remains insufficiently investigated, particularly within the kidney microenvironment.
Methods To evaluate the binding capabilities and role of FHR2, we analyzed its binding to HUVECs and key components of the glomerular basement membrane (GBM). We assessed FHR2's ability to inhibit C5b-9 cell deposition and evaluated C3b cofactor activity using serum from an aHUS patient with complete FHR2 deficiency1. We compared the patient serum effects with those from Normal Human Serum (NHS) containing standard FHR2 levels. Additionally, we supplemented the patient serum with recombinant FHR2 at a concentration of 140 ng. FHR2 proteins were expressed in HEK-293 cells and purified through affinity chromatography. Binding interactions and complement regulatory activities were evaluated using confocal microscopy, flow cytometry, ELISA, and Western blot analysis.
Results: FHR2 exhibited low-affinity binding to intact HUVECs but showed marked affinity to necrotic cells, with the binding site identified as SCR1-2. This differential binding suggests a role for FHR2 in distinguishing between healthy and damaged tissues, potentially influencing immune responses in the kidney microenvironment. Furthermore, FHR2 bound to all components of the glomerular basement membrane in a dose-dependent manner, particularly showing higher binding intensity for laminin-521, again with the binding site located within SCR1-2. Functionally, FHR2 supported FH in C3b cofactor activity and blocked C5b-9 deposition on cell surfaces. Supplementation of FHR2 at 140 ng/ml significantly reduced C5b-9 deposition on HUVECs in serum from the FHR2-deficient patient, indicating its potential to restore compromised complement regulatory functions.
Conclusion: In conclusion, FHR2 could be important for regulating the complement system in the kidney microenvironment, potentially distinguishing between healthy and damaged tissues. Its ability to support FH and inhibit C5b-9 deposition suggests possible therapeutic uses, but more research is needed to clarify its role and mechanisms.
References
1. Stea ED, Skerka C, Accetturo M, et al. Case report: Novel FHR2 variants in atypical Hemolytic Uremic Syndrome: A case study of a translational medicine approach in renal transplantation. Front Immunol. 2022;13:1008294. doi:10.3389/fimmu.2022.1008294
P-62
Convergent patterns of protein binding promiscuity of complement proteins and immunoglobulins
Mikel Rezola Artero1, Maxime Lecerf2, Lubka Roumenina1, Jordan Dimitrov2
1Centre de Recherche des Cordeliers, Paris, France. 2INSERM, Paris, France
Background
C1q and Factor H (FH) possess the capacity to bind to a multitude of discrete molecular targets in addition to their conventional partners. This property is instrumental in fulfilling their role as pattern recognition proteins and dictates their functional heterogeneity. It is also a typical feature of a fraction of circulating antibodies in all healthy individuals. These antibodies are referred to as polyreactive. Despite the recognised propensity of C1q and FH to bind to multiple targets, the mechanisms and scope of this binding remain to be fully elucidated.
Methods
In this study, human protein microarray technology was employed to evaluate the extent of protein recognition diversity of C1q, FH, C1s and three highly polyreactive human IgG antibodies. The binding to >17000 human proteins was assessed. Bioinformatics analyses explored identified targets, their evolutionary relationships, and physiochemical characteristics. Some of the interactions were also characterized by ELISA and SPR.
Results
We found that C1q and FH, contrary to C1s, exhibit remarkable promiscuity towards a substantial number of human proteins. Both complement proteins were found to recognise over 100 targets with a high intensity, belonging to both extracellular and intracellular compartments. While some of these binding partners have been previously identified, a significant number of novel targets are reported for the first time in this study. A notable finding was the observation that the range of binding promiscuity exhibited by C1q and Factor H was strikingly analogous to that of prominent human polyreactive antibodies. Furthermore, by molecular modelling and functional binding assays, it was demonstrated that both complement proteins and polyreactive antibodies used the same mechanism for achieving binding promiscuity. All these proteins possess positively charged binding surfaces and utilise electrostatic forces for indiscriminate target binding.
Conclusions
The present study delineates the prominent binding promiscuity of C1q and FH. The functional consequences of these newly identified interactions are worthy of further study, as they may highlight novel functions of C1q and FH. Importantly, an analogous pattern of binding promiscuity is shared between immunoglobulins and complement proteins, pointing to the existence of functional convergence in proteins that are evolutionarily unrelated. P-63
Impact of the A09/A15 epitope on structural assembly and function of C1q in the light of Systemic Lupus Erythematosus
Juliane Klehr1, Véronique Rossi2, Christine Gaboriaud2, Marten Trendelenburg3
1Department of Biomedicine, University of Basel, Basel, Switzerland. 2Université Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, Grenoble, France. 3Universty Hospital Basel, Basel, Switzerland
BACKGROUND
Systemic Lupus Erythematosus (SLE) is the archetype of an autoimmune disease characterized by the presence of various autoantibodies. Amongst others, autoantibodies are produced against C1q (anti-C1q), the recognition molecule of the classical complement pathway. C1q is a multi-functional protein also involved in immune cell modulation and apoptotic cell clearance. It consists of six heterotrimeric triple helices assembling into a bouquet-shaped octadecamer with collagen like (CLR) and globular head regions. Anti-C1q predominantly bind the CLR and particularly the epitopes A09 and A15 of the A chain. In this study, we investigate the arginine enriched sequence shared by A09 and A15 to elucidate the role of this domain on the structure and function of C1q.
We hypothesize that the A09/A15 epitope could be crucial for both, octadecamer assembly of C1q and immune cell modulation.
METHODS
We employed headless C1q (CLR_NC2) as a tool to alter the A09/A15 epitope by deletion mutagenesis and alanine scanning. CLR_NC2 mutants were subjected to mass photometry and nano differential scanning fluorimetry (nanoDSF) for stability measurements.
RESULTS
We generated truncated and single, double, and triple mutants in which arginine residues of the A chain were replaced by alanine in position 16, 19, 20, and 22. Lacking of the entire A09/A15 epitope or of arginine in position 19 result in misfolding of the CLR to low molecular weight assemblies. In contrast, single, double, and triple mutants of R16A, R20A, and R22A maintain the bouquet-shaped structure.
CONCLUSION
Arginine residues within A09/A15 play a minor role on the structural integrity of the CLR. While the arginine residue in position 19 is indeed indispensable for proper folding, arginines in position 16, 20, and 22 are replaceable and might hence be involved in the function of C1q.
References
I acknowledge the support of the BioEM lab, the Proteomics Core Facility, and the Biophysics Core Facility at the Biozentrum, University of Basel.
P-64
Characterization of disease-associated FHR-5 variants for their ligand binding and complement modulating capacity
Alexandra T. Matola1,2, Dániel Bencze1, Dávid Szakács1, Marcell Cserhalmi1, Barbara Uzonyi1,2, Mihály Józsi1,2
1Department of Immunology, ELTE Eötvös Loránd University, Budapest, Hungary. 2HUN-REN-ELTE Complement Research Group, Hungarian Research Network, Budapest, Hungary
Factor H-related 5 (FHR-5) belongs to the factor H protein family and plays a role in maintaining the balance of the complement alternative pathway activation and inhibition on host surfaces. Mutations in the CFHR5 gene were linked to eye and kidney diseases. In this study, we investigated the functional effects of five mutations reported in patients with membranoproliferative glomerulonephritis.
Wild type (wt) FHR-5 and mutants (P46S, V110A, G278S, R356H, M514R) were produced in the Sf9-baculovirus expression system and affinity purified. Interaction with disease-relevant host ligands, i.e. C3b, DNA, C-reactive protein (CRP), laminin, and MaxGel (extracellular matrix model) and complement deposition on immobilized FHR-5 were measured in ELISA. The kinetics of the C3b–FHR-5 interaction was determined by bio-layer interferometry. Binding to living endothelial cells and necrotic cells was analyzed by flow cytometry. The effect of FHR-5 on the fluid-phase C3 convertase activity was determined based on C3a generation. Complement-mediated rabbit and sheep red blood cell lysis in the presence of FHR-5 variants was tested in hemolytic assays.
FHR-5 P46S and M514R showed reduced binding to the host ligands, and weaker C3b binding compared to wtFHR-5. In addition, enhanced binding of FHR-5 V110A and G278S to MaxGel, and decreased R356H binding to laminin was revealed. All mutants bound less effectively to living endothelial cells compared to wtFHR-5, whereas G278S binding to necrotic cells was enhanced. Complement activation measured as C3, factor B, properdin and C5 deposition was reduced mostly by P46S and M514R mutations compared to wtFHR-5. We did not detect any effect of wtFHR-5 or the mutants on the fluid-phase C3 convertase activity. FHR-5 and the mutants did not alter complement-mediated hemolysis in near-physiological concentrations, but differences were measured at high concentrations.
Our data show that the mutations in the dimerization region (P46S) and at the C-terminus (M514R), although predicted to be benign, affect most of the interactions and functional properties of FHR-5. Mutations in the ligand binding middle region influence binding and activity variably. Our results shed light on the importance of the functional analysis of disease-associated mutations to understand the role of FHR-5 in kidney diseases.
References
Supported by grants from the National Research, Development and Innovation Office (OTKA K 146911), the Hungarian Research Network (HUN-REN, 0106307), the European Union (nr. 899163 SciFiMed project), and Kidneeds (Iowa, US).
P-65
Targeted binding and activation of native C3 without proteolytic cleavage induced by flip/flopped cell membranes
David Eikrem1, Vivek A. Manivel1, Camilla Mohlin2, Anna Adler1, Osama A. Hamad1, Claudia Dührkop1, Roland Ebert1, Yuji Teramura3,4,1, Kristina Nilsson Ekdahl1,2, Bo Nilsson1
1Uppsala University, Uppsala, Sweden. 2Linnaeus University, Kalmar, Sweden. 3National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. 4University of Tsukuba, Tskuba, Japan
Background: Formation of C3(H2O), allowing formation of the alternative pathway (AP) convertase C3(H2O)Bb, has been suggested to be the nidus of the AP. C3(H2O) has also been shown to bind to activated platelets, allowing binding to the opsonic receptors CD35 and CD11b/CD18. Here we have investigated C3, C3b and C3(H2O) for their ability to specifically bind to and to form AP convertases on various target cell surfaces such as activated platelets and apoptotic cells as alternative triggers to the unspecific fluid-phase AP convertase.
Methods: C3 and two C3(H2O) populations prepared in vitro were separated by Mono S ion exchange chromatography. These separations were utilised in determining the propensity for C3 to bind to and build a convertase on; activated and non-activated platelets, apoptotic cells, and phosphoserine and cholesterol containing liposomes. Furthermore, using monoclonal antibodies we tracked the exposure of neoepitopes upon C3 conformational change within these experiments and within platelet poor plasma clots.
Results: Unexpectedly, when exposed to activated platelets, native C3 bound and formed AP convertases most efficiently to platelets compared to C3b and all C3(H2O) forms. Native C3 also bound to apoptotic cells and liposomes which both exposed phosphatidyl serine and cholesterol, a property in common with activated platelets. The binding was enhanced by properdin and was inhibited by Annexin V on all surfaces. Binding of monoclonal antibodies demonstrated that the adsorbed native C3 had similar conformation as C3(H2O). Bound native C3 did not form covalent bonds with macromolecules at the cell membrane and was not cleaved to C3a and C3b.
Conclusion: Native C3 binds strongly to phosphatidyl serine and cholesterol containing cell membranes acquiring the ability to form C3 convertases and to bind to the phagocytic complement receptors 1 (CD35) and 3 (CD11b/CD18). This represents a new activation mechanism of native C3 that does not require proteolytic cleavage and the tick over of C3b.
P-66
Structural basis of complement C3 recognition by both convertases
Changhao Jia
Peking university, Beijing, China
The complement system plays a fundamental role in human immunity1,2, and its dysregulation is implicated in numerous diseases. Activation of the complement occurs through three main pathways: classical, lectin, and alternative; which converge at the central component, C3. The classical and lectin pathways utilize the C4b2a convertase to cleave C3 and initiate complement activation, while the alternative pathway employs the C3bBb convertase, which is further stabilized by properdin (P). The structural mechanisms governing C3 recognition by these C3 convertases remain incompletely understood. Recently, we determine the high resolution structures of two convertases with C3 engagement by cryo-Em and elucidated the mechanism of substrate C3 recognition by convertases. Alongside the high resolution structures of the C4b2 zymogen in the loading and activation states, these structural snapshots illustrate the sequential conformational changes during convertase maturation. These results offer unprecedented mechanistic insights into complement activation
P-67
Insights into potentiation of Factor H function by the Pneumococcal surface protein C (PspC): the structure of Factor H domains 8-14 in complex with PspC
Jannik Sichau1, Thomas Monecke2, Dierk Niessing2, Christoph Schmidt1
1Institute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University, Ulm, Germany. 2Institute of Pharmaceutical Biotechnology, Ulm University, Ulm, Germany
Background: Factor H (FH), an abundant plasma glycoprotein, restricts alternative pathway activation in the fluid phase and on host surfaces. FH is also exploited for therapeutic purposes, e.g., it may be recruited to biomaterial and cell surfaces via capturing agents. Alternatively, agents that potentiate the regulatory function of FH could be added to blood to introduce higher levels of complement regulation as therapeutic approach. One of the agents that can recruit FH to surfaces and simultaneously potentiates its activity is the N-terminal part of PspC (PspCN). It is known to bind FH with sub-nanomolar affinity and a surface patch surrounding complement control protein (CCP) domain 9 was identified as the binding site. However, it is still unknown how PspCN potentiates FH functions. We hypothesize that PspCN changes the conformation of FH CCPs 8-10 to induce a more active FH conformation.
Methods: We employed biophysical methods (surface plasmon resonance, spectral shift, X-ray crystallography) to characterize in detail the interaction between PspCN and FH. For these analyses, we expressed several FH domain fragments and mutational variants of PspCN. Additionally, alternative pathway haemolysis assays were used to investigate the functional consequences of FH potentiation.
Results: The crystal structure of the FH8-14:PspCN complex was solved at a resolution of 2.8 Å by molecular replacement. PspCN adopts a triple helix with helix 3 swapped between two molecules in a dimer in the crystal structure, with the main interface formed between FH CCP9 and PspCN helix 2. The complex structure was validated by mutating key side chains of PspCN and measuring the effect on the binding affinity. Comparison of the structure of FH8-14 (previously presented) with the complex structure of FH8-14:PspCN shows that the interdomain orientations between CCP9 and CCPs 8 and 10 change. It can be observed that in the apo structure pre-existing amino acid contacts are disrupted and new contacts are formed between CCP domain pairs 8/9 and 9/10 or between these domains and their respective linkers.
Conclusion: The combined biochemical data and the main change in the complex structure of FH8-14:PspCN provides a rational for testing how conformational changes can potentiate FH function.
P-68
Structural insights into how the membrane attack complex is recognised by the diagnostic antibody aE11
Bill Ho1, Charles Bayley-Jones1, Corinna Lau2, Tom Mollnes2, Bradley Spicer1, Michelle Dunstone1
1Monash University, Clayton, Australia. 2University of Oslo, Oslo, Norway
aE11 is a diagnostic antibody that is part of the international serum standard in detecting levels of terminal complement activation (Bergseth et al., 2013). Regular usage of this antibody in the past several decades to study levels of MAC in diseases. Interestingly, aE11 binds to a neoepitope that is presented in the pore but not the precursor components. However, the molecular mechanisms behind this binding modality remain elusive.
Here, the structure of aE11 bound to polyC9, a MAC mimetic, was solved by cryo-EM to 3.2 Å resolution. Interestingly, the MAC neoepitope is formed by the ancillary domains (TSP1, LDLRA) and part of the MACPF domain of two C9 subunits at the periphery of the pore. Further biophysical characterisation reveals that while aE11 stably binds to the neoepitope of the pore, it can also bind to a partial epitope on monomeric purified human C9. Taken together, the study provides the framework for antibody or neoepitope modification to study the MAC in different species and use as a potential therapeutic,
References
The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9. Bayly-Jones C, Ho BHT, Lau C, Leung EWW, D'Andrea L, Lupton CJ, Ekkel SM, Venugopal H, Whisstock JC, Mollnes TE, Spicer BA, Dunstone MA. Commun Biol. 2023 Jan 13;6(1):42.
P-69
Utilising induced pluripotent stem cells (iPSCs) to model the outer retina for complement gene therapy delivery
Sarah Hammadi1, Majlinda Lako1, Claire L Harris1,2, Magali Saint-Geniez3, David Steel1
1Newcastle University, Newcastle Upon Tyne, United Kingdom. 2Cardiff University, Cardiff, United Kingdom. 3Ophthalmology, Novartis Biomedical Research, Cambridge, Massachusetts, USA
Background: Age-related macular degeneration is a major cause of blindness worldwide. Although numerous therapies are currently in clinical trials, a critical challenge remains—the lack of a model that accurately replicates Bruch's membrane (BrM). This membrane is crucial in functional processes, particularly in gene therapy delivery. This study addressed this gap by establishing an ex-vivo decellularised model of the BrM-choroidal complex (dBrM), recellularised with induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE). Firstly, the ability of iPSC-RPE to form a functional monolayer on dBrM was assessed. Next, the transduction capabilities of adeno-associated type 2 vectors carrying GFP (AAV 2-GFP) and AAV 2 sCR1 Flag His tag were evaluated.
Methods: Human donor eyes were dissected and decellularised over two days using 1% Triton X-100, 0.1% SDS + 0.1M EDTA, and 10 U/ml DNase I. iPSC-RPE cells (Fujifilm, Cellular Dynamics) were cultured at a density of 78,000 cells on dBrM (0.50 cm²) and 45,000 cells on Matrigel (0.33cm2) for 21 days. Functional markers, including immunofluorescence imaging, VEGF secretion, transmission electron microscopy (TEM), and proteomic analysis, were assessed. On day 23, cells on Matrigel were transduced apically with AAV 2-GFP (Gyroscope Therapeutics, Novartis) at 5.63×10¹⁰, 9.00×10¹⁰ and 2.25×10¹¹ vg/ml, with GFP-positive cells quantified by flow cytometry. Final transduction of cells on dBrM with AAV2 sCR1 CCP1-11 Flag His (Gyroscope Therapeutics, Novartis) at 4.88×10¹⁰ vg/ml was followed by a fluid-phase co-factor assay and Western blot to assess complement function.
Results: At day 21 of culture, iPSC-RPE grown on dBrM formed confluent ZO1 positive monolayer and had significantly higher basal VEGF (1.45±0.018µg) compared to iPSC-RPE on Matrigel (0.24±0.035µg,p=0.0009,n=2). TEM shows typical iPSC-RPE morphology, including microvilli and melanosomes. Proteomic analysis detected key proteins such as SERPINF1 and TYRP1. The highest transduction with GFP achieved 82% GFP positive after 21 days of transduction with 2.25×10¹¹ vg/ml (n=2). Lastly, 22 days post AAV sCR1 CCP1-11 transduction, iPSC-RPE cells release functional sCR1 CCP1-11, with C3d bands (n=3).
Conclusions: We decellularised human BrM and recellularised with iPSC RPE. AAV2 sCR1 CCP1-11 successfully transduced iPSC RPE cells with functional sCR1 CCP1-11.
P-70
Moss-produced complement factor H (CPV-104) as a therapeutic regulator in complement-mediated diseases.
Paulina Dabrowska-Schlepp1, Todor Tschongov2, Swagata Konwar2, Karsten Häffner2, Mandy Hector3, Anne Wolf3,4, Thomas Langmann3,4, Andreas Busch1, Andreas Schaaf1
1Eleva GmbH, Freiburg, Germany. 2Department of Internal Medicine IV (Nephrology), Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany. 3Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany. 4Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
Background:
Complement-associated disorders are caused by the dysregulation of the complement system, especially its excessive activation. Most drugs that target the complement system are designed to inhibit the complement pathway at either the proximal or terminal levels. The use of a natural complement regulator such as factor H (FH) could provide a superior treatment option by rebalancing an overactive complement system. Utilizing Eleva‘s moss-based expression system allowed the generation of clinically relevant amounts of recombinant FH (CPV-104), which in vitro and in vivo characteristic was found comparable to that of its human counterpart[1]. We have investigated the therapeutic potential of CPV-104 focusing on C3 glomerulopathy (C3G) and age-related macular degeneration (AMD).
Methods:
CPV-104 was produced in moss suspension cultures cultivated in illuminated 500-L single-use stirred-tank reactors. In vivo long-term efficacy of CPV-104 was tested by repeated intravenous (IV) or subcutaneous (SC) administration of CPV-104 in FH deficient mice. To overcome the detrimental immune response in animals and enable the repeated treatment regime, we applied a protocol for depletion of CD20+ B-cells and CD4+ T-cells, preventing antibody formation without influencing the C3G phenotype. CPV-104 efficacy was also tested following intravitreal injection into wild-type mice undergoing light-induced acute retinal degeneration (AMD model).
Results:
The repeated intravenous CPV-104 administration showed the ability to lastingly resolve C3 deposits in FH-deficient mice[2]. Multiple SC injections also lead to prolonged FH availability in mouse sera, resulting in sustained elevation of serum C3 levels and reduced kidney C3 deposits. Further experiments in cynomolgus monkeys validated these findings, suggesting SC injections as another viable road of administration for CPV-104. Light-exposed wild type mice (AMD model) treated with CPV-104 showed strongly decreased microglia and Müller cell reactivity concomitantly with reduced pro-inflammatory cytokines compared to vehicle controls. Furthermore, light-induced retinal degeneration was attenuated in mice that received CPV-104.
Conclusion:
CPV-104 – the human FH analogue – has the potential to enable treatment of complement-associated human diseases by rebalancing rather than inhibiting the complement cascade.
References
[1] Tschongov et al. Front. Immunol., doi: 10.3389/fimmu.2024.1383123 [2] Tschongov et al. Front. Immunol., doi: 10.3389/fimmu.2025.1535547
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Hypothalamic C3aR Activation by Astrocyte-Derived Complement C3a Drives Obesity and Cardiometabolic Dysfunction
Carolin Gragoll1, Elias Rawish1, Timo Rusack1, Michael Schneider1, Katharina Kurz1, Zouhair Aherrahrou2, Jörg Köhl3, Ingo Eitel1
1Medical Clinic II, University Hospital Schleswig-Holstein (UKSH), Lübeck, Germany. 2Institute for Cardiogenetics, University of Lübeck, Lübeck, Germany. 3Institute for Systemic Inflammation, University of Lübeck, Lübeck, Germany
Background
Obesity is a major risk factor for metabolic syndrome and heart failure with preserved ejection fraction (HFpEF). While lipid-driven hypothalamic inflammation via NFkB has been linked to obesity, its impact on hypothalamic regulation of energy balance remains unclear. Given the growing interest in complement inhibitors as potential treatments, this study evaluates the role of hypothalamic complement activation in cardiometabolic syndrome and HFpEF, paving the way for the use of complement receptor antagonists in cardiometabolic diseases.
Methods
In a preventive approach, C57BL/6J (WT) mice were fed a high-fat diet (HFD) and treated with either the C3aR antagonist JR14a or a vehicle. Additionally, hypothalamus-specific C3aR Knockout (C3aR flox x LepR cre) mice received HFD. In a therapeutic setting, WT mice were first made obese before receiving JR14a or vehicle. Key metabolic parameters, including glucose and insulin tolerance, leptin resistance, and diastolic function, were assessed. Flow cytometry was used to measure intracellular C3 levels in astrocytes and microglia, while in-vitro studies with mHypoE-N46 cells investigated how C3a influences leptin-driven AgRP release.
Results
HFD-induced obesity led to increased hypothalamic C3a and C3b expression, impaired insulin sensitivity and central leptin resistance. Mice with a hypothalamus-specific C3aR KO gained significantly less weight compared to WT mice and maintained leptin and insulin sensitivity. Treatment with JR14a prevented diet induced obesity, improved insulin and leptin sensitivity, and reduced body weight in already obese mice. Echocardiography showed improved diastolic function and reduced left ventricular wall thickness in JR14a-treated mice. Astrocytes were identified as the main source of hypothalamic C3 in obesity. In-vitro, C3a inhibited leptin-mediated suppression of orexigenic AgRP secretion in hypothalamic appetite-regulating neurons.
Conclusion
This study demonstrates that diet-induced obesity is linked to hypothalamic activation of the complement C3a/C3aR axis, contributing to metabolic dysfunction and HFpEF. Inhibiting C3aR in the hypothalamus protects against obesity, revealing for the first time the role of the hypothalamic complement system in energy regulation. The novel C3aR antagonist JR14a shows promise as a potential therapeutic agent for cardiometabolic syndrome and HFpEF, supporting its progression to early clinical trials.
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CSL040 ameliorates primary graft dysfunction in pre-clinical models of lung transplantation
Zhenxiao Tu1, Mark Biondo2, Adele Barr2, Paolo Rossato2, Sandra Wymann3, Ingela Vikstrom2, Adriana Bazmorelli2, Darius Fratalia4, Carl Atkinson1
1Northwestern University, Chicago, USA. 2CSL Ltd, Melbourne, Australia. 3CSL Biologics, Bern, Switzerland. 4University of Florida, Gainesville, USA
Background: Complement activation is a key driver of inflammation following lung transplantation and contributes to the development of primary graft dysfunction (PGD). Excessive complement activity leads to endothelial damage, increased vascular permeability, and immune cell infiltration, worsening lung injury post-transplant. Given this here we assessed the therapeutic efficacy of CSL040, a novel soluble CR1-based complement therapeutic, as a means to reduce the severity of PGD, thereby improving lung transplant outcomes.
Methods: To assess CSL040 therapeutic efficacy, we utilize two rodent models (mouse and rat) of lung transplantation. For clinical relevance we utilized a brain-dead donor (BD) allogeneic lung transplant model. Donor lungs were procured from BD donors (Fischer and Balb/c), cold stored for 18 hours, prior to orthotopic transplantation into allogeneic recipients (Wistar and C57Bl/6). CSL040 was administered immediately post-transplant (0 hrs.) and at 24 hrs. post transplantation, at a dose of 30mg/kg. Grafts were harvested at 48 hrs. post-transplant and assessed using the American Thoracic Society recommendations. ATS guidelines were devised to ensure consistency in preclinical lung research and to improve translational potential to human disease. Specifically, three domains were assessed: lung histopathology (e.g., alveolar damage, inflammatory cell infiltration, edema), gas exchange (e.g., arterial blood gases), and inflammation (MPO). Additionally, the impact on local complement activation was assessed by immunohistochemistry for C3d.
Results: CSL040 was similarly effective in both rat and mouse lung transplant models. CSL040 provided effective protection from PGD as determined by significantly reduced histological evidence of injury, improved lung function as measured by PaO2, and reduced inflammation, as shown by reduced immune cell infiltration into the transplanted lung (MPO+ cells). Analysis of complement deposition showed that CSL040 treatment was associated with a significant reduction in C3d, in both species.
Conclusion: Taken together here we demonstrate, using two rodent lung transplant models that incorporate the clinically relevant BD donor injury, that CSL040 significantly improved transplant outcomes. These findings highlight CSL040 as a promising therapeutic strategy for enhancing lung transplant success and warrant further investigation in clinical settings.
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Pre-clinical characterization of CSL040, a soluble complement receptor 1 (CR1) fragment
Sandra Wymann1, Tanja Ruthsatz2, Anjan Bongoni3, Anup Nair4, Yun Dai4, Marcel Mischnik5, Helen Cao4, Jennifer McCrae3, David Leong4, Elena Velkoska4, Daniel Schu5, Kirstee Martin4, Barbara Dietrich2, Peter Cowan3, Tony Rowe4, Adriana Baz Morelli4, Matthew Hardy4
1CSL Biologics Research Center, Bern, Switzerland. 2CSL Behring GmbH, Vienna, Austria. 3Immunology Research Centre, St. Vincent's Hospital, Melbourne, Australia. 4CSL Ltd, Melbourne, Australia. 5CSL Behring Innovation GmbH, Marburg, Germany
Background
Human Complement Receptor 1 (CR1) is a potent regulator of complement both in vitro and in vivo. It functions by binding to its ligands C3b and C4b and inhibiting the classical, lectin and alternative pathways of the complement cascade via the mechanisms of decay acceleration activity and co-factor activity.
Methods
We have generated and characterized CSL040, a soluble version of CR1 containing the long homologous repeat (LHR) -A, -B and -C domains.
Results
CSL040 exhibits significantly greater in vitro and in vivo inhibition of all three complement pathways compared to sCR1/TP10, the full extracellular domain of sCR1. Extensive pharmacokinetic (PK) and pharmacodynamic (PD) assessments of CSL040 in rats and cynomolgus monkeys have been performed, with a demonstrated relationship between the PK properties and asialo content of recombinant protein preparations. The contribution of each LHR domain to C3b and C4b binding and complement pathway activity was comprehensively assessed. Dose-dependent attenuation of tissue damage by CSL040 was shown in two renal mouse models of disease: warm renal ischemia-reperfusion injury, and glomerulonephritis. In addition, the systemic toxicity, toxicokinetics and PD effects of CSL040 were assessed in two-week intravenous (IV) bolus studies in Han Wistar rats and cynomolgus monkeys at daily doses of 50, 150 and 500 mg/kg. CSL040 was demonstrated to be safe and well tolerated; the NOAEL for rats and cynomolgus monkeys is 500 mg/kg.
Conclusions
The pre-clinical program demonstrated that CSL040 has a favorable safety profile and supported the clinical development of CSL040 into First-in-Human studies (NCT0593758).
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The Power of Fusion: Tuning Pharmacokinetic and Pharmacodynamic Properties of Complement-Modulating Peptides and Proteins Via Antibody Engineering
Florian Meyer1, Stephanie Vogt1, Aleksandra Blagojevic1, Alexander John Lander1, Peter Rüthemann1,2, Sofia Pinheiro1, Said Rabbani1, Daniel Ricklin1
1Molecular Pharmacy, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland. 2Computational Pharmacy, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
Despite an impressive increase of complement-targeted therapeutics in the clinic, medical needs remain and dosing regimens of drugs and candidates are not ideal. Latter is particularly true for peptides and small proteins, which feature high potency and selectivity but face challenges regarding pharmacokinetic (PK) properties. Among PK-improving strategies, fusion of proteins/peptides to the crystallizable fragment (Fc) of IgG4 immunoglobulins provide an elegant option to enhance plasma half-life and potentially modulate target binding without activating complement. In this study, we generated Fc-fusion constructs with a peptide of the compstatin family and the leech-derived protein gigastasin, and evaluated the impact on complement inhibition and other properties.
Genes encoding compstatin analogue Cp01-V3I or gigastasin were cloned into the pFUSE-hIgG4-Fc2 vector and transfected into HEK293F cells for expression. The fusion proteins were purified using Protein-A affinity chromatography. Complement inhibition was measured with an in-house ELISA and/or chromogenic substrate assays. For the compstatin peptibodies, C3b binding was analyzed using surface plasmon resonance (SPR).
In the case of gigastasin, Fc-fusion enabled mammalian expression that could hitherto not be achieved for the non-tagged protein. Fc-Gigastasin potently inhibited complement initiation pathways albeit with reduced activity; interestingly, the impact was more pronounced for the lectin than the classical pathway. Similarly, the inhibitory potency of compstatin peptibodies in vitro was reduced when compared to the free peptides, yet SPR indicated an enhanced target residence on surface-bound C3b.
This proof-of-concept study demonstrates that Fc-fusion of complement-inhibiting proteins/peptides provides a suitable strategy to tune therapeutic properties. Although the initial fusion constructs showed somewhat lower activities, they remained potent inhibitors and activity may be regained by optimizing fusion formats. Importantly, our findings indicate that Fc-fusion may facilitate the production of therapeutics and impact target and/or location selectivity. Finally, it is expected that PK-properties of Fc-fusions are improved; their increased molecular size reduces renal clearance, while FcRn-mediated recycling may further extend half-life. In subsequent stages, it will be important to explore the in-vivo profiles of fusion constructs to assess their impact on both pharmacodynamic and -kinetic properties, optimize formats accordingly, and compare them to other PK-enhancing strategies such as lipidation.
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Rapid Complement Inhibition with the C5 Inhibitor Crovalimab: Timing Analysis Using Animal Model and COMPOSER Trial Data
Bradley P. Dixon1,2, Cristian Brocchieri3, Leigh Beveridge4, Muriel Buri3, Niels Janssen3, Patty Leon4, Yoshinori Tsuboi5, Simon Buatois3
1Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA. 2Children's Hospital Colorado, Aurora, CO, USA. 3F. Hoffmann-La Roche Ltd, Basel, Switzerland. 4Genentech, Inc., San Francisco, CA, USA. 5Chugai Pharmaceuticals Co., Ltd., Tokyo, Japan
Background: Atypical haemolytic uremic syndrome (aHUS) is treated with complement C5 antibody therapy, which effectively inhibits complement-mediated thrombotic microangiopathy (TMA) and improves renal function, but regular intravenous (IV) infusions can be burdensome. Crovalimab is a novel C5 antibody, designed for subcutaneous (SC) self-injection every 4 weeks (Q4W).
The 4-part, Phase I/II COMPOSER trial (NCT03157635) evaluated crovalimab in paroxysmal nocturnal haemoglobinuria (PNH), which is a disease driven by uncontrolled complement activation. In COMPOSER, crovalimab maintained disease control and was well tolerated after a median exposure of 3 yrs.
Crovalimab is under evaluation in adult and pediatric patients with aHUS in the ongoing Phase III trials, COMMUTE-a (NCT04861259) and COMMUTE-p (NCT04958265).
Due to the rapidly progressing nature of aHUS, patients require rapid suppression of complement activation upon diagnosis. Data from in vivo models and COMPOSER were used to determine the time to complete complement inhibition after the first crovalimab IV dose.
Methods: Part 2 (n=10) and Part 4A (n=8) of COMPOSER enrolled patients with PNH who were naive to complement inhibition. Part 2 patients received crovalimab 375 mg IV on Day (D) 1, 500 mg IV on D8, 1000 mg IV on D22 and 170 mg SC weekly from D36 for 20 wks. Part 4A patients received an optimised crovalimab dosing regimen of 1000 mg IV on D1, 340 mg SC on D2, 8, 15 and 22, and 680 mg SC Q4W from D29 onwards for 20 wks. Crovalimab concentration, free C5 and complement activity were measured using validated assays.
Results: In treatment-naive patients from COMPOSER Parts 2 and 4, mean free C5 concentration dropped to below 1 µg/ml, indicating a high level of target engagement within 1-6 hours from first IV dose. Also, inhibition of terminal complement activity was reached within 1 hour, with CH50 values near or below the LLOQ (10 U/ml). Complete complement blockade was generally maintained long term, up to Wk 20, in both Parts 2 and 4, regardless of dose.
Conclusion: Crovalimab induced a complete, rapid and sustained blockade of terminal complement activity within hours of first dose.
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Effects of Complement C3/C3b Inhibition on Control of Malignant Effusions and Neutrophil Phenotypes in Patients with Recurrent Epithelial Ovarian Cancer: Interim Analysis of a Phase 2 Clinical Trial
Emese Zsiros1, ANM Nazmul Khan2, Thejaswini Giridharan1, Sora Suzuki1, Celia DeJohn1, Suzanne Hess1, Katherine Mager1, Sarah Werner1, Karen McLean1, Nicole Gaulin1, Peter Frederick1, Igor Puzanov1, Martin Kolev3, Pascal Deschatelets3, Brahm Segal2
1Roswell Park Comprehensive Cancer Center, Buffalo, USA. 2Moffitt Cancer Center, Tampa, USA. 3Apelllis Pharmaceuticals, Waltham, USA
Background:
Malignant effusions (ME) can promote metastatic seeding and are challenging to manage. Using ME from patients with epithelial ovarian cancer (OC) and other solid tumors as authentic components of the tumor microenvironment (TME), we observed that ME reprogram neutrophils to acquire complement-dependent T cell suppressor function characterized by inhibition of T cell proliferation and cytokine and metabolic responses. Pegcetacoplan (APL-2) is a C3 and C3b inhibitor approved for paroxysmal nocturnal hemoglobinuria; its use in cancer is novel. The rationale for C3/C3b inhibition includes abrogation of neutrophil recruitment and suppressor function in TME, inhibition of neutrophil extracellular traps (NETs), and reduction of vascular leak driving ME accumulation. We designed a phase 2 trial of pegcetacoplan-based therapy in patients with recurrent OC and persistent MEs (NCT04919629).
Methods:
This trial is conducted at Roswell Park. It includes 2 sequential safety lead-in cohorts: (i) pegcetacoplan alone for 2 weeks followed by addition of pembrolizumab (n=3), and (ii) pegcetacoplan + pembrolizumab + bevacizumab (n=3). Subsequent randomized phase cohorts include: (i) bevacizumab (standard of care), (ii) pegcetacoplan + pembrolizumab, and (iii) pegcetacoplan + pembrolizumab + bevacizumab. Primary endpoints are safety and control of ME. Blood, ME, and tumor tissue are collected at baseline and on therapy.
Results:
Pegcetacoplan–based regimens were well-tolerated to date. Among nine evaluable patients, the best observed response has been stable disease (SD) in seven, including two patients who have not required therapeutic drainage of MEs for over 40 weeks. Pegcetacoplan had no impact on total white blood cell or absolute neutrophil counts but partially abrogated the capacity of MEs to induce neutrophil suppressor function and significantly reduced serum markers of NETosis.
Conclusions:
In this interim analysis, complement C3/C3b inhibition with pegcetacoplan demonstrates a favorable safety profile and a preliminary signal of ME control in recurrent OC. Immunologic studies suggest that the clinical benefit may be associated with diminished neutrophil suppressor function and NET formation. Further enrollment and long-term follow-up will clarify the therapeutic potential of this novel approach.
References
This work is supported by NIH R01CA267690. Apellis provided pegcetacoplan and Merck provided pembrolizumab in support of the clinical trial.
P-77
Characterization and efficacy of a C2-blocking antibody in a rat kidney transplant model
Eline Haspeslagh1, Laura Bracke1, Jolien Delaere1, Elisabeth de Zeeuw2, Karen De Winter1, Yasmine Driege1, Tim Delahaye1, Erik Hack2, Kevin Budding1,2, Daniëlle Krijgsman2, Inge Van de Walle1
1argenx, Ghent, Belgium. 2University Medical Center Utrecht, Utrecht, Netherlands
Background
Delayed graft function (DGF) following kidney transplantation is associated with inferior short- and long-term transplant outcomes and is not preventable by current therapies. The complement pathway has been proposed as a target for DGF prevention, as it majorly contributes to ischemia-reperfusion injury (IRI) underlying DGF. We addressed the impact of blocking the classical (CP) and lectin (LP) complement pathways at the level of complement factor C2, thereby leaving the alternative (AP) complement pathway intact, in a rat model of kidney transplantation.
Methods
Recipient rats received a kidney exposed to a long cold ischemia time and were treated intravenously with anti-C2 monoclonal antibody (mAb) or placebo control in the peri-transplantation period. Serum creatinine levels and blood urea were analyzed at Day 3 post-transplant. Transplanted kidneys from surviving animals were histologically examined at Day 14. As the involvement of the three distinct complement pathways in IRI is species dependent, we further assessed the relative importance of the CP/LP in IRI-induced complement activation on different human endothelial cells in vitro using a hypoxic chamber.
Results
Blocking C2 activity significantly improved serum creatinine and blood urea nitrogen levels 3 days post-transplant compared with those kidneys treated with placebo. Furthermore, kidney health at 2 weeks post-transplant improved, as indicated by a reduced incidence and/or severity of IRI-related histopathological findings, including tubular regeneration, kidney enlargement, and discoloration, which were associated with reduced kidney weight. In several human endothelial cell types, C3 fixation was consistently dependent on C2 presence and could be prevented by anti-C2 mAb treatment in a dose-dependent manner.
Conclusions
We confirmed in preclinical models that C2 is a suitable target to prevent complement-induced IRI and subsequent DGF after kidney transplant.
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Exploring the Role of the Terminal Complement Pathway in the DSS-Induced Colitis Model of Inflammatory Bowel Disease
Cedric Cui, Titaya Lerskiatiphanich, John Lee, Trent Woodruff
School of Biomedical Sciences, The University of Queensland, Brisbane, Australia
Background Inflammatory bowel disease (IBD) is a chronic condition characterised by inflammation in the gastrointestinal tract. Evidence suggests that dysregulation of the complement system, particularly downstream of C5 cleavage, plays a significant role in colitis pathogenesis. While the C5a–C5aR1 axis has been implicated in driving colonic inflammation, the broader roles of the terminal complement pathway, including the membrane attack complex (MAC), in modulating inflammation and tissue damage remain poorly understood. Here, we investigated C5a receptors and the MAC in experimental colitis to better define their respective contributions to disease progression and resolution.
Methods Using a dextran sulfate sodium (DSS)-induced colitis model, we assessed disease severity in wild-type and complement-deficient mice, including C5aR1-/-, C5aR2-/-, C5aR1/2 double knockout, and C9-/- strains. Over a 12-day experimental timeline we measured body weights, clinical disease scores, colon length, and performed histological analysis. This comprehensive approach allowed us to evaluate the respective contributions of C5a receptors and the MAC in both inflammatory processes and tissue protection in the context of colitis.
Results C5aR1-deficient mice exhibited partial protection from DSS-induced colitis, evidenced by reduced weight loss and improved clinical scores. Remarkably, C5aR2-deficient and C5aR1/2 double knockout mice displayed even stronger protection, with minimal weight loss and markedly reduced colon tissue damage in response to DSS treatment. In contrast, C9-deficient mice developed significantly exacerbated disease, highlighting the crucial role of the MAC in mitigating tissue damage in this model.
Conclusion These findings demonstrate that C5a signalling via both C5aR1 and C5aR2 contributes to inflammation and disease progression, while the MAC plays a protective role in mitigating tissue damage in DSS-induced colitis. Our results suggest that therapeutic strategies targeting C5a receptors, while preserving MAC activity, could offer a refined approach for treating bowel inflammation. Further studies are needed to fully elucidate the precise mechanisms by which terminal complement components modulate colitis and to explore potential clinical implications for complement-based therapies in IBD.
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Targeting C5a/C5aR1-mediated NETosis restores immunotherapy response in STK11-mutant lung cancer
Ajona Daniel, Janire Debersaques, Yaiza Senent, Ana Remirez, Cristina Sainz, Luis Montuenga, Juan Dubrot, Beatriz Tavira, Ruben Pio
Cima Universidad de Navarra, Pamplona, Spain
Background: Lung tumors harboring co-occurring KRAS and STK11 mutations are associated with an inert tumor immune microenvironment (TME) and exhibit resistance to PD-1/PD-L1 immune checkpoint inhibitors. We aimed to uncover novel immunotherapeutic vulnerabilities in this tumor subtype.
Methods: Using CRISPR-based gene editing, we generated Stk11-knockdown cells from Lacun3, a lung adenocarcinoma cell line with a KrasG12D mutation and wild-type Stk11 and Tp53. The effects of Stk11 knockdown were assessed both in vitro and in vivo. Transcriptomic changes between parental and Stk11-deficient Lacun3 cells were investigated by bulk RNA sequencing (RNAseq). The effects on the tumor microenvironment (TME) were analyzed through single-cell RNA sequencing (scRNAseq) and flow cytometry following subcutaneous inoculation of the cells in syngeneic Balc/c mice. Tumor growth was monitored to evaluate therapeutic responses.
Results: While in vitro proliferation was unchanged, Stk11-knockdown cells exhibited enhanced tumor growth in vivo. scRNAseq and flow cytometry revealed an immunosuppressive TME in Stk11-deficient tumors characterized by reduced T-cell infiltration and increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Transcriptomic analysis identified the complement system as a significantly dysregulated pathway. Genes involved in complement initiation (C1qb, C1r, Masp1, Masp2, C2 and Cfp) and soluble inhibitors (C4bp and Cfh) were downregulated, whereas complement effectors (C3 and Hc, the gene for C5), and membrane-bound inhibitors (Cd46, Cd55 and Cr1l) were upregulated in Stk11-deficient Lacun3 cells. Stk11-knockdown tumors showed elevated C5a levels and increased expression of C5aR1 on PMN-MDSCs. The C5a/C5aR1 axis has been implicated in the induction of NETosis (a form of programmed cell death in PMN-MDSCs that contributes to tumor progression). Accordingly, enhanced NETosis, determined as areas of co-localized expression of Ly-6G and citrullinated histone H3, was observed in Stk11-knockdown tumors. Therapeutic intervention with NETosis inhibitors (the PAD4 inhibitor GSK484 or DNase) or a C5aR1 antagonist (PMX205), in combination with PD-L1 blockade, significantly reduced tumor growth.
Conclusion: Stk11 deficiency promotes an immunosuppressive TME in lung tumors through complement dysregulation and C5a/C5aR1-driven NETosis. Targeting this axis restores sensitivity to immune checkpoint blockade, and offers a promising therapeutic strategy for patients with KRAS/STK11-mutant lung cancer.
References
Acknowledgements: This study was supported by the Alberto Palatchi Foundation and the Spanish Ministry of Health-Instituto de Salud Carlos III (FIS)-Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” (PI23/00573).
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HDM-FH treatment during static cold storage protects against ischaemic injury in a simulated kidney transplant model.
Beth Gibson, Chloe Connelly, Samuel Tingle, Grace Mallett, Harriet Denton, Georgios Kourounis, Madison Cooper, Saimir Luli, Lucy Bates, Marnie Brown, Sam lee, Balaji Mahendran, Emily Thompson, Colin Wilson, Neil Sheerin, Kevin Marchbank
Newcastle University, Newcastle-upon-Tyne, United Kingdom
Organs undergoing transplantation are subject to ischaemic damage following reperfusion in the recipient. Complement is a key driver of ischaemic reperfusion injury (IRI) and therefore an important target for drug treatment. Our research group previously designed a homo-dimeric minimal-FH (HDM-FH; PMID: 29588430) based on factor H, the major negative regulator of the alternative Complement pathway and the C3 amplification loop. A previous study using an ex vivo porcine renal transplant model applied HDM-FH during 6 hours of normothermic machine perfusion (NMP) with autologous blood. Subsequent analysis showed that HDM-FH delivered via NMP both controlled Complement activation and reduced ischaemic damage. Following these data we have now assessed HDM-FH in a paired human kidney model of simulated clinical transplantation. HDM-FH in University of Wisconsin (UW) preservation solution or UW alone was delivered to kidneys via cold flush and the organs maintained in cold static storage (SCS) for 12.5 hours. Kidneys were then flushed to remove unbound drug and attached to an NMP for reperfusion with red blood cells and human serum with active complement, mimicking the first 6 hours following transplantation. Use of a fluorescently-conjugated HDM-FH, the in vivo imaging system (IVIS) and standard immunofluorescent microscopy confirmed that the drug binds to human kidney at 4⁰C when delivered via organ preservation solution. As with our previous large animal model, treatment with HDM-FH reduced Complement activation in human kidneys. Delivery of HDM-FH spiked UW via SCS decreased perfusate levels of C3a, C5a and sTCC and urinary C5a when compared to UW alone. Further to this HDM-FH treated kidneys also showed decreased ischaemic damage downstream of this complement inhibition. Overall cellular apoptosis was reduced and we observed increased levels of the pro-survival protein Klotho, indicating reduced tubular epithelial cell damage. Endothelial cell activation was also decreased, with increased expression of S1P1, a marker of improved endothelial barrier integrity. In conclusion, application of HDM-FH in the preservation solution via cold flush and static cold storage appears to be a simple and effective drug delivery route to reduce Complement activation. This would protect transplanted kidneys from IRI.
P-81
Brain-penetrant anti-C7 therapy reduces neurodegeneration and microgliosis in an Alzheimer’s mouse model
Jack Reddaway1, Jacqui Nimmo1, Ryan Bevan1, Maarten Dewilde2,3, Bart De Strooper4,5, B. Paul Morgan1, Wioleta Zelek1
1UK Dementia Research Institute Cardiff, School of Medicine, Cardiff University, Cardiff, United Kingdom. 2Therapeutic and Diagnostic Antibodies, Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium. 3PharmAbs, the KU Leuven Antibody Center, KU Leuven, Leuven, Belgium. 4KU Leuven and VIB Leuven, Leuven, Belgium. 5UK Dementia Research Institute, University College London, London, United Kingdom
Background: Previously, we demonstrated that brain-penetrant complement C7 blocking antibody (mAb) mitigates brain inflammation, rescues synapse loss and improves cognition in Alzheimer’s disease (AD) mouse model (AppNL-G-F; PMID: 39215579), implicating membrane attack complex (MAC) in driving neuroinflammation in the model. Here, we extend this study, investigating the impact of anti-C7-mediated MAC inhibition on neuronal and glial health and function in the model.
Method: AppNL-G-F mice (6-9 months old, n= 12) were treated systemically with the recombinant brain penetrant mAb 73D1 anti-C7 mAb fused with nanobody (Nb62) targeting the transferrin receptor (TfR), to enable brain delivery (Nb62-r-mAb). The control AppNL-G-F group (n= 12) received the same C7-blocking antibody (73D1) without the brain-penetrant shuttle (control-r-mAb). Harvested brains were examined using immunofluorescence to detect neuronal degeneration (Fluoro-jade C), plaque pathology (Amylo-Glo) and microgliosis (CD11b and P2RY12). To measure impact on serum markers of neurodegeneration, the Alamar NULISAseq mouse panel (120 neurodegeneration-associated markers) was applied to serum samples from treated mice.
Results: No differences were observed between Nb62-r-mAb treated and control mice in Aβ plaque number despite reduced Aβ in brain homogenates (TBH; p= 0.047) and surprisingly, plaque size was greater in Nb62-r-mAb treated mice (p=0.024). Immunohistochemical analysis of hippocampus revealed fewer homeostatic (P2RY12-positive) and reactive (CD11b-positive) periplaque microglia in Nb62-r-mAb-treated mice (p= 0.023, p= 0.0449 respectively), indicating reduced microgliosis. Fluoro-Jade C IHC revealed fewer degenerating neurons in Nb62-r-mAb–treated mice (p= 0.005), demonstrating a neuroprotective effect. In support of these findings the Alamar NULISAseq mouse panel identified multiple serum markers of neurodegeneration as being altered between Nb62-r-mAb treated and control mice.
Conclusion: Inhibiting C7/MAC in the brain of APPNL-G-F mice had minimal effect on amyloid plaques but reduced microgliosis and neuronal degeneration, providing further support for the therapeutic potential of brain-penetrant complement-inhibiting drugs for neuroprotection downstream of amyloid accumulation.
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Development of novel peptide ligands with improved selectivity and pharmacological properties for complement system receptors
Richard Clark, Joshua Dent, Declan Gorman, Xaria Li, Xiaosa Wu, Cedric Cui, Jenny Fung, John Lee, Johan Rosengren, Trent Woodruff
School of Biomedical Sciences, The University of Queensland, Brisbane, Australia
The complement system is a key component of the innate immune response and contributes to protection against infection and recovery from injury. Key components of the complement system are the anaphylatoxins C3a and C5a, which acts via there receptors, C3aR (C3a) and C5aR1 and C5aR2 (C5a), to modulate a range of inflammatory responses. However, aberrant regulation of this system is implicated in a broad range of inflammatory-based diseases including many neurodegenerative conditions. A number of small peptide-based agonists and antagonists of the complement receptors, primarily based on the C-terminus of C3a and C5a, have previously been developed, but ligands with improved selectivity, pharmacokinetic and pharmacodynamic profiles, are desirable. In this work, we describe the design and characterisation of next-generation complement peptide ligands that address some of the limitations of the currently available molecules, including selective biased and unbiased agonists of C5aR1 (EC50s = 6 – 60 nM, >900-fold selectivity) and a potent and selective full agonist of C5aR2 (EC50 = 13 nM, >1000-fold selectivity). These are short linear peptides and are therefore simple and economical to produce so may be valuable and accessible tools for further elucidating the physiological role of the complement receptors, and as potential drug leads.
P-83
AAV-mHDM-FH: Safety and expression profile in two murine models of hyper-complement activation
Grace Mallett1, Isabel Pappworth1, Grant Logan2, Ian Alexander2, David Kavanagh3,1, Kevin Marchbank1,3
1Newcastle University, Newcastle-upon-Tyne, United Kingdom. 2Childrens Medical Research Institute, Sydney, Australia. 3National Complement Therapeutic Centre, Newcastle-upon-Tyne, United Kingdom
C3 glomerulopathy (C3G) and atypical haemolytic uraemic syndrome (aHUS) are rare kidney disorders characterized by the dysregulation of the alternative pathway (AP). Unlike aHUS, current treatment options for C3G are limited. Factor H (FH) plays a key role in regulating the AP, and mice lacking FH develop a disease similar to C3G. We previously developed mouse homodimeric minimal FH (mHDM-FH) as a gene therapy using an adeno-associated virus (AAV) vector incorporating a liver-specific promoter to rebalance the AP activation. We previously noted that a single dose of AAV-mHDM-FH (1 x 1011) restored complement regulation in Cfh-/- mice by 21 days post inoculation, however this was associated with a strong anti-HDM-FH response, which was markedly reduced in Cfh+/- mice.
In the current study, we focused on long-term evaluation of AAV-mHDM-FH in both Cfh+/- mice and C3N/N mice (the C3 D1115N gain-of-function mouse model of aHUS). At six months post-inoculation, C3N/N mice treated with AAV-mHDM-FH showed significantly improved survival compared to AAV-GFP controls. However, the therapeutic effect was partially limited due to reduced binding affinity of mHDM-FH to the mutant C3 D1115N protein. These findings indicate that while AAV-mHDM-FH retains some functionality in C3 mutation-driven disease, its efficacy may not match therapies targeting the terminal complement pathway.
In Cfh+/- mice, no signs of disease or safety concerns were observed over the six-month period. Importantly, increased serum levels of both C3 and FH were detected, suggesting improved control of the amplification loop in these mice. AAV transgene expression remained detectable in the liver at six months, an encouraging result for long-term efficacy.
Across both experimental time points, anti-AAV antibody responses were detected in Cfh+/- mice. These responses peaked at 21 days post-inoculation but were present at relatively low levels after 6 months. This pattern aligns with observations in patients receiving AAV-based gene therapies. Despite the presence of anti-AAV antibodies, the 6 month data showed transgene expression and no safety concerns, leaving the extent to which these antibodies impact therapeutic efficacy still uncertain. Looking ahead, we aim to enhance long-term vector expression by exploring the use of alternative promoters tailored to different cellular targets.
References
1. Yang Y, Denton H, Davis O R, Smith-Jackson K, Kerr H, Herbert A P, Barlow P N, Pickering M C and Marchbank K J An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy J Am Soc Nephrol 2018 29(6):1649-1661
P-84
Targeting C5a Inflammation in ALS: Ex Vivo Findings from PMX205 Phase Ib Trial
Jenny Fung, John Lee, Trent Woodruff
School of Biomedical Sciences, the University of Queensland, Brisbane, Australia
Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, with growing evidence suggesting that inflammation, particularly via complement activation, contributes to disease progression. C5a-C5aR1 signalling is the major inflammatory byproduct of complement activation. Preclinical studies have demonstrated that C5aR1 blockade with PMX205, a selective C5aR1 antagonist, can mitigate neuroinflammation in ALS models. PMX205 is under investigation in Phase I human trials in ALS patients. Here, we evaluated the effects of PMX205 in ALS patients by examining cytokine and immune response markers in blood samples, utilizing standard cytokine assays and advanced proteomic profiling.
Methods: Blood samples from eight ALS patients were collected before, and 1-2 hours after, PMX205 administration (0.2 or 0.4 mg/kg administered subcutaneously). Whole blood was then stimulated with vehicle (PBS) or C5a (10nM or 100nM) for 24 hours. Plasma cytokine levels were measured using the BioLegend® LEGENDplex™ Cytokine Bead Array (13 analytes). For a more comprehensive analysis, Somalogic proteomic technology was employed to assess 1,500 inflammatory and immune-related markers.
Results: C5a stimulation at both 10 nM and 100 nM significantly upregulated IL-1β, IFNα, TNFα, IL-6, IL-8, and IL-10 in ALS patient blood samples. Paired blood samples taken from patients after PMX205 administration showed significantly reduced levels of these cytokine. Extended proteomic analysis revealed additional changes driven by C5a, with numerous cytokine and chemokine proteins showing significant upregulation, and modulation after PMX205 treatment. Comparing pre- and post-drug treatment levels highlighted a consistent reduction in key cytokines and chemokines, with the most notable changes in CCL and CXCL family members, and cytokines IL-6 and IL-8. These results underscore a robust pharmacodynamic effect of low-dose subcutaneously administered PMX205 in modulating C5a-driven inflammatory responses in ALS patients.
Conclusions: PMX205 effectively inhibits C5a-mediated inflammation in ALS patients, as evidenced by both cytokine and proteomic analyses in blood samples stimulated ex vivo. ALS patients administered PMX205 had significantly reduced pro-inflammatory cytokines and chemokines. These findings provide critical pharmacodynamic support for further clinical evaluation of longer-term dosing of PMX205 in larger ALS cohorts. Future analyses using more detailed proteomics could provide deeper insights into the broader immune modulation effects of PMX205.
P-85
Inhibition of the complement component C5 and the Toll-like receptor molecule CD14 prevents systemic and local kidney inflammation in mice experiencing brain death.
Tom Eirik Mollnes1,2, Neeltina Jager3, Petra Ottens4, Camilla Schjalm5, Zwanida Veldhuis6, Henri Leuvenink6, Søren Pischke7
1Nordland Hospital Trust, Bodø, Norway. 2University of Oslo, Oslo, Norway. 3Department of Anesthesiology, University of Groningen,, Groningen, Netherlands. 4Department of Surgery, University of Groningen,, Groningen, Netherlands. 5Department of Immunology, University of Oslo, Oslo, Norway. 6Department of Surgery, University of Groningen, Groningen, Netherlands. 7Department of Anesthesiology and Intensive Care, Oslo University Hospital, Oslo, Norway
Background:
Brain death (BD) induces a systemic inflammation impairing donor organ quality. Complement and Toll-like receptors (TLRs), with the key co-receptor CD14 molecule, are key innate recognition immune systems. We hypothesized that dual inhibition of complement (C5) and TLRs (CD14) will prevent BD-mediated innate immune inflammation.
Methods:
BD was induced in mice either untreated, treated with a C5 inhibitor, a CD14 inhibitor, or both. Blood and kidneys were collected after three hours. Cytokines were analyzed using enzyme-immuno assays and qPCR.
Results:
In plasma, a substantial increase in interleukin-6 (IL-6), KC (IL-8 analogue), IL-12, monocyte chemoattractant protein (MCP-), macrophage inflammatory protein MIP-α, MIP-β, eotaxin, RANTES and G-CSF (median 90-fold increase) were observed in BD animals compared to sham (all p<0.01). In kidneys, BD substantially induced IL-6, KC, TNF, MCP-, P-Selectin, and VCAM-1 (all p<0.01). In plasma, C5 and CD14 inhibition, either single or in combination, virtually abolished all cytokines in the BD animals (>90% for six cytokines and 70-90% for three) (all p<0.01). In kidneys, the effect of inhibition was similar (>90% for IL-6 and KC and 60-80% for TNF and MCP- (all p<0.01). We are currently investigating the cytokine response in other organs from these mice, including heart, lungs and liver.
Conclusion: Single and combined inhibition of C5 and CD14 efficiently prevented BD-induced systemic inflammation and reduced local kidney inflammation. These data urge for clinical studies using this therapeutic approach on deceased BD donors to enhance donor organ quality.
P-86
Compstatins abolish pathogenic fibroblast function associated with inflammatory maladaptation of tissues
Jasna Friscic1, Sven Geisler2, Rebecca Lee3, Merita Rumano3, Dimitrios C Mastellos4, John D Lambris3, Markus H Hoffmann1
1University of Lübeck, Institute of Systemic Inflammation Research, Lübeck, Germany. 2University of Lübeck, Cell Analysis Core Facility, Lübeck, Germany. 3Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. 4National Center for Scientific Research 'Demokritos', Athens, Greece
Background: Chronic inflammatory diseases (CIDs) are characterized by hyperactivated local tissue. This tissue sensitization is caused by tissue-resident structural cells such as fibroblasts or epithelial cells that become metabolically and functionally primed during frequent or long-lasting episodes of inflammation. This cellular maladaptation is often driven by the activation of intracellular complement components (also known as the complosome), which consequently metabolically invigorates cells and permits them to fully utilize their inflammatory potential.
Methods: Primary human synovial fibroblasts (SFs) derived from the joints of individuals with established rheumatoid arthritis (RA) or SFs from healthy joints primed by repeated in vitro-stimulation with TNFα and ARPE-19 retinal epithelial cells were treated with compstatin derivatives. Uptake and subcellular localization of compstatins were analyzed by confocal microscopy employing antibodies against compstatin derivatives and cell organelles. Functional and metabolic profiling was performed in the presence or absence of human serum (providing an external source of C3) and with/without a C3-blocking antibody. Compstatin-induced transcriptomic changes in SFs were assessed by bulk RNA-Seq and downstream pathway analysis.
Results: Compstatins were readily taken up into fibroblasts and epithelial cells and partially colocalized with intracellular C3 in endosomes, autophagsosomes, and the endoplasmatic reticulum. Compstatins inhibited fibroblast function associated with inflammatory tissue priming in arthritis, such as adherence, matrix invasion, cytokine production, and migration. Gene set enrichment analysis suggested a compstatin-induced reduction of SF proliferation and activation, and changes in polyamines and cellular metabolism, which were reflected by metabolic flux analyses. In ARPE-19 cells, compstatins inhibited production of reactive oxygen species and generation of C3a. Antibody-mediated blockade of cell-secreted C3 inhibited uptake of compstatins and their capacity to blunt pathogenic cellular function.
Conclusion: Peptide-based C3- targeting therapeutics such as compstatins are attractive candidates for regulation of overshooting tissue inflammation in CIDs such as Rheumatoid arthritis and Age-related macular degeneration. Locally administered compstatins would block the activation of endogenously generated C3 in tissue-occupying cells, thus inhibiting or rolling back inflammatory maladaptation of tissues.
P-87
Mapping inhibitory epitopes on C7 with monoclonal antibodies
William MacIntosh-Smith1, Vinira Wijesuriya1, Jack Readdaway1, B Paul Morgan2, Wiola Zelek2
1Cardiff University, Cardiff, United Kingdom. 2Cardiff, Cardiff, United Kingdom
Background: Complement culminates in formation of the membrane attack complex (MAC), triggering cell activation or death through pore formation. MAC has been implicated in many inflammatory diseases, including dementias where MAC is involved in synaptic degradation and neuronal death that typify neurodegeneration. Hence, MAC formation is a favoured target to suppress neurodegeneration. C7 is an essential component of MAC assembly, presenting a viable target for therapeutic blockade of MAC. We showed that a brain-delivered blocking anti-C7 mAb rescued pathology in a mouse AD model (PMID 38485063). Here we describe a panel of anti-human C7 mAbs, including C7 function-blockers and non-blockers, and their use to map epitopes on C7 that block MAC assembly.
Methods: mAbs were generated from C7-deficient mice immunised with human C7 using hybridoma technology. Classical pathway haemolysis and reactive lysis (RL) assays using purified components were used to assess capacity to block MAC assembly. Binding to C7 and C5b67 complex was tested using ELISA. Epitope identification and binning was conducted using competition ELISA and surface plasmon resonance. Cross-species reactivity of mAbs was assessed using western blot.
Results: Haemolysis assays differentiated the C7-blocking (8 clones) and non-blocking (2 clones) mAbs. Blocking mAb activity was assessed in RL assays; stepwise assembly in RL assays verified if blocking mAbs inhibited MAC assembly by preventing binding of C7 to C5b6. ELISA and SPR demonstrated all mAbs bound strongly to C7 alone, and in preformed C5b67 complex. All mAbs bound cynomolgus monkey C7 in WB, while most additionally detected C7 in plasma from mouse and rat. SPR analysis and competition ELISA revealed four distinct inhibitory epitopes, defined as epitope 1 (mAb 4A5), epitope 2 (mAb 54A4), epitope 3 (mAb 22G4) and epitope 4 (mAbs 5F3, 5D11, 6E5, 9D3, 3F6). Non-blocking mAbs did not share any epitope with blocking mAbs.
Conclusion: Blocking of C7 to C5b6 was verified among all MAC blocking anti-C7 mAbs; nevertheless, they bound (at least) four distinct epitopes on C7 to cause this block. The presence of distinct blocking epitopes on C7 provides insight into MAC assembly and informs optimal targeting strategies for blocking MAC assembly in disease.
References
PMID 38485063
P-88
The enigmatic role of C5aR2 in diabetic kidney disease
Inez Trambas1, Adrienne Laskowski1, Amalia Khayyira1, Arpeeta Sharma1, Poh Yi Gan1, Trent Woodruff2, Melinda Coughlan1, Sih Min Tan1
1Monash University, Melbourne, Australia. 2The University of Queensland, Brisbane, Australia
Background
Diabetic kidney disease (DKD) is the primary cause of kidney failure globally. Existing treatments only slow, but do not stop, DKD from progressing to kidney failure. We have previously shown that complement activation, specifically the C5a-C5aR1 pathway, plays a pathogenic role in DKD. However, C5a also signals via a second receptor, C5aR2, and its role in DKD is currently unknown. We will investigate the role of C5aR2 in the DKD using C5aR2-/- mice.
Methods
Diabetes was induced in 6-week-old wildtype C57BL6/J and C5aR2-/- mice by five daily injections of low dose streptozotocin (STZ; 55mg/kg), and followed for 20 weeks. 24-hour urine was collected for the assessment of albuminuria, a marker of kidney function. Mitochondrial function was analysed using the Seahorse XF Bioanalyzer. Spectral flow cytometry was employed to immunophenotype renal and splenic immune cell populations. Gene expression and kidney structure were analysed using qPCR and histological staining, respectively.
Results
Wild-type diabetic mice exhibited increased urine output, water intake and blood glucose, and renal functional decline consistent with diabetes, however, the ablation of C5aR2 did not have a significant impact on these metrics. In contrast to our previous findings in C5aR1-/- mice, deletion of C5aR2 had no effect on albuminuria and renal fibrosis in diabetic mice. qPCR analyses revealed a significant increase in inflammatory genes such as MCP- and TNFa in diabetes, but the deletion of C5aR2 had little impact on these markers in diabetic mice. Mitochondrial functional assays revealed dysfunction in complex II-mediated respiration in C5aR2-/- mice. Furthermore, spectral flow cytometry revealed a slight increase in CD11b+ monocytes in the spleen and kidneys of C5aR2-/- mice, and interestingly a significant decrease in C5aR1+ monocytes in the diabetic kidney in comparison to WT diabetic mice. However, these changes did not result in an improvement in kidney function or structural damage in diabetic C5aR2-/- mice.
Conclusion
While further studies are required to elucidate additional metabolic changes associated with this enigmatic receptor in the diabetic setting, our novel findings suggest that C5aR2 does not play a pathogenic role in this preclinical model of DKD.
P-89
Motor neuron disease C9orf72 dipeptides mediate neurotoxicity in human brain organoids through activation of C5aR1
Yuzhihan Cao1, Giovanni Pietrogrande2, Selin Pars2, Richard Clark1, Ernst Wolvetang2, Trent Woodruff1, Eduardo Albornoz1
1School of Biomedical Science, University of Queensland, Brisbane, Australia. 2Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia
Background: Motor neuron disease (MND) is a progressive neurodegenerative disorder characterized by motor neuron loss and neuroinflammation. The C9orf72 hexanucleotide repeat expansion is the most common genetic cause of familial MND, leading to the accumulation of poly-Gly-Ala (poly-GA) dipeptide repeats (DPRs). These aggregates trigger neuroimmune responses, yet their effects on human microglia-neuron interactions remain poorly understood. This study investigates the role of complement C5a receptor (C5aR1) and inflammasome activation in poly-GA-mediated neuroinflammation using a human brain organoid co-culture model containing microglia (MDMi-BO).
Methods: Human monocyte-derived microglia-like cells (MDMi) were incorporated into iPSC-derived brain organoids (BOs) to establish the MDMi-BO model, recapitulating features of the neuroimmune environment of the human brain. Poly-(GA)10 DPRs were synthesized and applied to MDMi-BOs to assess neurotoxicity and associated innate immune activation. IL-1β release was quantified via ELISA, while phosphorylated C5aR1 (pC5aR1) and inflammasome markers were analysed using immunocytochemistry. The contribution of C5aR1 to neurotoxicity and neuroinflammation was examined using the selective inhibitor PMX205.
Results: Poly-(GA)10 induced robust IL-1β secretion in MDMi-BOs, accompanied by increased pC5aR1, NLRP3 and ASC expression in microglia, indicating complement and inflammasome activation. Immunofluorescence analysis revealed neuronal loss, supporting the role of microglia in DPR-induced neurotoxicity. Pharmacological inhibition of C5aR1 with PMX205 reduced pC5aR1 expression, and partially suppressed IL-1β production. Notably, PMX205 significantly prevented neuronal damage mediated by poly-(GA)10 DPRs.
Conclusion: The MDMi-BO model provides a physiologically relevant system to investigate neuroimmune interactions in neurodegenerative diseases such as MND. Poly-GA aggregates trigger complement and inflammasome activation in microglia, leading to neuronal loss in MDMi-BOs. C5aR1 inhibition attenuated DPR-mediated neuronal loss and IL-1β production, suggesting that complement activation may amplify inflammasome signalling to drive neuronal toxicity. These findings highlight the potential of targeting complement activation and microglial C5aR1 signalling to mitigate neurodegeneration in MND.
P-90
Exploring the rules for classical complement pathway activation by antibody pairs with co-dependent oligomerization
Vitalijs Ovcinnikovs
Genmab BV, Utrecht, Netherlands
HexElect is an antibody format designed to selectively activate the classical complement pathway on cells expressing two different surface antigens. This selectivity is achieved through use of engineered IgG1 antibody pairs with co-dependent Fc hetero-oligomerization. However, the rules for simultaneous engagement and clustering of IgG-bound antigens in a manner compatible with complement activation are currently unclear. To address this, we investigated the role of antigen pair expression and their natural proximity in classical complement pathway activation by HexElect antibodies, using C3b deposition as a readout (C3b). We probed every possible pairing of 50 cell surface proteins with two antibody clones each, in a full factorial design. For each target, mRNA- and protein expression using bulk RNAseq and quantitative flow cytometry were measured, and target pair co-localization was determined using proximity ligation assay (PLA). In both PLA and C3b assays, approximately 10-15% of all target combinations resulted in a detectable signal, and when comparing individual targets there was significant overlap between the readouts. However, at the target-combination level, correlation was much lower (R=0.55). Aiming to understand antibody-induced interactions, we then explored which parameter was best able to predict the magnitude of C3b deposition. Applying symbolic regression machine learning models, we found that surface protein expression was the best predictor (R=0.82), followed by PLA (R=0.55) and gene expression (R=0.39). Combining PLA and/or expression did not lead to a significant increase in performance, implicating protein expression as the main driver of antibody-induced interactions, and subsequent activation of complement (C3b).
P-91
Mechanism of action of bi-specific antibodies recruiting endogenous complement inhibitors for targeted local complement inhibition in the context of rheumatoid arthritis
Leendert Trouw, Haiyu Wang
Leiden University Medical Center, Leiden, Netherlands
Background
The complement system is well known as a defense mechanisms against infections, but also contributes to tissue damage in many diseases. In several autoimmunediseases, autoantibodies accumulate locally in target organs. In rheumatoid arthritis (RA), autoantibodies like anti-carbamylated protein antibodies (anti-CarP) can trigger excessive complement activation, leading to the continuous tissue damage. Complement inhibitory drugs have been developed, however, these drugs work systematically, which results in increased risk of infections. Therefore, we aim to make bi-specific antibodies that can inhibit complement locally while leaving the systemic complement activity intact.
Methods
Bi-specific antibodies (bsAbs) were produced by controlled Fab-arm exchange. In bsAbs, one arm binds to a disease relevant antigen, like carbamylated proteins and the other arm binds to endogenous complement regulators, like factor H (FH) or C4b-binding protein (C4BP). Antibodies targeting HIV, DNP or biotin were also produced as controls.
Results
We previously showed that bsAbs targeting model antigen DNP and FH or C4BP strongly inhibited all three pathways of complement activation. Now we generated disease relevant bsAb based on, RA related, anti-CarP antibodies, again with anti-FH or anti-C4CP. Also anti-CarP bsAbs could achieve complement inhibition in a local and antigen specific fashion. In the presence of complement activating anti-CarP autoantibodies from RA patients, the anti-CarP bsAbs could also inhibit complement activation. Next we recently performed studies on the mechanisms of action of the bsAbs. We observed that the endogenous levels of FH and C4BP are sufficient. The mechanism of action studies revealed that bsAbs that recruit FH or C4BP can function as inhibitors multiple times, while bsAbs based on e.g. eculizumab only work once.
Conclusion
BsAbs targeting autoantigens can achieve complement inhibition, providing a new therapeutic approach for the local treatment of autoimmune diseases.
P-92
Regulation instead of inhibition: a triple fusion protein of complement regulators (TriFu) is superior in protecting animals in a rat xenotransfusion model when compared to C5-inhibition
Susa Savukoski, Alina Dettner, Arthur Dopler, Christoph Q. Schmidt, Markus Huber-Lang, Marco Mannes
Ulm University Medical Center, Ulm, Germany
Background:
The use of complement inhibitors has revolutionized the treatment of complement-mediated diseases, leading to the development of numerous inhibitors targeting different proteins of the cascade. However, recent mechanistic insights have unraveled scenarios in which stoichiometric inhibitors can be bypassed, raising concerns about their efficacy in certain clinical settings. A novel concept involves the combination of key functional domains from three naturally occurring regulators into a single molecule – referred to as a triple fusion molecule (TriFu) – which has demonstrated promising effects in vitro. However, in vivo evidence of its efficacy has been lacking until now.
Methods:
In a xenotransfusion model mimicking a mismatched erythrocyte transfusion, human red blood cells (hRBC) were transfused into Wistar rats which naturally have pre-formed antibodies against hRBCs leading to classical pathway activation. Blood samples were collected at multiple time points before euthanization at two hours post-transfusion. Blood and organ samples were analyzed between the groups which included: no treatment, treatment with TriFu, C5 inhibition (OmCI) or cobra venom factor (CVF) for de-complementation prior to transfusion.
Results:
Transfusion of hRBC resulted in massive intravascular hemolysis, as indicated by increased plasma levels of lactate dehydrogenase and hemoglobin. This hemolysis was only slightly reduced by C5 inhibition but was markedly attenuated by TriFu, as confirmed by flow cytometric quantification of remaining hRBC. C3 opsonization of hRBC was most pronounced under C5 inhibition, whereas TriFu effectively prevented C3 opsonization to a level comparable to that after de-complementation with CVF. Under hemolytic conditions, neutrophil counts and inflammatory cytokines (TNF, IL-6, IL-18, MCP-) were substantially elevated in peripheral blood. TriFu and CVF effectively reduced these inflammatory responses to control levels, whereas OmCI did not. Hemolysis-dependent C3 deposition in liver tissue was completely abrogated by TriFu, highlighting its potential for organ protection.
Conclusion:
De-complementation with CVF and complement regulation by TriFu effectively stopped intravascular hemolysis, leading to a marked reduction in a wide range of systemic inflammatory signs. In contrast, conventional C5 inhibition was less effective, suggesting the superiority of TriFu-mediated complement regulation over C5 inhibition strategy.
P-93
Complement Biomarker Responses to Factor B and C3 Inhibition in C3 Glomerulopathy
Kameron Kruger, Anna Carmen, Dingwu Shao, Carla M Nester, Richard JH Smith, Yuzhou Zhang
University of Iowa-MORL, Iowa City, USA
Background
C3 glomerulopathy (C3G) is a rare renal disorder characterized by complement dysregulation of the alternative pathway (AP). Emerging AP inhibitors include: (1) iptacopan, an oral factor B inhibitor that selectively binds to the enzymatic center of factor B, blocking its activity and preventing downstream complement activation; and (2) pegcetacoplan, a targeted inhibitor of C3/C3b that prevents the cleavage of C3 and subsequent formation of the C3 and C5 convertases. While both inhibitors have demonstrated clinical efficacy in reducing proteinuria and stabilizing renal function, their pharmacodynamic effects on complement biomarkers remain incompletely characterized.
Methods
We evaluated complement biomarker profiles in 14 C3G patients treated with iptacopan (n = 6; age 19–67 years) or pegcetacoplan (n = 8; age 14–36 years). Serum and plasma samples collected before and after treatment were analyzed using a comprehensive in-house complement biomarker panel to monitor longitudinal changes.
Results
Both iptacopan and pegcetacoplan effectively inhibited AP activation, as evidenced by suppression of AP-specific activation assays and reductions in complement activation fragments (C3a, C3d, and Bb). These changes indicate complement inhibition at the level of the C3 convertase. Consequently, plasma C3 levels increased in both treatment groups, though the magnitude of increase differed: iptacopan-treated patients generally reached low-normal C3 (normal reference range: 90–180 mg/dL), whereas pegcetacoplan-treated patients often exhibited markedly elevated plasma C3, substantially exceeding normal limits. Additionally, highly elevated properdin levels were observed in pegcetacoplan-treated patients but not in those treated with iptacopan. Both therapies also reduced C5 convertase activity, as demonstrated by normalization of plasma C5 and soluble C5b-9. Notably, neither drug impacted circulating autoantibody levels. Together, these findings highlight distinct pharmacodynamic profiles and illustrate the dynamic complexity of complement regulation in C3G.
Conclusion
Iptacopan and pegcetacoplan robustly inhibit AP activation, offering promising therapeutic strategies for C3G. Larger longitudinal studies are needed to define biomarker predictors of treatment response and guide personalized complement-directed therapy.
References
Supported in part by National Institutes of Health R01 DK110023.
P-94
AON-D21 – Complement C5a-Neutralizing Human/Mouse Cross-Reactive L-aptamer from Bench to Phase 2 Clinical Trial
Kai Hoehlig, Antonio Perez, Simone Sell, Sven Klussmann, Axel Vater
Aptarion Biotech AG, Berlin, Germany
Using mirror-image in vitro selection by exponential enrichment (SELEX), nuclease-resistant aptamers targeting human and mouse C5a were identified. These aptamers bind C5a and inhibit signaling through both C5a receptors. A crystal structure provides insights into the contact points between aptamer and C5a.
Non-clinical studies of the initially identified L-aptamer (NOX-D19) and its affinity-enhanced variants (NOX-D20, NOX-D21/AON-D21) have demonstrated efficacy in several mouse models of human diseases, including bacterial pneumonia (with and without mechanical ventilation), syngeneic lung cancer (in combination with anti-PD1 therapy), cholesterol crystal embolism, and asthma sensitization.
Following preclinical development in mice and marmoset monkeys, a single- and a multiple ascending dose trial were conducted in healthy volunteers. Pharmacokinetic/pharmacodynamic (PK/PD) modeling enabled the design of a dosing regimen for repeated administration aimed at sustained C5a inhibition.
Currently, AON-D21 is being evaluated in the Phase 2 ADCAP trial, which is assessing its safety and efficacy in 150 ICU-admitted patients with severe community-acquired pneumonia (viral or bacterial) across 28 sites in five European countries. Initial pharmacodynamic results confirm inhibition of circulating C5a in treated patients. The final study results are expected in 2026.
References
This will be a review of preclinical and clinical work of a currently active complement therapeutics program with multiple references.
P-95
Eculizumab in severe HELLP syndrome – a case report
Noora Korkalainen1, A. Inkeri Lokki2,3, Mikko Haapio4, Jenni Heikkinen-Eloranta1
1Women’s Hospital, HUS Helsinki University Hospital and University of Helsinki, Helsinki, Finland. 2Department of Bacteriology and Immunology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 3; Heart and Lung Center, HUS Helsinki University Hospital and University of Helsinki, Helsinki, Finland. 4HUS Abdominal Center, HUS Helsinki University Hospital and University of Helsinki, Helsinki, Finland
Background:
Preeclampsia complicates 2-8% of pregnancies globally and is one of the leading causes of maternal and perinatal mortality worldwide. Preeclampsia is characterized by newly-onset hypertension and proteinuria during second half of pregnancy, or in the absence of proteinuria, subjective maternal symptoms. Preeclampsia may lead to fetal growth restriction, prematurity, and it may cause organ failure in liver and kidneys of the mother, leading to significant long-term effects for the mother and the newborn. HELLP syndrome is a pregnancy-associated TMA disorder that affects 0.5-0.9% of pregnancies.
Case presentation:
Forty-one-year-old primigravida was referred at gestational age H29+4 to the Helsinki University Hospital Maternity clinic because of elevated blood pressure (130-169/100-107mmHg). Antihypertensive medication was started with follow-up visits. In the evening of 34+0 she presented severe upper abdominal pain, and the blood pressure was extremely elevated at 213/120 mmHg. The platelet level was decelerating from 146 to 91, ALT was elevated, and lactate dehydrogenase was elevated. An urgent cesarean section was performed at 34+1 weeks at 2 a.m. because of compromised maternal status. Female infant (1570g; -2,5 SD) was born. After the cesarean section, the mother`s platelets and Hb were low. Urine protein excretion peaked being 7g/24h in the morning. By the afternoon, mother’s condition declined. Hemolysis was clearly observed, and there was disturbance in the coagulation. Coombs test was negative, serum complement C3 and C4 were low. Due to the extremely severe course of illness, eculizumab was given on the second postpartum day. On the third postpartum day, the patient`s situation was improving. She received an additional dose of eculizumab on the seventh postpartum day.
Results:
The patient had severe preeclampsia and HELLP. Although genetic testing for cm-TMA (complement mediated TMA) remained negative, the clinical course (severe liver injury and mild kidney injury) and response to eculizumab treatment suggested that our patient has pregnancy-induced cm-TMA.
Conclusion:
The differential diagnosis between HELLP and cm-TMA often lies in spontaneous recovery of HELLP patients usually on third postpartum day. However, here the course of illness was extremely severe, and hence, eculizumab treatment was given resulting in a full recovery of the patient.
P-96
Tumor-derived Factor B Drives Tumor Growth and anti-PD-1 resistance in STK11-mutant Lung Adenocarcinoma
Sora Suzuki1, Bojidar Kandar1, ANM Nazmul Khan1,2, Thejaswini Giridharan1, Han Yu1, Kayla Catalfamo1, Prashant Singh1, Ryan Bushey3, Elizabeth Gottlin3, Michael Campa3, Edward Patz3,4, Daniel Ajona5, Ruben Pio5, Brahm Segal1,2, Edwin Yau1
1Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA. 2Moffitt Cancer Center, Tampa, FL, USA. 3Duke University School of Medicine, Durham, NC, USA. 4Grid Therapeutics LLC, Durham, NC, USA. 5Cima Universidad de Navarra, Pamplona, Spain
Background: Loss of function STK11 mutations occur in about 15-20% of lung adenocarcinoma (LUAD) and are associated with immunotherapy failure and worse survival. We observed that STK11-mutant LUAD overexpresses complement C3, and high C3 mRNA expression was associated with worse survival. Using syngeneic Kras-mutant LUAD (CMT167), we observed that knockout of STK11 rendered tumors resistant to anti-PD-1, and knockout of C3 in STK11KO tumors (CMT167-STK11KO-C3KO) resulted in markedly reduced tumor growth. Similar results regarding STK11-KO occurred in the Lacun3 LUAD model, supporting generalizability. The effect of C3-KO was not observed in nude mice or CD8-depleted WT mice, indicating that tumor-derived C3 promotes immune evasion. RNA-sequencing comparing STK11KO-C3WT vs. STK11KO-C3KO tumor showed that C3 increases expression of multiple complement genes including complement Factor B (FB), which is required for the alternative pathway (AP) C3 and C5 convertases. The role of tumor cell-intrinsic FB in anti-tumor immunity is unknown. We hypothesized that tumor-derived FB promotes growth and anti-PD-1 resistance in STK11-mutant tumors.
Methods: We used CRISPR-Cas9 RNP to generate CMT167-STK11KO tumor cells with FB deletion (CMT167-STK11KO-FBKO). To evaluate the effect of tumor-derived FB in vivo, we compared the subcutaneous tumor growth of CMT167-STK11KO-FBKO and CMT167-STK11KO LAUD cells in syngeneic immunocompetent mice. Iptacopan is an oral FB inhibitor approved for paroxysmal nocturnal hemoglobinuria. We evaluated whether iptacopan would overcome anti-PD-1 resistance of STK11KO tumors in mice.
Results: Knockout of FB in STK11KO tumors resulted in robust inhibition of growth in immunocompetent mice compared to STK11KO-FBWT tumors. While single-agent treatment with anti-PD-1 or iptacopan did not significantly affect STK11KO-FBWT tumor growth, combination anti-PD-1 and iptacopan therapy significantly delayed tumor growth and extended survival.
Conclusions: Our results extend our prior findings on tumor-derived C3 promoting immune evasion in STK11-mutant LUAD and identify tumor-derived FB in promoting tumor growth and anti-PD-1 resistance. These data provide rationale for an early phase clinical trial of iptacopan to enhance anti-PD-1 effectiveness in patients with STK11-mutant LUAD.
P-97
Analysis of Human Factor H-Related Gene and Protein Expression in Rheumatoid Arthritis Synovium Identifies a Novel Mechanism Promoting Dysregulated Complement Pathway Activation
Nirmal Banda1, Larry W. Moreland1, Kevin D. Deane1, Dmitri Simberg1, Robert I. Scheinman1, Rachel M. Frank1, Michael R. Clay1, Jennifer A. Seifert1, Rachel Lau2, Costantino Pitzalis2, Myles J. Lewis2, V. Michael Holers1
1Colorado University at Anschutz Medical Campus, Aurora, USA. 2Queen Mary University of London, London, United Kingdom
Complement activation plays an important role in models of human rheumatoid arthritis (RA), but its role in human disease is unknown. Complement factor H (CFH) is a negative regulator of the alternative pathway (AP). In addition to CFH, five human complement factor H-related (CFHR) proteins largely function ex vivo as positive regulators of the AP. The role of FH and FHRs in early RA (eRA, <1 year) and chronic RA is unknown.
We compared bulk CFH and CFHR mRNA expressions in both the human rheumatoid arthritis (RA) synovium and blood from the Pathobiology of Early Arthritis Cohort (PEAC) (n = 87) and chronic RA (n = 208) studies. Bulk RNA-seq data from PEAC were analyzed to determine the expression of CFHR mRNAs in the synovium and blood from three histologic pathotypes. CFHR mRNA levels comparing gender, as well as presence of anti-cyclic citrullinated peptide (CCP) antibodies and rheumatoid factor (RF), were also examined. CFHR and CFH proteins along with immune cells were detected in the synovium (n = 9) using multiplexed immunohistochemistry (MIHC).
Using MIHC, all CFHR and CFH proteins were present in RA synovium. With regards to regional synovial localization, CFH was almost undetectable or expressed at low levels in the synovial lining but abundant in sub-synovial lining areas. In contrast, CFHR proteins were co-localized in the synovial lining area along with complement C3. In three pathotypes, no differences were found in the level of expression of CFHR mRNAs. In the synovium, a significant negative correlation was observed between CFH mRNA levels and the disease activity score. In synovium and blood, no differences were observed in the expression of CFHR mRNA levels comparing gender, or presence of anti-CCP antibodies or RF. In RA patients, there was a significant and positive correlation between CFHR3 synovial mRNA level, anti-CCP, and RF, but a negative correction between CFH and X-ray joint space narrowing, a marker of disease severity.
In sum, due to the low levels of CFH but the presence of CFHRs, the synovial lining might fail to be protected from complement-mediated attack, and CFHR3 may play a particularly important pathogenic role.
P-98
Renal Pathology as a Predictor of Complement Dysregulation in C3 Glomerulopathy
Jillian Hall1, Lauren Fergus1, Tina Liu1, Monica Hall1,2, Patrick Walker3, Richard Smith1,2, Carla Nester1,2
1Molecular Otolaryngology and Renal Research Laboratories, Iowa City, USA. 2University of Iowa Hospitals and Clinics, Iowa City, USA. 3Arkana Laboratories, Little Rock, USA
Background:
C3 Glomerulopathy (C3G) is characterized by complement dysregulation and resulting C3 deposition in glomeruli. We reviewed the characteristics of the baseline kidney biopsy in a cohort of patients with C3G to determine whether biopsy features of activity and chronicity at presentation correlate with complement dysregulation across the disease course.
Methods:
Patient data from the University of Iowa’s C3G Natural History Study were used. Criteria for entry included baseline native biopsy diagnosis of C3G, complement biomarkers within 1 year of diagnostic biopsy, and at least one additional complement panel drawn greater than one year later. Patients with a history of dialysis, transplant, or anti-complement therapy were excluded. Significance was assessed using Pearson correlation coefficients with two-tailed p values (95% confidence); results were considered statistically significant when p-values were less than 0.05.
Results:
Of 59 subjects, 35 (59.3%) presented with an activity score of ≥ 9 while 12 (20.3%) presented with a chronicity score of ≥ 4, the a priori determinants of increased risk for progression to renal failure. A high activity score was associated with a high soluble C5b-9 (p = 0.009) and a negative slope over time (p = 0.042, R = -0.283). In patients >18 years of age, a high activity score was also associated with an increased Ba slope over time (p = 0.03, R = 0.579). In patients who presented <12 years of age, the index chronicity score correlated with a sustained increase of C5 nephritic factor (p = 0.023, R = 0.532).
Conclusion:
Some complement biomarkers correlate with indices of renal pathology in C3G. Specifically, a more severe activity score correlates with a high soluble C5b-9 and a progressive increase in Ba, consistent with complement activity playing a role in renal inflammation. The relationship between an increased chronicity score and sustained elevation of C5 nephritic factor in the pediatric population points to a longer-term effect of ongoing terminal pathway activity.
References
References: Kidney Int. 2018 Apr;93(4):977-985, Clin J Am Soc Nephrol. 2022 Jul;17(7):994-1007, Am J Kidney Dis. 2021 May;77(5):684-695.e1. Acknowledgements: NIDDK: R01 110023
P-99
Complement C3 and Complement Factor H affect glomerular IgA deposition in IgA Nephropathy
Xianzhi Li1,2, Xiaohan Yuan1,2, Xinran Ni1,2, Lijun Liu1,2, Sufang Shi1,2, Jicheng Lv1,2, Hong Zhang1,2, Li Zhu1,2
1Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China. 2Peking University Institute of Nephrology, Beijing, China
Background:
IgA nephropathy (IgAN), characterized by glomerular deposits of IgA, is the most common primary glomerulonephritis worldwide. This IgA deposition triggers complement activation, leading to kidney damage. Genome-wide association studies (GWAS) have identified the CFH-CFHRs region as a susceptibility locus for IgAN. Our previous work has shown that genetic variants in this region affect glomerular C3 deposition in IgAN patients, influencing renal injury. This study aims to investigate the interactions between complement factor H (CFH), complement C3, and IgA deposition in IgAN.
Methods:
A total of 1,584 biopsy-proven IgAN patients were included, with 326 patients also having circulating CFH levels, to explore the correlation between C3, CFH with glomerular IgA deposition intensity. Additionally, IgAN models were created using repeated intraperitoneal injections of complete Freund’s adjuvant (CFA) and Lactobacillus casei cell wall extract (LCWE) in both C3 knockout mice (C3-KO-IgAN) and wild-type mice (WT-IgAN) to study the role of C3 in IgAN. CFH supplementation was given to WT-IgAN mice (5 mg/kg intraperitoneally every 72 hours for 3 weeks) to evaluate its therapeutic effects.
Results:
In IgAN patients, lower circulating levels of CFH and C3—indicators of complement overactivation—were significantly associated with increased glomerular IgA deposition. CFA and LCWE led to glomerular IgA deposition, mesangial proliferation, and immune cell infiltration in both WT-IgAN and C3-KO-IgAN mice. In WT-IgAN mice, glomerular C3 deposition accompanied with IgA deposition. Interestingly, while C3-KO-IgAN mice showed no C3 deposition and a trend toward reduced urinary albumin-to-creatinine ratio (ACR), they had significantly increased glomerular IgA deposition and macrophage infiltration. Treatment with CFH in WT-IgAN mice resulted in lower circulating IgA and IgA-IgG levels, increased circulating C3 levels, and decreased glomerular IgA and C3 deposition, alongside reduced macrophage infiltration and inflammatory cytokines, and a significant reduction in urinary ACR.
Conclusion:
Our findings indicate that C3 knockout, while inhibiting complement activation and reducing urinary ACR, paradoxically exacerbates glomerular IgA deposition and inflammatory cells infiltration. CFH supplementation offers renal protective effects by concurrently reducing IgA deposition, complement activation, inflammation, and glomerular injury. These results suggest that CFH supplementation may represent a promising therapeutic strategy in complement-targeted therapies for IgAN.
P-100
Age-Dependent Plasma Factor D Levels and Their Clinical Implications in Complement-Mediated Kidney Diseases
Angela Nelson, Yuzhou Zhang, Cecelia Fierce, Zachary Lynch, Christopher Culek, Carla Nester, Richard Smith
Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, Iowa City, USA
Background
Complement Factor D (FD), also known as adipsin, is a serine protease essential for alternative pathway (AP) activation. Among complement proteins, it circulates at the lowest plasma concentration (1–2 mg/L in adults). As FD is primarily synthesized by adipocytes and catabolized through renal tubules, we sought to test two hypotheses. First, that FD levels in healthy children are significantly lower than those in adults reflecting differences in body mass index (BMI), and second, that FD levels in patients with chronic kidney disease are elevated independent of BMI.
Methods
Plasma FD levels in EDTA samples were quantified using an ELISA (Hycult). A cohort of 159 healthy controls (age range: 1–65 years) without known complement disorders or inflammatory conditions was analyzed to establish normal reference levels, which were compared to FD levels in 145 subjects (age range: <1–82 years) with complement-mediated renal disease. FD levels were examined in relation to BMI and renal function.
Results
In healthy controls, FD levels strongly correlated with age (r = 0.83): in persons ≥20 years of age, the mean FD concentration was 1.07 mg/L (median: 1.12 mg/L, ±1 SD: 0.78–1.36 mg/L), while in persons <20 years of age, the mean FD level was significantly lower at 0.58 mg/L (median: 0.62 mg/L, ±1 SD: 0.41–0.75 mg/L). Among patients with complement -mediated kidney disease, FD levels correlated with chronic kidney disease (CKD) as measured by serum creatinine (r = 0.82) but did not correlate with age (r=0.25). In the cohort with CKD, there was no correlation between FD levels and BMI (r = 0.29).
Conclusion
This study defines age-specific FD reference ranges to improve the assessment of complement activity and kidney function in pediatric patients. In addition, we show that as kidney function deteriorates, FD levels increase independent of BMI. Given its role in AP activation, this increase in FD may contribute to complement dysregulation further exacerbating local kidney injury.
References
Supported in part by National Institutes of Health R01 DK110023.
P-101
Mice lacking MASP- and/or MASP- are protected from the development of retinal degeneration in a murine model of NaIO3-induced dry age-related macular degeneration (AMD)
Takeshi Machida1, Tomoko Omori1, Yumi Ishida1, Tetsuju Sekiryu2, Hideharu Sekine1
1Department of Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan. 2Department of Ophthalmology, Fukushima Medical University School of Medicine, Fukushima, Japan
Background:
Age-related macular degeneration (AMD) is one of the causes of visual impairment in the elderly, and overactivation of the alternative pathway (AP) is thought to be associated with the pathogenesis of the disease. In the later stage of AMD, it progresses to neovascular AMD with choroidal neovascularization or dry (or atrophic) AMD without choroidal neovascularization. MASP- and MASP- are essential complement factors for the physiological activation of the lectin pathway (LP) and AP, respectively. To clarify whether the LP is involved in the pathogenesis of dry AMD and further investigate the involvement of the AP, we analyzed the retina of a sodium iodate (NaIO3)-induced murine dry AMD model using mice lacking MASP- and/or MASP-.
Methods:
To induce dry AMD-like retinal degeneration, wild-type, MASP--deficient (MASP--/-), MASP--deficient (MASP--/-), or MASP-/3-double-deficient (MASP-/3-/-) C57BL/6J mice were injected with 20 mg/kg of NaIO3 through a tail vein. The eyes were then removed, and retinal histopathological analysis was performed using H&E, immunofluorescence, and TUNEL staining. C3 activation levels in protein extracts from retinal tissue were measured by Western blot analysis using anti-mouse C3 polyclonal antibody.
Results:
Wild-type mice showed retinal degeneration, including depigmentation and disruption of the retinal pigment epithelium, atrophy of the photoreceptor layer, and thinning of the outer nuclear layer after NaIO3 injection. In contrast, those pathological changes were significantly attenuated in MASP--/-, MASP--/-, and MASP-/3-/- mice. Photoreceptor cell death and retinal C3 activation were observed in NaIO3-injected wild-type mice, whereas those pathological changes were significantly attenuated in NaIO3-injected MASP--/- and MASP-/3-/- mice. On the other hand, those pathological changes or retinal MBL-A or C4 deposition levels in NaIO3-injected MASP--/- mice were comparable to those in NaIO3-injected wild-type mice.
Conclusion:
Our results indicate that MASP- plays a pivotal role in C3 activation in the retina most likely via AP activation leading to the development of retinal degeneration in the NaIO3-induced murine dry AMD model. Our results also indicate that MASP- plays a role in the development of NaIO3-induced retinal degeneration in this murine model, although it remains unclear whether its role in the retinal degeneration is through LP activation.
P-102
The Complement C3a Receptor Regulates the Tumour Microenvironment
Barbara Rolfe, Stefan Sonderegger, Nadya Panagides, Crystal McGirr, Jamileh Nabizadeh, Xaria Li, Trent Woodruff
The University of Queensland, Brisbane, Australia
Background:
The complement system is an essential part of the innate immune system, regulating inflammation, facilitating immune defence and maintaining tissue homeostasis. While critical to immune function, inappropriate or excessive complement activation contributes to pathological inflammatory conditions including cancer. Our prior research has demonstrated a role for the complement protein C3a and its receptor, C3aR, in tumour growth.
Results:
We showed that inhibition of C3aR signalling limited tumour growth in murine models of melanoma, colon and breast cancer by inhibiting tumour associated macrophages, mobilising neutrophils and promoting T cell infiltration of the tumour. A central role for neutrophils was demonstrated by antibody depletion experiments that reversed the tumour inhibitory effects observed in C3aR-deficient (C3aRKO) mice; tumour-infiltrating CD4+ T cells were returned to control levels, suggesting that these newly mobilised neutrophils may promote T cell infiltration of the tumour. However, flow cytometric analysis showed that in mice, C3aR is expressed primarily by macrophages and monocytes but not by neutrophils, T cells or dendritic cells, indicating that the effects of C3aR signaling on neutrophils and T cells must be indirect. Further investigation showed that C3a treatment of bone marrow-derived macrophages (BMDM) from WT but not C3aRKO mice induced calcium mobilisation and phosphorylation of ERK and AKT. Interestingly, although C3a alone had no effect on production of pro-inflammatory cytokines/chemokines by BMDM, C3aR signalling modulated the production of TLR-induced chemokines such as CCL2 and CXCL2.
Conclusion:
We propose that C3aR signalling intrinsically promotes tumour growth by regulating macrophage TLR-induced production of chemokines and cytokines, which in turn determine the immune composition of the tumour microenvironment.
P-103
Cell-intrinsic complement C3 and FB promote acute kidney injury
Anne Grunenwald, Idris Boudhabhay, Victoria Poillerat, Sara Tozoni, Nicolas Merle, Lubka Roumenina
Centre de Recherche des Cordeliers, Paris, France
Background:
Acute Kidney Injury (AKI) remains a major clinical challenge with no specific treatments. While the complement system is known to contribute to AKI, current therapeutic approaches targeting the cascade have limited efficacy. Complement proteins also function intracellularly, but their role in AKI remains largely unexplored.
Methods:
We investigated the role of cell-intrinsic and intracellular complement in rhabdomyolysis-induced AKI (RIAKI) using both patient samples and a mouse model. RIAKI was induced by glycerol injection in wild-type, C3⁻/⁻ and FB⁻/⁻ mice. Complement was pharmacologically inhibited at multiple levels (C3, FB, C5, C5aR1). Immune infiltration and complement deposition were assessed by multiplexed immunofluorescence and flow cytometry. In vitro, proximal tubular cells (PTCs) were silenced for C3 or FB and analyzed for proliferation, viability, and pathway activation. Phenotype rescue experiments with purified proteins were conducted to distinguish autocrine cell-autonomous from intracellular mode of action of C3 and FB in vitro. RNA sequencing was used to assess molecular changes in mouse kidneys and cell lines.
Results:
Despite clear overexpression and also reabsorption of C3 and FB in renal tubules and recruitment of C5aR1⁺ myeloid cells, pharmacologic inhibition of complement failed to protect against RIAKI. In contrast, C3⁻/⁻ and FB⁻/⁻ mice were protected, showing reduced tubular injury, enhanced heme detoxification, altered cell cycle progression, and attenuated inflammation. These findings were supported by transcriptomic analyses of both mouse and human kidneys. In PTCs, silencing C3 or FB impaired proliferation and enhanced detoxification. The proliferation phenotype was independent of extracellular presence of C3 or FB, indicating an intracellular mode of action. In vitro, and in mice in our experiments and by re-analyzing human kidney snRNAseq datasets, C3 expression appeared to regulate mTOR and Myc pathway activation, while FB was linked to ROS-induced DNA damage and inflammatory signaling.
Conclusion:
Our study reveals that cell-intrinsic and intracellular C3 and FB, rather than circulating complement proteins, drive early kidney injury in RIAKI. The failure of systemic complement inhibitors, despite protection in knockout models, underscores the need to shift therapeutic focus toward cell-intrinsic and intracellular complement pathways. Targeting cell-autonomous complement activity may represent a more efficient strategy for AKI treatment.
P-104
Different patterns of complement activation markers are present in acute and chronic adult primary ITP patients
Zoltán Prohászka1, Nóra Veszeli1, Justin Pieter Oosterlee2, Jasmin Rast2, Johanna Gebhart2
1Semmelweis University, Budapest, Hungary. 2Medical University of Vienna, Vienna, Austria
Primary immune thrombocytopenia (ITP) is an autoimmune disease with shortened half-life, decreased production of platelets, and resulting thrombocytopenia and bleeding. The pathogenesis involves B- and T-cell mediated processes and the development of platelet autoantibodies, leading to platelet destruction. Furthermore, complement activation appears to be a key contributor to the pathomechanism, though its triggers and extent remain uncertain. Recently, sutimlimab, a humanized monoclonal antibody targeting C1s, has been tested for the management of chronic/refractory ITP.
To better understand complement activation in ITP, serum and EDTA-plasma samples of acutely presenting and chronic ITP patients and healthy controls were selected from the Vienna ITP Biobank, a prospective cohort study of adult primary ITP patients (EC No. . The aim was to perform a detailed complement biomarker profiling with emphasis on classical pathway biomarkers C1s-C1-inhibitor, C4a, C4d, together with alternative and terminal pathway components, regulators and activation markers. Samples were assessed for complement biomarker determinations by ELISA or turbidimetry.
We examined samples of 12 acutely presenting and 11 chronic ITP patients compared to 30 healthy controls. Statistical analysis, using healthy controls’ results as reference, showed that acute ITP patients have increased C4d and C3a split product and terminal pathway activation marker sC5b-9 levels. Chronic ITP patients showed elevated C1s-C1-inhibitor complex, C4d and C4a concentrations, supporting the involvement of classical pathway. Lectin pathway marker MASP--C1-inhibitor complex, and alternative pathway marker Bb levels did not differ between controls and patients.
Our results confirm previous observations about the presence of complement activation in ITP, and indicate that in some patients, autoantibodies activate the classical pathway. This is further underlined by the novel observation of high C1s-C1-inhibitor complex levels in chronic patients. Furthermore, there are notable differences in complement profiles between disease phases, with C5a and sC5b-9 elevated in acute and C5a and C4a in chronic patients, possibly suggesting variations in the underlying pathways and pathophysiology. Such research will potentially offer novel tools for patient stratification to achieve effective treatment of ITP.
P-105
Characterization of inherited C3 deficiency – a patient case
Mari Humalajoki1, Pirkka Pekkarinen1, Merja Helminen2, Marjo Vuorela3, Petteri Arstila1, Seppo Meri1, Inkeri Lokki1
1University of Helsinki, Helsinki, Finland. 2Tampere University Hospital, Tampere, Finland. 3Päijät Häme Central Hospital, Lahti, Finland
Background:
Primary human complement deficiencies are rare conditions which affect functions of the immune defense. Central complement component C3 deficiencies manifest in early childhood mostly as recurring bacterial infections. The severity of the conditions showcase the importance of the human complement system in antimicrobial defense and human immunity. Overall, there are only about 30 reported cases of total C3 deficiency worldwide. This study introduces two Finnish siblings with no functional C3 in the plasma of either the proband or their sibling. Primary C3 deficiency was suspected first in the elder sibling due to relapses of severe pneumococcal infections (meningitis, sepsis, pneumonia) under the age of two. We set out to study the genetic basis of the deficiency.
Methods:
The proband's C3 gene was sequenced and results validated with a minigene splicing assay and Whole Exome Sequencing. Total C3 protein deficiency was analyzed by immunoblotting of serum and peripheral blood mononuclear cell (PBMC) lysate samples. Comprehensive clinical data was obtained from patient physicians.
Results:
C3 deficiency was confirmed by immunoblotting of plasma and PBMC cell lysates, as well as by complement activation tests. Sera from patients lacked complement activity. In genetic studies, we observed a maternal truncation at exon 9 of the human C3 gene and a paternal indel disrupting a splice site motif at exon 29 leading to an exon skipping event. The skipping event creates a conformation change in the TED/CUB domain, and results in functional depletion of C3. Together, the variants create a case of compound heterozygosity resulting in total C3 deficiency.
Conclusions:
Compound heterozygosity leads to a complete deficiency of mature, functional C3 through premature truncation and an exon skipping event. The lack of C3 in serum was first described in Pekkarinen et.al. (2013) and the recent study confirms the genetic mechanism of the deficiency.
References
Pekkarinen, P.T., Vaali, K., Jarva, H., Kekäläinen, E., Hetemäki, I., Junnikkala, S., Helminen, M., Vaarala, O., Meri, S., Arstila, T.P., 2013. Impaired intestinal tolerance in the absence of a functional complement system. Journal of Allergy and Clinical Immunology 131, 1167–1175. https://doi.org/10.1016/j.jaci.2012.09.004
P-106
Antibodies in the Healthy Population Influence Convertase Formation
Christopher Culek, Carla Nester
University of Iowa, Iowa City, USA
Background
We reported a large proportion of healthy college students carry autoantibodies that influence alternative pathway convertase formation on C3b-coupled surface plasmon resonance (SPR) assays.1 These C3 convertase autoantibodies (C3CAbs), were distinct from Nephritic Factors (Nefs), the anti-convertase autoantibodies enriched in C3 glomerulopathy patients, in two important ways: 1) they were not detected by routine Nef assays and 2) they did not amplify complement. In this study, we expand the sample population size and diversity and broaden the characterization by SPR.
Methods
Plasma were collected from 141 university hospital medical visits. Inclusion criteria were no evidence of infection, autoimmune, or inflammatory disorders and no evidence of glomerular disease. Samples were selected to cover ages 1 to 75+ years old. All other demographic data was blinded.
Polyclonal IgG was purified from plasma samples using Melon Gel (ThermoFisher) and dialyzed into 10mM MgCl2 HBS-EP buffer. IgG was evaluated by SPR in three ways. First, antibody influence on convertase formation was measured as described in the published manuscript.1 Second, convertase-stabilizing function was tested in a DAF-dependent manner based on previously described techniques.2 Finally, stabilizing function at 37C was measured. Samples were compared to Nef+ IgG and sham negative control monoclonal antibodies.
Results
Convertase formation was positively influenced by IgG from all age groups with no trend by age. Proconvertase formation and anti-C3b antibodies were also detected and partially correlated to convertase formation (R2= 0.49 and 0.21, respectively). The median estimated half-life of the convertase-IgG complex at 37C was 187 seconds for the Test population, statistically greater than reagent convertase (90 seconds) but less than Nef+ control (2400 seconds). DAF-dependent stability assay data showed statistical differences between the populations with Tukey’s multiple comparisons of p= <0.0001 between Sham, Test, and Nef Ig. The test population varied widely in their stability measurements.
Conclusions
This study supports our original findings. C3CAbs were detected in the “healthy” population regardless of age. These antibodies appear to influence the formation of the convertase with low impact on convertase stability. This low impact may explain their absence in routine Nef detection methods and their minimal impact on complement activity.
References
1- PMC10695638 2- PMC3608896
P-107
Baseline and Longitudinal Associations between serum C3/C4 levels, tissue complement biomarkers, and IgA Nephropathy in South Asians.
Suceena Alexander1, Anita Meter2, Rajanbabu Franklin1, Neetu Prince1, Sanjeet Roy1, Mohamed R. Daha2, Jonathan Barratt3, George T John1
1CMC, Vellore, India. 2University of Groningen, Groningen, Netherlands. 3University of Leicester, Leicester, United Kingdom
Background: Approximately 30–40% of patients with IgA nephropathy (IgAN) in India present with proteinuria and renal impairment, resulting in a dismal 10-year renal survival rate of 35%. Complement therapeutics are changing the treatment landscape for IgAN. This study aimed to determine the association between complement components and traditional biomarkers in IgAN in South Asians.
Methodology: The GRACE-IgANI cohort consisted of 200 prospectively recruited patients with well-characterized baseline and longitudinal outcomes. Serum C3 and C4 levels were measured using endpoint nephelometry. Tissue C3 was stained with immunofluorescence, while Tissue C3d, C4d, and C5b-9 were stained immunohistochemically. We assessed the relationship between the complement components and both baseline and longitudinal outcomes.
Results: Severe clinical manifestations, such as the presence of hypertension and eGFR <60 ml/min/1.73 m2 were associated with significantly lower serum C3 levels and serum albumin ≤ 4 g/dL and not with proteinuria. Lower serum C3 levels was significantly associated with higher chronicity MEST-C scores (S1, T1/T2) and global glomerulosclerosis (GS>33%) in biopsy. Serum C3 levels did not correlate with serum IgA levels, but higher serum C3 levels correlated significantly with increased mesangial IgA deposition. Increased mesangial C3d deposition reflected severe IgAN disease at baseline, as it correlated with increased MAP, proteinuria, decreased serum albumin, and decreased eGFR. The mean positivity of C3d increased with higher chronicity in biopsy, evidenced by S1 and T1/T2 and with GS>33%. Like C3d, an increase in mesangial C4d was seen with higher SBP, lower serum protein levels, and reduced eGFR, although the association was weaker than with C3d. In terms of histopathological factors, the average C4d positivity rose when GS exceeded 33%. In our cohort, mesangial deposition of terminal complement components (C5b-9) did not exhibit any clinical correlations. Additionally, serum C3/C4 levels were not associated with the mesangial deposition of C3, C3d, C4d, or C5b-9.
Conclusion: Significantly lower serum C3 levels, higher levels of serum C4, and increased tissue C3d and C4d levels were observed in high-risk IgAN patients at baseline. At the three-year follow-up, the combination of lower C3 and higher C4 levels predicted worse renal outcomes in IgAN patients.
References
Medjeral-Thomas NR, Cook HT, Pickering MC. Complement activation in IgA nephropathy. Semin Immunopathol. 2021;43(5):679-690. doi:10.1007/s00281-021-00882-9 Vivarelli M, Barratt J, Beck LH, et al. The role of complement in kidney disease: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference. Kidney Int. 2024;106(3):369-391. doi:10.1016/j.kint.2024.05.015
P-108
The role of C3 in steatotic liver disease
Adrian Iliescu, Dien Nguyen, Robert Byrne, Gareth Fenn, Thomas Pembroke, Timothy Hughes, You Zhou
Cardiff University, Cardiff, United Kingdom
Background: Metabolic dysfunction-associated fatty liver disease (MASLD) is caused by excess fat in the liver and is becoming a major health problem due to the rise in obesity rates. There are no approved treatments in the UK and the disease costs the NHS in excess of £17 billion per year. The complement system is a key player in inflammation and innate immunity. In addition to its immune role it has been shown to impact lipid transport around the body. This study explored the role of complement component 3 (C3), in the pathogenesis of MASLD.
Methods: In vitro, the liver cell line Huh-7 was challenged with fatty acids. Western blotting, qPCR, Oil Red O and immunofluorescence staining for key markers were used to dissect the role of C3 during lipid uptake into these cells. In vivo, a model for MASLD using CDAHFD (Choline-Deficient, L-Amino Acid-Defined, High-Fat Diet) for 14 weeks in wild type and C3 deficient mice was utilised. Outputs measured included: Lipid deposition (Oil Red O), inflammation (H&E) and fibrosis (Picrosirius Red). Triglyceride and free fatty acid content were assessed and qPCR used to measure expression levels of key genes involved in lipid handling and inflammation.
Results: In liver cells, mRNA and western blotting showed that C3 expression was increased following lipid challenge which surprisingly also induced expression of complement factor D. Inhibition of C3 or factor D with Compstatin and Danicopan respectively, reduced lipid accumulation and C3 activation. In vivo, C3 deficient mice showed less hepatic lipid deposition but displayed fibrosis. The complement receptors C3ar, C5ar1 and C5ar2 were upregulated in the liver.
Conclusion: Our results suggest a role for C3 in hepatic lipid accumulation during MASLD. This warrants further investigation to delineate the mechanisms involved and assess whether the complement system could be therapeutically targeted to slow the onset and progression of MASLD.
References
Dr Lewis Watkins (Training), Dr Jacqui Nimmo (Antibody provision), Miss Kirsten Bailie (Training), Prof Paul Morgan (Guidance, training, antibody provision) , Dr Wioleta Zelek (reagent provision) and Dr Svetlana Evans (Antibody provision).
P-109
Comprehensive functional analyses of 110 complement Factor B variants provide novel diagnostic and structural insights
Hector Martin Merinero1,2, Andrea Reparaz Suevos2, Renee X. Goodfellow1, Samantha Blain1, Laura Garcia Robles2, Juan Guzman Caldentey2, Sonsoles Martin Santamaria2, Santiago Rodriguez de Corodba2, Richard J. H. Smith1, Yuzhou Zhang1
1Molecular Otolaryngology and Renal Research Laboratories, University of Iowa, Iowa City, USA. 2Centro de Investigaciones Biologicas Margarita Salas, CSIC, Madrid, Spain
Background: Complement-mediated hemolytic uremic syndrome (CM-HUS) is a life-threatening thrombotic microangiopathy driven by complement dysregulation, primarily due to pathogenic variants in genes encoding complement components and regulators. Accurate classification of these variants is critical to optimize the medical utility of genetic analyses. Among all CM-HUS-associated genes, variants in CFB, encoding factor B (FB), are particularly challenging due to their structural and functional complexity.
Methods: We collected 110 CFB variants found exclusively in gnomAD (n=39), CM-HUS patients (n=16), or both (n=55), and expressed them with a C-terminal His-tag in mammalian cells. Using purified FB protein, assembled C3-convertases were tested for overall on-surface and fluid-phase activity, affinity for C3b, and stability/resistance to FH-regulation using ELISA, immunofixation electrophoresis and hemolytic assays. In-silico predictions (CADD and REVEL) were compared to functional data to assess accuracy in classifying novel CFB variants.
Results: We validated the robustness of our methodology by finding the same functional consequences for 16 CFB variants reported previously. In total, of the 110 variants, 63 (57.3%) altered FB expression or function in-vitro and were classified as pathogenic (gain-of-function, n=14; loss-of-function, n=29; no expression, n=20). Of the 16 variants reported only in CM-HUS patients, gain-of-function was overrepresented (43.8%) as compared to gnomAD-exclusive variants where loss-of-function (41.0%) and no-expression (23.1%) were more frequent. We describe the mechanisms by which variants affect FB function, including interference with C3 pro-convertase formation, impediments to FD cleavage, and increased C3-convertase stability/resistance to regulation by complement regulators. Notably, functional data on variants located in the serine protease domain, combined with detailed in-silico molecular dynamic analyses, provided novel structural insights into the interaction between the C3-convertase and its C3-substrate.
Conclusions: We provide the most comprehensive functional report of FB variants to date, demonstrate that gain-of-function variants in CFB predispose to CM-HUS, and show that evidence of association with CM-HUS cannot be predicted with current in-silico software. Notably, the location of these variants in FB supports current understanding of the conformational changes and interactions described during C3-convertase assembly and activity but suggests there is a knowledge gap with respect to the mechanism by which complement regulators accelerate C3-convertase decay.
References
This work was supported in part by funds from Kyle and Kelsey Duncan (Wyatt’s White Christmas); we thank those patients who have made this research possible. The work was also supported by Alexion Pharmaceuticals, Inc. We acknowledge the contribution to this work of Guillermo del Angel and William Mowrey.
P-110
A new screening approach to identify autoantibodies dysregulating the C3bBb convertase in C3 glomerulopathy and immunoglobulin-mediated membranoproliferative glomerulonephritis
Julia Roquigny1, Marie-Sophie Meuleman2, Sophie Chauvet1,2, Veronique Fremeaux-Bacchi1,2
1HEGP, Paris, France. 2Centre de Recherche des Cordeliers, Paris, France
Background:
In C3 glomerulopathy (C3G) and primary immunoglobulin-mediated membranoproliferative GN (Ig-MPGN) are characterized by alternative pathway (AP) dysregulation, often driven by autoantibodies that stabilize the AP C3 convertase (C3NeF). While C3NeF are particularly prevalent in pediatric patients, their clinical relevance remains unclear. Further challenges arise from their heterogeneity and the variability of detection methods, complicating characterization and clinical interpretation
Methods:
Our study included 27 patients (78% of children) diagnosed with C3G (n=19) or Ig-MPGN (n=8). To specifically study anti-C3bBb autoantibodies functions, only patients with protein G-purified IgG tested negative for anti-C3b and anti-Factor B were selected. We evaluated patients’ IgG functions towards C3bBb using the gold standard assay based on hemolysis of sheep erythrocyte bearing C3bBb, and novel Luminex-based assays that specifically measure the Bb subunit of C3bBb bound to magnetic beads. By luminex, we investigated whether purified patients’ IgG to directly stabilize or increase the formation of the AP C3 convertase (C3bBb).
Results:
Twenty-one patients were positive for C3NeF by hemolytic test. By Luminex, the C3bBb formation was enhanced in the presence of 15 patients’ IgG, while a stabilization effect was observed with 12 patients’ IgG. Notably, 9 patients’ IgG carried a dual function of formation and stabilization. The effect of formation and stabilization were strongly correlated (R²= 0.63), whereas no correlation was observed with IgG-mediated hemolytic activity. Interestingly, the presence of double-function anti-C3bBb was always associated to low C3 levels, but only the level of C3bBb formation was correlated to plasma C3 consumption. The C3NeF hemolytic activity showed no association with C3 nor soluble C5b-9 levels. The patients with double function anti-C3bBb antibodies had a worse renal survival than those negative (P=0.02). After a median follow-up of 87 [52–119] months, 6/27 patients (22%) progressed to end-stage kidney disease. Patients with dual-function anti-C3bBb antibodies had significantly worse renal survival (p=0.02).
Conclusion:
The direct functional assessment of IgG against the C3bBb convertase provided better associations with C3 dysregulation and the kidney outcome than the traditional hemolytic assay. Our findings highlight the diversity of IgG-mediated mechanisms towards C3bBb, that drive the phenotype of C3G and Ig-MPGN.
P-111
The Alzheimer’s disease-associated complement receptor 1 variant CR1*2 enhances glial phagocytosis of relevant targets and impacts neuroinflammation
Nikoleta Daskoulidou, Bethany Shaw, Wioleta Milena Zelek, Bryan Paul Morgan
UK Dementia Research Institute at Cardiff University, Cardiff, United Kingdom
Background:
Genome-wide association studies (GWAS) in Alzheimer’s disease (AD) identify the variants in the CR1 gene among the top genetic hits. CR1 encodes the C3b receptor complement receptor 1 (CR1; CD35) and the AD-associated variant, CR1*2, encodes a longer isoform containing an additional functional domain. We previously demonstrated CR1 expression on glial cells and reported preliminary evidence that expression of CR1*2 impacted glial phagocytosis. Here we extend these studies, examining expression levels of the CR1 isoforms, impact on phagocytosis of relevant targets and modulation of glial activation and inflammation.
Methods:
Induced pluripotent stem cell-derived microglia and astrocytes were developed from donors homozygous for either CR1*1 or CR1*2 alleles as described. CR1 expression at mRNA and protein level was measured using quantitative RT-PCR and western blotting respectively. Phagocytic activity was assessed in real-time on the Opera Phenix analyser using pHrodo-labelled E.coli bioparticles, AlexaFluor 488-conjugated amyloid-β aggregates, and pHrodo-labelled human synaptoneurosomes, with or without serum opsonisation. Glial activation and inflammatory responses were assessed by measuring cell-specific activation markers and inflammatory cytokines at mRNA and protein levels, unstimulated and at intervals after addition of the different phagocytic targets.
Results:
Cell surface expression, assessed using a mAb binding a single epitope in each, was significantly higher for CR1*1 compared to CR1*2 on both microglial and astroglial lines. Both CR1*1 and CR1*2-expressing glial lines displayed weak phagocytic activity against all tested targets, markedly enhanced by opsonisation with human serum. In contrast, opsonisation with Factor I-depleted serum resulted in only modest enhancement. Factor I depletion to block C3b processing to iC3b markedly reduced serum enhancement, supporting a collaboration between CR1 and the iC3b receptors CR3 and CR4 in glial phagocytosis. Both microglia and astrocytes expressing the AD risk variant CR1*2 showed significantly higher phagocytic activity for all serum-opsonised targets compared to CR1*1-expressing lines.
Conclusion:
We demonstrate that complement opsonisation is critical for effective glial phagocytosis and that CR1 works in collaboration with CR3/4 to mediate this. Despite lower cell expression, the AD-associated CR1*2 variant enhances phagocytosis of complement-opsonised material by microglia and astrocytes and alters the activation and inflammatory responses of these cells to phagocytic challenges.
P-112
Doxycycline inhibits MMP- and inactivates microglia to reduce PNN breakdown and preserve motor neurons in SOD1(G93A) mice.
Sang Won Cheung, Margreet Ridder, Peter Noakes
University of Queensland, St Lucia, Australia
Background:
Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated PNN breakdown and the contributing cellular factors in the SOD1G93A transgenic ALS mouse model. Compared with wild-type controls, we observed PNN breakdown around α-motor neurons in the ventral horn of between onset and mid-stage disease SOD1G93A mice. This was observed with increased microglia expressing matrix metallopeptidase-9 (MMP-), an endopeptidase degrading PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Following PNN breakdown, α-motoneurons of onset SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death.
Methods:
To test whether MMP- expressing glia were degrading PNNs and rendering α-motor neurons vulnerable to oxidative stress, we administered doxycycline (625mg/kg), a potent MMP- inhibitor and microglial inactivator, via feed in WT and presymptomatic SOD1G93A mice (P30) until early mid-stage (P100) and end-stage (P140). We conducted immunofluorescence and confocal microscopy to target proteins of interest, and MMP- zymography to assess MMP- activity. Analysis of body weight and hindlimb grip-strength was also conducted.
Results:
Doxycycline administration significantly reduced microglial activation as assessed through morphology and number in the ventral horn of P100 SOD1G93A mice and decreased PNN breakdown. In addition, doxycycline administration reduced the amount of PNNs phagocytosed by microglia and reduced overall MMP- activity in the early mid-stage SOD1G93A mice. Preliminary analysis of C5, which may act as a ‘eat-me’ signal for microglia, shows overall reduction in the ventral horn of doxycycline treated SOD1G93A mice. Hindlimb grip-strength data showed a slight increased between onset and mid-stage in doxycycline treated SOD1G93A mice compared to vehicle treated SOD1G93A mice.
Conclusion:
These results suggest that MMP- and activated microglia degrade PNNs around α-motor neurons, resulting in their eventual death in ALS disease, and doxycycline may slow this pathology.
P-113
Analysis of circulating immune complexes in serum of patients with ANCA-associated glomerulonephritis
TAKASHI ODA, Iroha Okano, Youwa Takahashi, Sachiko Iwama, Rie Fujii, Takahiro Uchida, Muneharu Yamada, Tadasu Kojima
Tokyo Medical University Hachioji Medical Center, Hachioji, Japan
Background: We have reported that circulating immune complexes (CICs) are detected at high rates in the serum of patients with ANCA-associated glomerulonephritis (AAGN) and complement activation through classical pathway plays an important role in the disease (Renal Failure 2022). However, the nature of CIC and its role in AAGN are unknown. In this study, we extracted CIC and analyzed its components and complement activation potential.
Methods: The sera of four AAGN patients in whom the presence of CIC had been confirmed were evaluated. An immunoadsorbent column was prepared using anti-rheumatoid factor monoclonal antibody (mRF antibody: provided by Nissui Pharmaceutical Co., Ltd.), and CIC was extracted from the patients' sera by immunoprecipitation. The extracted CIC were analyzed for its components using Western Blot. Furthermore, the complement activation potential of the extracted CIC was evaluated in vitro by measuring the concentration of various complement component products, such as C4a and C5b-9, using ELISA, after allowing the extracted CIC to react with normal human serum.
Results: Specific bands were detected by Western blotting with monoclonal anti-myeloperoxidase (MPO) antibody and anti-human IgG antibodies in all samples evaluated. In addition, in an evaluation of the complement activation potential of extracted CIC using ELISA, the concentration of complement activation components, such as C4a and soluble C5b-9, increased when they were reacted with isolated CIC, and this concentration increase was completely suppressed by adding the chelating agents of either EDTA or EGTA.
Conclusion: CIC in the serum of patients with AAGN were found to be composed at least in part of MPO and IgG (presumably anti-MPO IgG antibody=MPO-ANCA). It was further found that the isolated CIC has the potential to activate the complement system probably via the classical pathway. These results support the concept that complement activation through classical pathway by immune complex of MPO and MPO-ANCA plays important roles in AAGN.
References
Reference: 1). Kojima T. et al. Renal Failure. 44:1, 714-723 (2022) 2). Kojima T. et al. Front. Med. 9:1031445 (2022)
P-114
Beyond Systemic Complement Dysregulation: The Active Contribution of Renal Cells to Complement Dysregulation in C3 Glomerulopathy
EMMA DILETTA STEA1, Francesca Montenegro2, Francesca Giannuzzi2, Picerno Angela2, Sallustio Fabio2, Loreto Gesualdo2
1Unit of Nephrology, Dialysis and Transplantation, Fondazione IRCCS Policlinico San Matteo, PAVIA, Italy. 2Nephrology, Dialysis and Transplantation Unit, Department of Precision and Regenerative Medicine and Ionian Area (DiMePRe-J), University of Bari Aldo Moro, BARI, Italy
Background
C3 glomerulopathy (C3G) is a rare renal disorder characterized by the overactivation of the complement alternative pathway in the “fluid phase”, which leads to the deposition of C3 and its fragments within the kidney, resulting in a poor prognosis and a high rate of progression to end-stage renal disease. Despite its clinical significance, there is a lack of studies exploring C3G from a molecular perspective, complicating the identification of effective therapeutic strategies, which are currently nonexistent. Due to this knowledge gap, we aimed to determine whether the kidney is merely a passive observer or an underestimated participant in the pathology.
Methods
We isolated CD133-/CD24+ renal cells and from the urine of five patients with C3G and five healthy subjects and performed RNA sequencing and Biological pathways enrichment analysis and network analysis. Additionally, we utilized CD133+/CD24+ renal progenitors from patients and healthy subjects (HS) to create renal spheroids and cultured them to serum (20%) from C3G patients and HS under baseline conditions and after complement depletion.
Results
We identified 483 differentially expressed genes in renal cells from patients with C3G and healthy subjects. A significant modulation of the complement system in C3G renal cells emerged from the pathway analysis. Compared to HS, C3G renal cells seem to sustain the renal production of several complement components, such as C2, C3, C4A, C4B, CFB, and MASP1, which were highly upmodulated (FDR< 0.001 and LogFC>3). Moreover, we found the SERPINF2 gene, encoding for alpha-2-antiplasmin, that increase the complement activity, overexpressed (LogFC 4.33, FDR 0.009). It was also directly linked to IL1B gene, the central node of the inflammatory network. In immunofluorescence, the spheroids from C3G patients displayed strong expression of C3, regardless of the serum to which they were exposed (serum from HS, complement-depleted serum from C3G patients, and active serum), reinforcing the finding of local production.
Conclusion
Our findings suggest that renal cells in C3G patients actively contribute to complement hyperactivation, challenging the notion of passive involvement. This insight may represent a breakthrough in understanding the molecular mechanisms of C3G and could pave the way for developing new therapeutic targets.
P-115
Complement is dysregulated in Huntington’s disease in mouse and man.
Jacqui Nimmo, Kyle Fears, Kok Yung Lee, Paul Morgan, Thomas Massey, Wioleta Zelek
UK Dementia Research Institute Cardiff, Cardiff, United Kingdom
Background:
Huntington’s disease (HD) is a heritable monogenic neurodegenerative disease, stemming from CAG repeat expansion in the huntingtin gene (HTT) encoding for mutant huntingtin (mHTT). Symptoms of HD include cognitive impairment and loss of motor function, which becomes progressively severe in later stages. Precisely how mHTT causes disease progression and loss of synapses in the striatum is unclear but evidence of complement involvement was reported. Here we used two well-studied HD mouse models to explore complement dysregulation in serum and brain. Findings were validated in human HD brain.
Methods:
Total brain homogenates (TBH) and sera were harvested from seven hemizygous R6/1 mice (~five months old) with eleven matched controls, and thirteen heterozygous HdhQ150 mice (16-20 months old) with three matched controls. Complement C1q, activation products C3b/iC3b/C3c, and terminal complement complex (TCC) were measured in TBH (expressed as µg/mg total protein) and sera. Postmortem human brain caudate nucleus samples from HD donors (Vonsattel classification 2 and 3; moderate stage) and cognitively healthy age and sex matched controls (n=10) were obtained from Cambridge Brain Bank. Tissues were sectioned and stained for immunohistochemistry (IHC) detection of C1q, C3 fragments and TCC.
Results:
R6/1 HD mice displayed significantly elevated levels of C1q (P=0.0451), C3b/iC3b/C3c (P=0.0012), and TCC (P<0.001) in TBH compared to matched controls, indicating complement dysregulation. These mice also showed significantly increased serum levels of C1q (P=0.0132), C3 fragments (P=0.0206), and TCC (P=0.005). In contrast, no significant differences in C1q, C3 fragments and TCC were detected in HdhQ150 HD model mice compared to matched controls either in TBH or serum, suggesting that complement activation is less pronounced in this model of HD involving older mice. Human postmortem brain tissue analysis demonstrated increased C1q (P=0.0011) and C3 fragment (P=0.0433) deposition in the HD caudate nucleus compared to matched controls, demonstrating complement dysregulation in this critical brain region in HD.
Conclusion: We show that complement is dysregulated in brain and serum of a rapidly progressive HD mouse model and in the caudate nucleus of HD brains, implicating complement in HD pathology and supporting further investigation into the impact of therapeutically targeting complement in HD.
References
Wilton, D.K., Mastro, K., Heller, M.D. et al. Microglia and complement mediate early corticostriatal synapse loss and cognitive dysfunction in Huntington’s disease. Nat Med 29, 2866–2884 (2023). https://doi.org/10.1038/s41591-023-02566-3
P-116
A multi-omic pipeline identifies complement as a driver of age-dependent progression in a model of multiple sclerosis
Kevin Champagne-Jorgensen1, Kennedy Hoven1, Judy Zhu1, Joyt Patel1, Ikbel Naouar1, Michelle Zuo1, Alexandra Florescu1, Annie Pu1, Maia Goldberg1, Vivian Xie1, Cassandra Wong2, Zhen-Yuan Lin2, Anne-Claude Gingras2, B. Paul Morgan3, Jennifer Gommerman1, Valeria Ramaglia1,4
1University of Toronto, Toronto, Canada. 2Lunenfeld-Tanenbaum Research Institute, Toronto, Canada. 3UK Dementia Research Institute, Cardiff, United Kingdom. 4Krembil Brain Institute, Toronto, Canada
Age is the strongest predictor of multiple sclerosis (MS) progression, but the reasons for this are poorly understood. We recently described an experimental autoimmune encephalomyelitis (EAE) model that replicates aspects of age-dependent MS progression, including leptomeningeal inflammation and subpial hippocampal pathology. Here, we sought to develop an experimental and computational pipeline to identify and therapeutically target neuroimmune pathways that moderate disease progression in EAE mice. To this end, we performed single-cell RNA sequencing (scRNA-Seq) of leptomeninges from young or old mice at EAE initiation, peak, and recovery (for young mice) vs chronic (for old mice) disease phases. In parallel, we developed a novel approach to terminally collect up to 30 μL of pure cerebrospinal fluid (CSF) from individual young vs old mice across disease stages. Using data-independent acquisition LC-MS/MS we analyzed the global proteome of individual mice and resolved >2300 proteins, which varied systematically in abundance between young and old mice throughout EAE. Integrating scRNA-Seq data with complementary CSF proteome and immunofluorescence imaging, we identified production of complement C3 mRNA in the leptomeninges, C3 protein accumulation in the CSF and C3 activation in the hippocampus as a prominent marker of aged EAE disease. Using an adeno-associated viral (AAV) approach to overexpress the C3 inhibitor Crry at sites of C3 activation in the EAE hippocampus, we found that inhibition of C3 activation in old but not young mice resulted in milder disease. These data suggest that C3 activation in EAE is a mechanism driving age-divergent disease worsening in mice. Using imaging mass cytometry and downstream analysis pipeline, C3 activation products were also found on oligodendroglia in the hippocampus of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and hippocampal demyelination. Taken together, our data identifies complement as a driver of age-dependent progression in EAE that is relevant to the human disease.
P-117
Modulation of Complement-Mediated Inflammation in the mdx Mouse Model of Duchenne Muscular Dystrophy by PK007
Sai Yarlagadda1, Chynna-Loren Sheremeta2, Innis Hooper1, Mark Smythe2, Peter Noakes1, Miranda Grounds3
1The University of Queensland, Brisbane, Australia. 2Institue for Molecular Bioscience, Brisbane, Australia. 3The University of Western Australia, Perth, Australia
Background: Duchenne Muscular Dystrophy (DMD) is a progressive neuromuscular disorder marked by chronic inflammation and muscle degeneration. The mdx mouse model recapitulates key pathological features of DMD, including elevated complement system activation. In particular, complement component C5 has been implicated in exacerbating muscle inflammation and injury. PK007, an inhibitor of hematopoietic prostaglandin D synthase (HPGDS), has demonstrated anti-inflammatory effects in dystrophic models and may influence complement-related pathways.
Methods: Four-week-old mdx mice were treated with PK007 or vehicle control over a 10-day treatment period (postnatal 18 days to postnatal 28 days). Skeletal muscles, including the gastrocnemius (GA), tibialis anterior (TA), and extensor digitorum longus (EDL), were collected for molecular and histological analyses. Bulk RNA sequencing was performed to evaluate transcriptomic changes, with a particular focus on complement-related genes. For protein-level assessment, C5 expression was evaluated by immunofluorescence using a monoclonal anti-C5 antibody. Tissue sections were imaged, and fluorescence intensity was quantified to assess differential expression between treatment groups.
Results: RNA-seq analysis revealed a downregulation of C5 transcript levels in PK007-treated muscles compared to vehicle controls. Immunofluorescence analysis demonstrated a reduction in C5 protein expression in dystrophic muscle following PK007 treatment.
Conclusion:
These findings suggest that PK007 may attenuate complement-mediated inflammation in DMD by downregulating the expression of complement component C5. In combination with its previously observed anti-inflammatory effects, PK007 may contribute to reduced muscle damage in dystrophic muscle, supporting its further investigation as a promising therapeutic candidate in the treatment of muscular dystrophy.
References
Rosenberg AS, Puig M, Nagaraju K, Hoffman EP, Villalta SA, Rao VA, Wakefield LM, Woodcock J. Immune-mediated pathology in Duchenne muscular dystrophy. Sci Transl Med. 2015 Aug 5;7(299):299rv4. doi: 10.1126/scitranslmed.aaa7322. PMID: 26246170; PMCID: PMC5951380.
P-118
Targeted Treatment With Pegcetacoplan for Adolescents With C3G or Primary (Idiopathic) IC-MPGN in the VALIANT Phase 3 Trial
Marina Vivarelli1, Gema Ariceta2, Yael Borovitz3, Bradley P Dixon4, Larry A Greenbaum5, Cristoph Licht6, Antonio Mastrangelo7, Nabil Melhem8, Naoya Fujita9, Nicole Van de Kar10, Dean Wallace11, Li Li12, Luis López Lázaro13, Johan Szamosi13, Carla M Nester14
1Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. 2Hospital Vall d'Hebron, Barcelona, Spain. 3Schneider Children's Medical Center, Petah-Tikva, Israel. 4University of Colorado School of Medicine, Aurora, USA. 5Emory School of Medicine, Atlanta, USA. 6The Hospital for Sick Children, Toronto, Canada. 7Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. 8Evelina London Children's Hospital, Guys & St Thomas' NHS Foundation Trust, London, United Kingdom. 9Aichi Children's Health and Medical Center, Aichi, Japan. 10Radboudumc Amalia Children's Hospital, Nijmegen, Netherlands. 11Royal Manchester Children’s Hospital, Manchester, United Kingdom. 12Apellis Pharmaceuticals, Inc., Waltham, USA. 13Swedish Orphan Biovitrum AB, Stockholm, Sweden. 14University of Iowa, Stead Family Children’s Hospital, Iowa City, USA
Background: C3 glomerulopathy (C3G) and primary (idiopathic) immune complex membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases where uncontrolled C3 activation results in excessive glomerular deposition of C3 breakdown products, often diagnosed in adolescence or early adulthood. Current treatments have significant side effects and approximately 20% of children progress to kidney failure within 10−15 years of diagnosis. Pegcetacoplan, a C3/C3b inhibitor, targets the central complement pathway components to directly inhibit overactivation of C3 breakdown. In the overall VALIANT (NCT05067127) population, pegcetacoplan led to significant proteinuria reduction and estimated glomerular filtration rate (eGFR) stabilization vs placebo in C3G or primary IC-MPGN patients. Here, we report results for adolescents (12–17 years).
Methods: VALIANT, a randomized, double-blind, placebo-controlled Phase 3 trial, included adolescents (≥12 years) and adults with biopsy-proven C3G or primary IC-MPGN in native or post-transplant kidneys and proteinuria >1 g/day. In adolescents without a baseline biopsy, eligibility criteria included ≥1 of the following: increased plasma soluble C5b-9, decreased serum C3, presence of hematuria, or presence of C3 nephritic factor. Patients were randomized 1:1 to pegcetacoplan (subcutaneous infusion twice weekly) or placebo for 26 weeks in the RCP. Biopsies were optional for adolescents. The primary endpoint was the log-transformed ratio of urine protein-to-creatinine ratio (UPCR) at week 26 vs baseline. Key secondary endpoints included the proportion of patients achieving a composite renal endpoint criterion (≥50% UPCR reduction and ≤15% eGFR reduction), ≥50% UPCR reduction, and eGFR change from baseline.
Results: In total, 28 adolescents were randomized to pegcetacoplan and 27 to placebo. Pegcetacoplan led to significant and clinically meaningful UPCR reductions at week 26, with a 74.5% relative reduction in proteinuria in the pegcetacoplan vs placebo arms (95% CI 58.5, 84.3; nominal P<.0001). A greater proportion of adolescents in the pegcetacoplan vs placebo arm achieved the composite renal endpoint (57.1% vs 3.7%; nominal P=.0016) and ≥50% UPCR reduction (71.4% vs 3.7%; nominal P=.0002). Pegcetacoplan was associated with clinically meaningful eGFR stabilization.
Conclusions: In VALIANT, pegcetacoplan was well tolerated in adolescents with C3G and primary IC-MPGN and was the first treatment to induce meaningful proteinuria reduction and eGFR stabilization in this population.
P-119
Assessing C3 Nephritic Factor Function Using Luminex-Based Formation and Stabilization Assays
Cecelia Fierce, Christopher Culek, Renee X Goodfellow, Yuzhou Zhang, Richard Smith, Carla Nester
University of Iowa, Iowa City, USA
Background
C3 Nephritic factors (Nefs) are autoantibodies that bind to and stabilize the C3 convertase, thereby enhancing complement activity and driving glomerular diseases such as C3 Glomerulopathy (C3G) 1,2. Recently, a Luminex method was developed to evaluate Nefs3. Utilizing this technique, the authors proposed that Nefs influence both convertase formation and stabilization. In this study, we compare the Luminex-based assay to hemolytic-based and surface plasmon resonance (SPR) assays to assess the formation/stabilization model of Nef activity.
Method
A test population of polyclonal IgG samples were evaluated for C3Nef activity by hemolysis (n=21)1 and for convertase formation and stability by SPR analysis (n=21)4. Samples were then applied to the Luminex Formation (n=21) and Stability assays3 (n=12).
Results
Nef hemolytic scores ranged from 10% (0+) to 100% (4+). Linear regression between hemolysis and Luminex assays was statistically significant: Luminex Stability-Hemolysis yielded R2 = 0.46, F= 8.6, p= 0.015; Luminex Formation-Hemolysis yielded R2 = 0.29, F= 4.3, p= 0.013. ANOVA test with Dunn’s multiple comparisons for Luminex data grouped by hemolytic score (0, 1+, or 4+) showed statistically significant difference between 0 and 4+ Nefs by both Stability (p= 0.0003) and Formation (p= 0.0012) assays but no significance between 0 and 1+ Nefs by either Luminex assay.
Linear regression between Luminex Formation assay and SPR assay analysis showed significance to the SPR “Formation” report point (R2= 0.62, F= 29.3, p= <0.0001) and SPR-based stability analysis (R2=0.51, F=18.85, p=0.0004). The Luminex Stability assay correlated weakly with the SPR Stability analysis but did not reach significance (p= 0.15).
Conclusion
Luminex Formation correlated better with the SPR Formation report point, while Luminex Stability showed better correlation to the hemolytic stability assay. However, the overall correlations were lower than anticipated, which may point to 1) the small sample size; 2) more nuanced interactions between polyclonal IgG and the C3 convertase; and/or 3) the need for further optimization of the Luminex and SPR methodologies.
References
1) PMID: 22223606 2) PMID: 35734939 3) PMID: 39325562 4) PMID: 22854646
P-120
VALIANT: Randomized, multicenter, double-blind, placebo-controlled, phase 3 trial of pegcetacoplan for patients with native or post-transplant recurrent C3G or primary (idiopathic) IC-MPGN
Carla M Nester1, Andrew S Bomback2, Gema Ariceta3, Yahsou Delmas4, Bradley P Dixon5, Daniel P Gale6, Larry A Greenbaum7, Seung Hyeok Han8, Nicole Isbel9,10, Christoph Licht11, Antonio Mastrangelo12, Masashi Mizuno13, Maria Izabel Neves de Holanda14, Matthew C Pickering15, Giuseppe Remuzzi16, Nicole Van De Kar17, Marina Vivarelli18, Patrick D Walker19, Dean Wallace20, Daniel Zecher21, Li Li22, Zhongshen Wang22, Luis López Lázaro23, Johan Szamosi23, Fadi Fakhouri24
1University of Iowa, Stead Family Children’s Hospital, Iowa City, USA. 2Columbia University Irving Medical Center, New York, USA. 3Hospital Vall d'Hebron, Barcelona, Spain. 4Service de Néphrologie, Hôpital Pellegrin, CHU Bordeaux, Bordeaux, France. 5University of Colorado School of Medicine, Aurora, USA. 6University College London, London, United Kingdom. 7Emory School of Medicine, Atlanta, USA. 8Yonsei University College of Medicine, Seoul, Korea, Republic of. 9Princess Alexandra Hospital, Brisbane, Australia. 10The University of Queensland, Brisbane, Australia. 11The Hospital for Sick Children, Toronto, Canada. 12Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. 13Nagoya University Graduate School of Medical Sciences, Nagoya, Japan. 14Hospital Federal de Bonsucesso, Ruschel Medicina, Rio de Janeiro, Brazil. 15Imperial College, London, United Kingdom. 16Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy. 17Radboudumc Amalia Children's Hospital, Nijmegen, Netherlands. 18Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. 19Arkana Laboratories, Little Rock, USA. 20Royal Manchester Children’s Hospital, Manchester, United Kingdom. 21Regensburg University Hospital, Regensburg, Germany. 22Apellis Pharmaceuticals, Inc., Waltham, USA. 23Swedish Orphan Biovitrum AB, Stockholm, Sweden. 24Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
Aims: C3 glomerulopathy (C3G) and primary immune complex-membranoproliferative glomerulonephritis (IC-MPGN) are complement-mediated diseases driven by C3 dysregulation with excessive accumulation of C3 breakdown products in the kidney. Pegcetacoplan (PEG) a C3/C3b inhibitor, targets the central components of the complement pathway, directly inhibiting C3 overactivation and preventing further deposition of C3 breakdown products in the glomeruli. VALIANT (NCT05067127) is the first Phase 3 trial investigating PEG in a broad cohort, including adolescents (≥12 yrs) and adults with native or post-transplant recurrent C3G or primary IC-MPGN.
Methods: VALIANT is a randomized, multicenter, double-blind, placebo (PBO)-controlled trial evaluating PEG efficacy and safety. 124 pts were randomized to PEG (n=63) (twice weekly subcutaneous infusion) or PBO (n=61) for 26 weeks (wks). The primary endpoint was log-transformed UPCR ratio at wk. 26 vs baseline, assessing proteinuria reduction vs PBO. Key secondary endpoints at wk. 26 were a composite renal endpoint (proportion of pts achieving ≥50% UPCR and ≤15% eGFR decline), proportion of patients achieving ≥50% UPCR reduction, C3G histologic index activity score change (adjusted LS mean change), reduced C3c renal biopsy staining of ≥2 OOM, eGFR change, (LS mean change), mL/min/1.73 m2. Safety was assessed by treatment-emergent adverse events (TEAE) frequency and severity.
Results: The primary endpoint was met, with PEG demonstrating a 68.1% (95% CI: –76.2, –57.3) mean UPCR reduction vs. PBO at wk. 26 (p<0.0001). Results were consistent across disease type, age, and transplant status subgroups. PEG also led to robust reductions in C3c staining and clinically meaningful eGFR stabilization vs PBO. Treatment-emergent AE frequency and severity were similar between arms. None of the 4 serious infections (3 PEG; 1 PBO) were attributed to encapsulated bacteria.
Conclusion: PEG is the first therapy to achieve significant and clinically meaningful reductions in proteinuria (68.1% vs. PBO), C3c staining and eGFR stabilization in pts ≥12 yrs with C3G or primary IC-MPGN. PEG was well tolerated with no new safety signals observed.
P-121
Metabolic changes in the retinal pigment epithelium of patients with complement factor H haploinsufficiency.
Rayne Lim1, Bree Nelson1, Emily Zhao1, Sheldon Niernberger1, Jianhai Du2, Jennifer Chao1
1University of Washington, Seattle, USA. 2West Virginia University, Morgantown, USA
Background: Age-related macular degeneration (AMD) is a retinal disease that causes degeneration in the macula – the central part of the retina crucial for visual acuity. The complement system is a well-established contributor to the etiology of AMD, specifically via heightened complement activity at the retinal pigment epithelium (RPE). High penetrant mutations in the complement factor H (CFH) gene are of special interest because it is associated with early onset macular drusen (EOMD), a phenotypically severe subtype of AMD.(1) Recently, we reported a novel CFH SNP in an EOMD family, with A>G substitution at the intron splice site before exon 4, resulting in nonsense mediated decay of the CFH gene. The RPE generated from patient derived induced pluripotent stem cell (iPSC) had 50% reduction of both the CFH and factor H-like protein 1 (FHL-1) protein, which resulted in an increased local complement activity.(2) To further understand non-canonical roles of CFH intracellularly, this study aims to assess the metabolic changes in the RPE during conditions of CFH/FHL-1 insufficiency.
Methods: iPSC RPE derived from control patients without retinal degeneration, EOMD patients and Crispr-corrected EOMD (cEOMD) isogenic clones were cultured and collected for targeted metabolomics, mitochondria respiration and glycolytic flux.
Results: RPE from EOMD patients were found to have reduced levels of intracellular glucose and glucose 6-phosphate compared to control RPE. Glycolysis stress test revealed increased glycolytic capacity and glycolytic reserve in the EOMD RPE, suggesting changes to glucose consumption. Glutathione to oxidized glutathione ratio (GSH:GSSG) indicative of oxidative stress showed higher oxidative state in the EOMD RPE, consistent with increases in basal and maximum respiration, ATP production, and spare capacity. In contrast, GSH:GSSG was reverted to that of control levels in the cEOMD RPE. EOMD RPE also had reduced tryptophan and kynurenine, which was increased in cEOMD RPE, highlighting alterations in the kynurenine pathway and de novo synthesis of NAD.
Conclusion: Oxidative stress, glucose consumption, mitochondria respiration and NAD synthesis were altered in an EOMD patient-derived RPE, suggesting CFH and FHL-1 to have roles in influencing RPE metabolism.
References
1. R. L. Taylor et al., Loss-of-Function Mutations in the CFH Gene Affecting Alternatively Encoded Factor H-like 1 Protein Cause Dominant Early-Onset Macular Drusen. Ophthalmology 126, 1410-1421 (2019). 2. R. R. Lim et al., CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration. Investigative Ophthalmology & Visual Science 65 (2024).
P-122
Distinct associations of the lectin pathway proteins MASP-, MASP-, and MAP- with clinical and serological profiles in Systemic Lupus Erythematosus
Linnea Lindelöf1, Elisabet Svenungsson2, Iva Gunnarsson2, Roland Ebert1, Peter Garred3, Oskar Eriksson1
1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. 2Division of Rheumatology, Department of Medicine, Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. 3Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, University Hospital of Copenhagen, Copenhagen, Denmark
Background The complement system plays an essential role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the role of the classical pathway. Recently, we demonstrated a link between the lectin pathway recognition molecule ficolin-3, hematological manifestations, and autoantibody profiles in SLE [1]. In the present study, we expanded the study to include the lectin pathway serine proteases MASP- and MASP-, along with the non-enzymatic splice product MAP-, proposed to be a lectin pathway regulator.
Methods Serum samples from 531 SLE patients and 322 matched population controls were collected at the Karolinska Institute (KI) in Stockholm, Sweden. The MASP-, MASP-, and MAP- concentrations were measured by in-house ELISAs. Logistic regression models adjusted for age and sex were used to explore associations between MASP-, MASP-, and MAP- serum concentrations and SLE disease manifestations and autoantibody specificities.
Results Serum MASP concentrations were significantly higher in patients with SLE compared to control individuals for both MASP- (p <0.0001), MASP- (p=0.0294), and MAP- (p=0.0001). Among these components, serum MAP- levels showed the strongest association with clinical manifestations, while associations were weaker for MASP- and MASP-. When comparing patients in the highest vs. the lowest MAP- quartile, significant associations were observed for cutaneous (discoid rash OR 2.9, p=0.004) and hematological (leukopenia OR 2.1, p=0.006; lymphopenia OR 1.9, p=0.020) SLE manifestations. Moreover, subjects in the highest MAP- quartile had a higher frequency of SLE-associated antinuclear antibodies (anti-Sm OR 3.7, p =0.001; anti-SSB OR 3.1, p = 0.001), but a lower frequency of anti-phospholipid antibodies (anti-β2GP1 IgG OR 0.51, p=0.074; anti-cardiolipin IgG OR 0.53, p=0.074; anti-cardiolipin IgA OR 0.30, p=0.005).
Conclusion Serum levels of MAP- are associated with disease manifestations in SLE and follow a pattern previously observed for ficolin-3. Our results demonstrate a consistent pattern of clinical and serological associations for lectin pathway components in SLE and further pinpoint a possible role for ficolin-3/MAP- complexes in SLE pathogenesis.
References
[1] Lindelöf L, Rantapää-Dahlqvist S, Lundtoft C, Sandling JK, Leonard D, Sayadi A, et al. A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile. Journal of Autoimmunity 2024;143:103166.
P-123
Charting the Path of Dendritic Cell Subsets in Allergic Asthma Using C5aR1 Fate Mapping Mice
Mareike Ohms, Elvira Ehlers-Jeske, Gabriele Köhl, Jörg Köhl
University of Lübeck, Lübeck, Germany
Background C5aR1 modulates cDC2 function during allergen sensitization, controlling their potency to induce Th2 responses. Previously, we found reduced MHC-II and CD40 expression in pulmonary C5aR1+ cDC2s as compared to C5aR1- cDC2s in BALB/c mice, associated with a reduced potency of C5aR1+ cDC2 to drive T cell proliferation. Importantly, the frequency of C5aR1- cDC2s increased following one time allergen exposure. Thus, we generated C5aR1 fate mapping mice to track the dynamics of different C5aR1+ and C5aR1-cDC2 subsets and assess their function in response to allergen sensitization.
Methods We created C57BL/6 tdTomatoC5ar1CreERT2R26FEYFP fate mapping, mice, which allow to define current (tdTomato) and historical (EYFP) C5aR1 expression upon tamoxifen administration. We used such mice to define the phenotype of pulmonary C5aR1+ and C5aR1- cDC subtests 24 hours after i.t. HDM/OVA exposure.
Results After HDM/OVA exposure, we identified three distinct populations of cDC2s in the lung: tdTomato+ (C5aR1+/-), tdTomato+EYFP+ (C5aR1+/+) and tdTomato- (C5aR1-) but no tdTomato-EYFP+ (C5aR-/+) cells. In contrast to BALB/c mice, the frequency of C5aR1+/- cDC2s increased, whereas the frequency of C5ar1- cDC2s decreased compared to PBS controls in C57BL/6 C5aR1 fate mapping mice. Interestingly, we found a complex expression pattern of cDC2 surface marker molecules in the different cDC2 subsets. In C5aR1+/- cells, the frequency of CD26, Ly6C and CD371 increased, whereas in C5aR1- cDC2s, the frequency of cDC2s expressing such markers either decreased or remained unchanged. Instead, in C5aR1- cDC2s, we found an increased frequency of cells expressing CX3CR1, CD64 or CD301. Of note, only in the group of C5aR1+/+ cDC2s, the frequency of Dectin-1+ cells increased. Currently, we are conducting functional studies with the phenotypically distinct C5aR1+ and C5aR1- cDC2 subsets to define their ability to activate naive CD4+ T cells.
Discussion The observed lack of tdTomato-EYFP+ cDC2s suggests that C5aR1- cDC2s do not lose C5aR1 expression after allergen exposure but lack C5aR1 already when recruited to the lung. The increased frequency of C5aR1+/- cDC2s in C57BL/6 as compared to the reduced frequency of C5aR1+ cDC2 in BALB/c mice may explain the lower sensitivity of C57BL/6 mice to develop pulmonary allergy.
P-124
Features of Circulating Factor H Immune Complexes in Complement-Mediated Diseases
Stephanie Cook1, Dingwu Shao1, Sydney Jellison1, Santiago Rodriguez de Cordoba2, Carla Nester1, Richard Smith1, Yuzhou Zhang1
1Molecular Otolaryngology and Renal Research Laboratories - University of Iowa, Iowa City, USA. 2Centro de Investigaciones Biologicas Margarita Salas - Consejo Superior de Investigaciones CIentificas, Madrid, Spain
Background
Complement factor H (FH) regulates the alternative pathway (AP) through two functions: the N-terminal domain mediates decay-accelerating activity (DAA) and cofactor activity (CA), while the C-terminal domain mediates binding to host surfaces. Factor H autoantibodies (FHAAs) can disrupt these functions, with the specific disease consequence of C3 glomerulopathy (C3G) or complement-mediated thrombotic microangiopathy (cm-TMA) reflecting FHAA binding site specificity. Enzyme-linked immunosorbent assays (ELISA) typically detect free FHAAs by binding to immobilized FH (FHAA ELISA) or circulating FH–anti-FH immune complexes (FHICs) using sandwich ELISA. However, free FHAAs can form complexes before or during testing, causing interference and false-negative results. The clinical role of FHICs remains unclear.
Methods
We screened 120 samples using FHAA ELISA, FHIC sandwich ELISA, and a newly developed lateral flow assay to identify patients with detectable free FHAAs or circulating immune complexes only. Based on these screening results, six samples (four cm-TMA and two C3G patients) underwent further characterization. FHICs were isolated using a protein G pull-down assay, and their specificity, affinity, and stability were analyzed.
Results
We observed significant heterogeneity in FH-FHAA affinity and epitope specificity profiles between patients with cm-TMA and those with C3G. Functional assays showed that FHAAs and FHICs impair FH regulatory functions in the fluid phase and on cell surfaces, enhancing AP activation.
Conclusion
Our findings demonstrate the complexity and clinical relevance of FHICs in complement-mediated diseases, while highlighting limitations of standard diagnostic tests. Because FHICs may impact disease severity and therapeutic response, further studies should focus on standardizing diagnostic algorithms to characterize FHAAs thereby facilitating appropriate personalized therapeutic interventions.
Acknowledgements
Supported in part by National Institutes of Health R01 DK110023.
References
Supported in part by National Institutes of Health R01 DK110023.
P-125
Detecting FH–Anti-FH Immune Complexes in MGRS-C3G
Sydney S Jellison1, Sarah M Roberts1, Samantha J Martin1, Stephanie N Cook1, Carla M Nester1, Andrea Reparaz2, Santiago Rodriguez de Cordoba2, Richard JH Smith1, Yuzhou Zhang1
1Molecular Otolaryngology and Renal Research Laboratories - University of Iowa, Iowa City, USA. 2Centro de Investigaciones Biologicas Margarita Salas - CSIC (Consejo Superior de Investigaciones CIentificas), Madrid, Spain
Background: C3 glomerulopathy (C3G) is a complement-mediated kidney disease driven by dysregulated alternative pathway (AP) activation. In some patients (typically >50 years), a diagnosis of monoclonal gammopathy of renal significance (MGRS) reflects the presence of small B-cell or plasma cell clones producing nephrotoxic monoclonal immunoglobulins. How these proteins contribute to disease (MGRS-C3G) remains a key knowledge gap. We hypothesize that Factor H autoantibodies (FHAAs), particularly when forming immune complexes (FHICs), contribute significantly to disease. We also believe traditional ELISA-based assays have limited sensitivity in detecting circulating FHICs, especially IgM-type FHAAs.
Methods: We evaluated a lateral flow assay (LFA) (1) consisting of three cassettes to detect both IgG- and IgM-type FHICs in serum from 58 C3G patients. Patients were categorized by FHAA ELISA results: Group A, FHAA >1000 Arbitrary Units (AU); n=10 (C3G = 8; MGRS-C3G = 2, including one IgM-only case); Group B, ambiguous/equivocal levels (200–1000 AU); n=14 (C3G = 10; MGRS-C3G = 4); Group C, MGRS-C3G lacking known acquired or genetic drivers but showing fluid-phase complement hyperactivity; FHAA <200 AU; n=33.
Results: Group A: LFA confirmed FHIC positivity in all 10 patients, including the IgM-only case. Six others were IgM co-positive, showing enhanced LFA sensitivity over ELISA. Group B: LFA detected FHICs in 11 of 14 patients (79%), clarifying ambiguous ELISA results. All four MGRS patients were strongly positive for IgG-type FHICs. No IgM-type positives were seen. Group C: Despite negative ELISAs, LFA detected FHICs in 12 of 33 patients (36%), including four weakly IgM-FHAA co-positive.
Overall, FHICs were found in 33 of 58 patients, including 18 of 39 MGRS-C3G cases.
Conclusion: In 18 of 39 MGRS patients (46%), FHAAs were detected as the monoclonal protein driving complement dysregulation. Conventional ELISAs identified only 6, likely due to FHIC formation masking detection. These findings underscore both the pathogenic role of FHAAs in MGRS-C3G and the diagnostic value of LFA. Large-scale prospective studies are warranted to confirm and extend these observations.
References
Supported in part by National Institutes of Health R01 DK110023. (1) Rodrıguez de Cordoba S et al. Front. Immunol. 15:1527016. doi: 10.3389/fimmu.2024.1527016
P-126
Complement C9-Mediated RBC Hemolysis Drives Microvascular Obstruction via Endothelial Necroptosis and Hemolyzed RBC Aggregation in COVID-19
Chia Lun (Mike) Wu1,2, Lining Arnold Ju1, Ethan Italiano1,2, Rocko Jarvis-Child1,2, Imala Alwis1,2, Rhyll Smythe1,2, Eduardo A Albornoz3, Jonathan Noonan4, Marie Portelli1,2, Marrisa Baptista1,2, Jessica Maclean1,2, Pashtana Norri1,2, Jinglu Yang1,2, John Lee3, James D McFadyen4,5, Alexandra Sharland1, Trent M Woodruff3, Andre Samson6,7, Amy Rapkiewicz8, Tessa Barrett9, Alan Pham5, Simone Schoenwaelder1,2, Yuping Yuan1,2, Shaun P Jackson1,2
1University of Sydney, Sydney, Australia. 2Heart Research Institute, Sydney, Australia. 3University of Queensland, Brisbane, Australia. 4Baker Heart and Diabetes Institute, Melbourne, Australia. 5The Alfred Hospital, Melbourne, Australia. 6Walter and Elize Hall Research Institute, Melbourne, Australia. 7University of Melbourne, Melbourne, Australia. 8New York University Winthrop Hospital, New York, USA. 9New York University, New York, USA
Background
Microvascular obstruction (MVO) is a major attributor to tissue injury in ischemic diseases, including acute myocardial infarction and severe COVID-19 with multi-organ failure. Thus far, RBCs have been widely regarded as passive bystanders in MVO—mechanically trapped within compromised vessels under disturbed flow and endothelial dysfunction, without actively contributing to the obstructive process.
Complement activation, particularly the terminal complement complex C5b-9, is a key mediator of vascular injury in ischemia-reperfusion (IR) and COVID-19-associated microangiopathy. While complement’s role in inflammation and endothelial damage is well recognized, its influence on RBC behavior, particularly in promoting RBC aggregation and leading to MVO in IR, remains poorly defined. Notably, our study suggests that C9-mediated RBC membrane fragmentation (hemolysis) directly contributes to MVO.
Methods
IR injury models of mouse gut, heart, and brain were used alongside COVID-19 and ischemic human tissues. Multiplexed immunofluorescence and multimodal imaging identified endothelial necroptosis (pMLKL, pRIPK1), C5b-9 deposition, and RBC fragmentation marked by intensified glycophorin A (CD235). RBC aggregation was modeled using a flow platform with necrotic human microvascular endothelial cell line 1 (HMEC-1) monolayers and complement inhibition.
Results
RBC microangiopathy was evident in the microvasculature of patients with myocardial infarction, gut ischemia, stroke, and septic or cardiogenic shock. Mechanistically, IR-induced MLKL-dependent endothelial necroptosis triggered C9-mediated RBC hemolysis. Hemolyzed RBCs became intrinsically adhesive, forming aggregates with surrounding intact RBCs and creating occlusive structures that drive MVO independently of platelets or fibrin. C5b-9 deposition was found spatially associated with necroptotic endothelium and lysed RBCs, particularly in COVID-19 tissues.
Genetic deficiency of C9 or endothelial MLKL significantly reduced RBC hemolysis, MVO, and tissue injury. In vitro, we recapitulated the RBC aggregation on necrotic HMEC-1 monolayers under IR relevant shear stress. Inhibition of C5b-9 formation using complement-depleted plasma or Cp40 (a C3 inhibitor) effectively prevented the RBC aggregation.
Conclusion
Our findings identify complement C9-mediated RBC hemolysis as a key driver of MVO, revealing a novel therapeutic target in ischemic vascular disease.
P-127
Senescent erythrocytes as a trigger of complement activation and implications for breakthrough haemolysis in Paroxysmal Nocturnal Haemoglobinuria patients
Sümeyye Erdemci-Evin1, Britta Höchsmann2,3, Hiba Ghaboosh1, Marco Mannes4, Hubert Schrezenmeier2,3, Christoph Q. Schmidt1
1Institute Institute of Experimental and Clinical Pharmacology, Toxicology and Pharmacology of Natural Products, Ulm University, Ulm, Germany. 2Institute of Clinical Transfusion Medicine and Immunogenetics, Ulm, Germany. 3German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen, Ulm, Germany. 4Institute of Trauma Immunology, Ulm University, Ulm, Germany
Background:
Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic stem cell disorder caused by the absence of GPI-anchored complement regulatory proteins, rendering red blood cells (RBCs) vulnerable to complement-mediated lysis. Although inhibitors targeting C5 (Eculizumab, Ravulizumab), C3 (Pegcetacoplan), and other upstream components (e.g., Iptacopan, Danicopan) have significantly improved clinical outcomes, patients can experience breakthrough haemolysis. For breakthrough haemolysis during C5 inhibition molecular mechanisms have been proposed while for breakthrough events during proximal complement pathway inhibition remain enigmatic. RBCs undergo structural and biochemical changes as they age. We hypothesized that RBC senescence may contribute to complement activation and thus provide an explanation for ongoing intravascular complement activation – even during complement inhibition.
Methods:
RBCs from healthy donors were fractionated by age using Percoll density gradient centrifugation. Isolated RBC populations were analyzed by flow cytometry and immunofluorescence microscopy for surface deposition of complement proteins (C3d, C4d) and immunoglobulin G (IgG). The expression of GPI-anchored complement regulators (CD59, CD55), and the immune adherence receptor CD35, was also assessed across age-defined fractions.
Results:
Senescent RBCs exhibited significantly higher levels of surface-bound C3d, C4d, and IgG compared to younger RBCs. In addition, we observed a gradual decrease in the expression of complement regulators CD59, CD55, and CD35 on aged RBCs. This age-associated loss of regulatory proteins may contribute to increased susceptibility to complement deposition. These findings suggest that aging RBCs progressively accumulate opsonins and lose protective features, predisposing them to recognition and clearance by phagocytes under physiological conditions.
Conclusion:
Our findings indicate that RBC senescence is associated with the gradual accumulation of IgG and complement fragments, along with a reduction in surface complement regulators such as CD59, CD55, and CD35. These changes likely represent a physiological mechanism for the immune recognition and clearance of aged erythrocytes Apart from infections, inflammatory conditions, or pregnancy also senescent erythrocytes may contribute to breakthrough haemolysis when PNH patients are treated with a complement inhibitor. These observations provide a mechanistic link between physiological RBC aging and complement-driven pathology in PNH,
P-128
Single-cell Transcriptomic Changes in Mouse Hearts during Collagen-Induced Arthritic Inflammation
Kathryn Hok1, Yi-Dong Li1, Stacey Mueller-Ortiz1, Aleksey Y. Domozhirov1, Marcos I. Restrepo2, Pooja Shivshankar1
1McGovern Medical School, Houston, USA. 2UTHealth San Antonio, San Antonio, USA
Chronic cardiac inflammation with fibrosis is a leading cause of heart failure at any age. Systemic inflammation in autoimmune rheumatoid arthritis (RA) doubles the risk of heart attack and heart failure. It is therefore imperative to identify early-stage mechanisms by which RA pathogenesis predisposes patients to cardiac dysfunction. Activated complement immune peptides, C3a and C5a, facilitate innate immune response in chemotaxis and amplify chronic inflammation. C5a via its cognate receptor C5ar1 signaling is implicated in exacerbating chronic inflammatory diseases. Because C5ar1 is expressed on myeloid and non-myeloid cells, including endothelial cells, we hypothesized that the C5a/C5ar1 signaling acts as a major molecular trigger to the pathogenesis of arthritic cardiovascular remodeling. In the model of type II collagen-induced arthritis (CIA) in WT C57B6/J mice with intradermal C5a challenge, we observed colocalization of markers of myocardial hypertrophy, Cav1 and Vegfr3, in the perivascular and myocardial areas. In unbiased endothelial function-specific transcriptome arrays, CD31+-endothelial cells of CIA+C5a mice showed upregulated novel inflammatory, angiogenesis, and prothrombotic mediators (collectively termed as RA mediators). C5ar1-signaling dependent modulation in inducible Nos (iNos) and endothelial-specific collagen (Col3a1) mRNAs were measured in WT-C5a challenged CD31+-endothelial cells. These findings intrigued us to investigate the single-cell specific transcriptomic changes in C5a-challenged CIA mouse hearts compared to those in PBS-control mouse hearts. Hypergeometric, KEGG, and reactome enrichment analyses revealed endothelial and cardiomyocytes clusters mapping to hypertrophic/dilated cardiomyopathy. We identified clusters of vascular and lymphatic endothelial cells and cardiomyocytes with common significant downregulation of atrial natriuretic peptide encoding gene Nppa, and the mitochondrial cytochrome C oxidases mt-Co1, Co2, Co3. Most importantly, significant upregulation of profibrotic Crispld2, Eprs1, and Mpp7 in the myofibroblasts clusters revealed the putative association of dampened energy metabolism in the endothelial cells and cardiomyocytes with myofibroblasts activation in C5a-induced inflammatory response arthritis-induced cardiac hypertrophy.
References
This work was supported by the NIH to P.S. (R01-AI158694). We acknowledge our former Chairman and Professor, Dr. Rick A. Wetsel, for his financial support (R01-AI025011) and his Chair funds to carry out this study.
P-129
Deep neutrophil phenotyping unveils the impact of complement activation in sickle cell pathophysiology
William Briones1,2, Seema Patel1,2, Maya Maarouf1,2, Jayre Jones1,2, David Archer1,2, Sean Stowell3, Rabindra Tirouvanziam2, Satheesh Chonat1,2
1Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, USA. 2Department of Pediatrics, Emory University School of Medicine, Atlanta, USA. 3Department of Pathology, Brigham and Women's Hospital, Boston, USA
Background:
Sickle cell disease (SCD) impacts millions globally. Acute chest syndrome (ACS) is a leading cause of admission to ICU and death among SCD patients. Our clinical preliminary data show that complement activation is elevated during ACS. Corroborating this, a murine model of SCD found that complement activation plays a role in driving ACS and associates with lung neutrophil extracellular trap (NET) formation (Chonat et al., STM 2025). Given these data, we hypothesized that hemolysis-mediated complement activation primes neutrophils to generate NETs, resulting in endothelial damage and ACS.
Methods:
Blood collected from SCD patients experiencing ACS (n=8) and at steady-state (n=7) was used to phenotype neutrophils and measure complement deposition on neutrophils using high dimensional flow cytometry. In addition, novel sickle cell (SS) mice deficient in complement component 3 (C3) (SSC3KO) were treated with hemin, and tested for complement activation and NET formation.
Results:
Consistent with the established role of complement in neutrophil activation in SCD mice, Unifold Multiparameter Approximation and Projection (UMAP) analysis demonstrated increased C3 deposition on neutrophils from ACS patients compared to those from patients at steady-state (3% vs. 0.6%, respectively; p<0.01). ACS patients were also found to have increased immature neutrophils compared to those at steady-state (3% vs. 0.2%, respectively; p<0.001), and that C3 deposition occurred on ~16% of immature ACS neutrophils. Moreover, of the immature ACS neutrophils, 58% had a phenotype of reverse transendothelial migration (neutrophils that had entered tissue but migrated back into circulation), with 26% of these cells having C3 deposition. While C3 deposition on mature neutrophils (3.4% ACS vs 0.8% steady-state; p<0.05) did not associate with altered complement regulatory protein expression, the majority of immature ACS neutrophils had decreased complement regulatory protein expression. Consistent with this, hemin-treated SSC3KO mice failed to develop ACS, and lacked NET formation and P-selectin expression in the lungs compared to hemin-treated wild type mice.
Conclusion:
These new findings suggest that neutrophil activation during ACS is modulated by systemic complement activation. Ongoing studies in our group are evaluating how complement recruits and primes neutrophils for NET formation and endothelial dysfunction using biochemical and genomic tools.
References
R01HL163637 (SC)
P-130
The conversion of native C3 to C3(H2O) is contact-driven facilitated by neutrophil extracellular traps
Sanjaya Kumar Sahu1, Priyan D Weerappuli2, John P Atkinson1, Alfred HJ Kim1
1Division of Rheumatology, Department of Medicine, Washington University, School of Medicine, St. Louis, USA. 2Department of Biomedical Engineering, University of Michigan, Ann Arbor, USA
The tick-over of C3 is a slow, spontaneous, and rate-limiting process of the alternative pathway (AP) of complement. It maintains a low level (~1%) of baseline activity, ensuring the system is primed for rapid and efficient responses. It has been speculated that certain highly negatively charged surfaces such as polystyrene or air bubbles can drive the conversion of C3 to C3(H2O). Various autoimmune diseases are characterized by AP activation (i.e. lupus and antiphospholipid syndrome) along with high rates of NETosis. We speculate that elevated AP activation in these conditions may be due to high neutrophil extracellular trap (NET) load, creating excessive negatively charged surfaces that can drive C3(H2O) formation. To test this, we first examined whether NETs are sufficient to induce native C3 to C3(H2O) conversion.
Bead-purified human neutrophils were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) incubated for 2–3 hrs. to induce NET formation, which was confirmed by immunofluorescence. DNA-histone mesostructures (DHMs) were also used, comprising of chromatin, histones, and trehalose, as “artificial” NETs with structural and biologic characteristics similar to isolated NETs (2). Native C3 was incubated with NETs or DHMs for 1, 2.5, or 5 minutes and C3(H₂O) was quantified via ELISA (1). 1 unit/μL of deoxyribonuclease I (DNase I) was used to digest NETs over 15 minutes at 370C.
We observed accelerated conversion (~1800-fold compared to an estimated 1% tickover rate)) of native C3 to C3(H₂O) when C3 was co-incubated with NETs or DHMs, which was abrogated when DNase was added to NETs or trehalose only was used as controls. The rate of conversion was rapid, as the maximally observed C3(H2O) was reached at the earliest timepoint (1 min) and did not increase with additional incubation. Surprisingly, the rapid kinetics of conversion using DHMs was preserved when performed at +4ºC.
These findings further support that the conversion of native C3 to C3(H₂O) can be contact-mediated, and that biologic substrates such as NETs can dramatically facilitate this conversion. We are currently confirming the interaction between native C3 and NETs through cryo-EM structural determination.
References
1) Development and Optimization of an ELISA to Quantitate C3(H 2 O) as a Marker of Human Disease. Front Immunol. 2019 Apr 4:10:703., PMID: 31019515 PMCID: PMC6458276 2) Extracellular Trap-Mimicking DNA-Histone Mesostructures Synergistically Activate Dendritic Cells. Adv Health Mater. 2019 Nov;8(22): e1900926., PMID: 31614077 PMCID: PMC6872909
P-131
The evaluation of clinical scoring system in complement mediated-TMA patients
Meng Tan, Ying Tan, Minghui Zhao
Nephrology Department, Peking University First Hospital, Beijing, China
Background
To date, distinguishing atypical hemolytic uremic syndrome (aHUS), also called Complement Mediated-TMA (CM-TMA), from other forms of thrombotic microangiopathies (TMAs) remains a significant challenge in clinical practice. Fadi's work proposed a scoring system incorporating clinical, biological, and pathological features, which aids in differentiating aHUS from other TMA forms, particularly those associated with malignant hypertension[1]. The aim of this study was to evaluate and validate the utility of this scoring system in our cohort.
Methods:
Between 2000 to 2021, patients were diagnosed renal TMA patients and diagnosed with CM-TMA in Peking University First Hospital were included . Patients were further categorized into those with typical aHUS (aHUS, n=18), malignant hypertension (MT, n=69), and pregnancy-related TMA (P-TMA, n=20). Complement activation was assessed in a subset of these patients using the following methods: (1) Human renal glomerular endothelial cells (HRGECs) were incubated with patient sera, and C5b-9 deposition was quantified using flow cytometry (FACS); (2) C5b-9 staining was evaluated in kidney biopsy specimens.
Results:
All patients were evaluated using Fadi's scoring system. The average score in the entire CM-TMA cohort was 2.44 ± 2.03. Specifically, the average scores were 1.64 ± 1.61 for MT patients, 3.39 ± 2.03 for aHUS patients, and 4.35 ± 1.57 for P-TMA patients. Among them, 26 patients underwent complement activation assessments. In the high-score group (score 3–6, n = 18), five patients (27.7%) exhibited C5b-9 staining in renal biopsies, six patients (33.3%) demonstrated positive C5b-9 formation in HRGECs, and nine patients (50%) showed negative results in both renal biopsies and HRGECs. In the low-score group (score -2 to 2, n = 8), four patients (50%) exhibited C5b-9 staining in renal biopsies, five patients (62.5%) demonstrated positive C5b-9 formation in HRGECs, and only two patients (25%) showed negative results in both renal biopsies and HRGECs.
Conclusion:
In our work, complement activation was confirmed by the presence of C5b-9 both in vivo and in vitro in CM-TMA patients who were assigned a low score according to Fadi's scoring system. In clinical practice, it is essential to utilize not only clinical features but also complement assessments to accurately differentiate CM-TMA from other conditions.
References
1.Fakhouri F, Schwotzer N, Frémeaux-Bacchi V. How I diagnose and treat atypical hemolytic uremic syndrome. Blood. 2023;141(9):984-995. doi:10.1182/blood.2022017860
P-132
Genetic C4 deficiency in lower extremity peripheral arterial disease
A. Inkeri Lokki1,2, Mirjami Laivuori3, Maarit Venermo3, Marja-Liisa Lokki4, Juha Sinisalo2
1Department of Bacteriology and Immunology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 2Heart and Lung Center, HUS Helsinki University Hospital and University of Helsinki, Helsinki, Finland. 3Department of Vascular Surgery, Helsinki University Hospital and University of Helsinki, Helsinki, Finland. 4Department of Pathology, University of Helsinki, Helsinki, Finland
Background
Atherosclerosis causes thickening and calcification of the arterial wall due to chronic inflammatory and thrombotic processes. Atherosclerosis affecting arteries of the lower limbs from the aortic bifurcation distally is called lower extremity peripheral arterial disease (LE-PAD). It is estimated that over 230 million people worldwide suffer from LE-PAD. Even asymptomatic LE-PAD patients have an increased risk for coronary artery disease (CAD) and cerebrovascular disease. Early detection and risk factor control are the key to improving the prognosis of LE-PAD patients. While complement activation is a known feature of atherosclerosis, little is known about its role in LE-PAD susceptibility and prognosis.
Methods
Biological samples and clinical characteristics from 97 femoral endarterectomy patients were collected at the Helsinki University Hospital between 2014-2017 (1). C4A and C4B copy number variation was determined by RT-qPCR (2). C4 deficiency was defined as <2 copies of C4A and/or C4B gene. Association of C4 deficiency to clinical presentation and follow-up data was tested.
Results
C4A/B deficiency is associated with local atherosclerosis (Fisher’s exact p=0.012) and survival in 7–10-year follow up (Χ2 p=0.021). C4B deficiency is associated with smoking (Χ2 p=0.012) and better 7–10-year survival (Χ2 p=0.007). Similarly, C4A deficiency is associated with protection from prior CAD incidents (Fisher’s exact p=0.034). Furthermore, C4A deficiency associates with lack of metaplastic bone in the plaque histology specifically in men (N=59, Fisher’s exact p=0.009) while not in women (N=35, Fisher’s exact p=1).
Conclusion
Risk factors for LE-PAD are known to differ between men and women, for example, smoking is the most important risk factor for men. While this study is underpowered to study all characteristics separately for male and female patients, we found clear indication that C4 deficiencies may have a sex-specific influence in the progression of LE-PAD. The protection of C4A deficiency from prior CAD incidents in LE-PAD corroborates our previous finding in a cohort of acute myocardial infarction. C4 deficient patients may experience less inflammation due to lower capacity of complement activation by the classical pathway.
References
1. Laivuori M et al. Osteoid metaplasia in femoral artery plaques is associated with the clinical severity of LE-PAD in men. Front Cardiovasc Med.2020;7:594192. 2. Paakkanen R et al. Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction. PLoS One.2012;7(6):e38813.
P-133
The C3-C3aR axis modulates trained immunity in alveolar macrophages
Alexander Earhart1, Rafael Aponte Alburquerque1, Marick Starick1, Aasritha Nallapu2, Lorena Garnica1, Ayse Naz Ozanturk1, Hrishikesh Kulkarni2
1Washington University in St Louis, St Louis, USA. 2UCLA, Los Angeles, USA
Background: Complement protein C3 is crucial for immune responses in mucosal sites such as the lung, where it aids in microbe elimination and enhances inflammation. While trained immunity - enhanced secondary responses of innate immune cells after prior exposure - is well-studied, the role of the complement system in trained immune responses remains unclear.
Methods: We investigated the role of C3 in trained immunity using a combination of in vitro studies using a model of trained immunity induced by heat-killed Pseudomonas aeruginosa (Pa), and in vitro, using models of heat-killed Candida albicans and heat-killed Pa.
Results: In vivo, trained wild-type mice showed significantly elevated pro-inflammatory cytokines and increased C3a levels upon a second stimulus, whereas C3-deficient mice exhibited a blunted cytokine response and heightened evidence of lung injury. Ex vivo, C3-deficient alveolar macrophages (AMs) displayed reduced chemokine and cytokine output after training, which was restored by exogenous C3 but not by C3a. Inhibiting C3aR, both pharmacologically and with a genetic C3aR knockout, prevented this restoration, indicating the necessity of C3aR engagement. Mechanistically, trained WT AMs demonstrated enhanced glycolytic activity compared to C3-deficient AMs - a defect corrected by exogenous C3 in a C3aR-dependent manner.
Conclusions: These findings reveal that C3 modulates trained immunity in AMs through C3aR signaling, affecting cytokine production and metabolic reprogramming, and highlight a novel role for C3 in trained immunity.
P-134
NOVEL FUNCTIONS OF CD59 ISOFORMS IRIS-1 AND IRIS-2 IN INSULIN-PRODUCING PANCREATIC β-CELLS
Thu-Ha Tran, Vanessa Kasbauer, Alexander Ekström, Vaishnavi Dandavate, Ben C. King, Anna M. Blom
Department of Translational Medicine, Lund University, Malmö, Sweden
CD59 is a well-known inhibitor of the membrane attack complex (MAC). Our previous studies identified that the intracellular CD59 isoforms, IRIS-1 and IRIS-2, play a crucial role in insulin exocytosis in pancreatic β-cells. Lacking glycosylphosphatidylinositol (GPI) anchor of the membrane-bound CD59, these intracellular variants interact with insulin granules and SNARE proteins, facilitating insulin release at different phases. However, the precise molecular mechanisms underlying their function remain unexplored. Pancreatic β-cell dysfunction is a major cause of type 2 diabetes, owing to the defects in its metabolism and insulin secretion machinery. This study employs CRISPR-Cas9 gene editing to generate CD59 knockout and introduce an overexpression of human IRIS-1 or IRIS-2 in rat β-cell lines INS-1 to investigate the molecular functions of these CD59 isoforms. The APEX-biotin proximity labeling method was used to identify novel binding partners of IRIS-1 and IRIS-2. Mass spectrometry analysis of biotin-labeled proteins can suggest the intracellular pathways regulated by these isoforms. Besides, RNA sequencing revealed that CD59 isoforms significantly influence pyruvate metabolism, a key determinant of β-cell function, as pyruvate fuels mitochondrial oxidative phosphorylation, triggering insulin secretion. In addition, glycogen accumulation, related to pyruvate metabolism in β-cells, is reported to cause metabolic dysfunction and reduced insulin secretion. Additionally, our findings indicate that IRIS-1 and IRIS-2 may regulate intracellular insulin transport through interactions with Noc2, an effector of exocytosis. Our study highlights the novel metabolic and intracellular trafficking functions of IRIS-1 and IRIS-2. Understanding the mechanisms by which these CD59 isoforms regulate insulin secretion could provide insight into the non-canonical roles of CD59 in insulin-secreting β-cells.
P-135
Complement C5aR1 Modulates Excitatory Synaptic Transmission in Cortical Pyramidal Neurons via a Calcium-Independent Mechanism
Sandra Parker, Mark Bellingham, Trent Woodruff
University of Queensland, Brisbane, Australia
Background: The complement system, traditionally considered a peripheral immune cascade, is now recognised for its role in central nervous system (CNS) physiology (Bohlson & Tenner, 2023). While complement proteins such as C1q and C3 facilitate synaptic pruning via microglial phagocytosis both physiologically and pathologically (Tillmon et al., 2024; Wang et al., 2020), the anaphylatoxin C5a and its receptor C5aR1 are also expressed in the healthy brain. Notably, C5aR1 has been found to colocalise with synaptic proteins at excitatory synapses in the hippocampus, suggesting a potential role in modulating synaptic function (Crane et al., 2009). However, its precise function in neuronal activity remains unclear. This study aimed to determine whether C5aR1 modulates excitatory synaptic transmission in cortical pyramidal neurons.
Methods and Results: To test this, we performed whole-cell patch-clamp recordings on layer 5 pyramidal neurons in the primary motor cortex of adult wild-type (WT) and C5aR1-knockout (C5aR1-KO) mice. We found that neurons lacking C5aR1 exhibited significantly longer inter-event intervals of spontaneous excitatory postsynaptic currents (sEPSCs), indicating a reduction in excitatory synaptic input. Pharmacological inhibition of C5aR1 in WT neurons using PMX53 similarly prolonged sEPSC inter-event intervals, suggesting that C5aR1 is necessary for maintaining normal excitatory transmission. However, exogenous application of C5a did not enhance sEPSC frequency, raising the possibility that C5aR1 may exert its effects through constitutive activity or a ligand-independent mechanism.
To determine whether C5aR1 modulates neurotransmitter release in a calcium-dependent manner, we recorded miniature excitatory postsynaptic currents (mEPSCs) and assessed paired-pulse facilitation (PPF). mEPSCs, which reflect action potential-independent neurotransmitter release, were significantly reduced following C5aR1 inhibition, suggesting that C5aR1 influences presynaptic neurotransmitter release. In contrast, PPF, a measure of presynaptic calcium-dependent vesicle release probability, remained unchanged, indicating that C5aR1 does not regulate excitatory transmission via classical calcium-dependent pathways.
Conclusions: These findings demonstrate that neuronally-expressed C5aR1 plays a functional role in maintaining excitatory synaptic input in murine cortical pyramidal neurons. Given the emergence of complement-targeting therapies for neurological disorders such as macular degeneration and neuromyelitis optica (West et al., 2024), understanding the neurophysiological role of C5aR1 is essential for assessing the potential impact of complement inhibition on normal brain function.
References
Crane, J. W et al., (2009). The C5a anaphylatoxin receptor CD88 is expressed in presynaptic terminals of hippocampal mossy fibres. Journal of neuroinflammation, 6, 1-10.Wang, C., et al., (2020). Microglia mediate forgetting via complement-dependent synaptic elimination. Science, 367(6478), 688-694.
P-136
Novel Metabolic Role of the Complement Activator MASP- in Regulating Pancreatic β-Cell Function
Gautham Sridharan1, Jian li2, Shira Tsour1, Viktor Von-Ehrlich Treuenstatt2, Chijioke Chukwudi2, Jin Byungchang2, Nirajan Shrestha2, Bukyung Kim3, Dan Berman1, Joseph Barry1, Quentin Bethune1, Elane Fishilevich1, Dhruv Desai1, Dhaval Oza1, Frederic Tremblay1, Margarette Stefater-Richards2, Carsten Dirksen4,5, Sten Madsbad4,5, Russell Goodman2, Jason Kim3, Peter Garred6,5, Peng Yi2, Nima Saeidi2,7
1Alnylam Pharmaceuticals, Cambridge, USA. 2Harvard Medical School, Boston, USA. 3Kosin University College of Medicine, Busan, Korea, Republic of. 4Hvidovre and Amager Hospital, Copenhagen, Denmark. 5University of Copenhagen, Copenhagen, Denmark. 6Rigshospitalet, Copenhagen, Denmark. 7Massachusetts General Hospital, Boston, USA
Background:
Hepatokines have recently emerged as significant regulators of systemic metabolism and hold promise as therapeutic targets for metabolic diseases, particularly type 2 diabetes (T2D). Mannose-binding lectin serine protease-2 (MASP-), traditionally known for its role in complement activation, was identified as a hepatokine whose expression significantly decreases following Roux-en-Y gastric bypass (RYGB), a highly effective treatment for obesity and T2D. Thus, we hypothesized that MASP- might have non-canonical roles in glucose metabolism apart from what is regarded as the functions of the complement system.
Methods:
We utilized high-resolution mass spectrometry-based proteomics to profile plasma samples from rodents and MASP- serum measurements from humans following RYGB surgery. Hepatic Masp2 expression was silenced in mouse models of obesity and T2D using GalNAc-siRNA technology to assess impacts on glucose metabolism, insulin secretion, and pancreatic β-cell function. Glucose tolerance tests, hyperinsulinemic-euglycemic and hyperglycemic clamps, as well as glucose-stimulated insulin secretion (GSIS) assays were employed to evaluate physiological responses. RNA sequencing and pathway enrichment analyses were conducted on isolated pancreatic islets to identify the underlying molecular mechanisms.
Results:
Plasma proteomic analysis and serum measurement identified MASP- as significantly reduced post-RYGB in both rodents and humans, associated with improved glycemic control. Liver-specific silencing of Masp2 in obese and diabetic mice markedly improved glucose tolerance and increased insulin secretion from pancreatic β-cells without altering peripheral insulin sensitivity or body weight. Mechanistically, Masp2 silencing alleviated endoplasmic reticulum (ER) stress in β-cells, enhancing insulin trafficking and secretion. Further experiments demonstrated that recombinant human MASP- directly impaired insulin secretion in isolated mouse and human islet cells, without activating the canonical complement system. These observations suggest a previously unrecognized, direct regulatory role independent of complement activation.
Discussion:
Our study identifies MASP- as a novel hepatokine with critical non-canonical roles in β-cell function and glucose homeostasis. The sustained improvement in glycemic control following hepatic Masp2 knockdown, coupled with alleviated ER stress and enhanced insulin secretion, positions MASP- as a promising therapeutic target for T2D treatment. These findings offer new insight into hepatokine biology and open avenues for novel diabetes therapies utilizing clinically validated RNA interference platforms.
P-137
Pregnancy complications in C5aR2-deficient mice are caused by an altered uterine immune cell network
Katja Kähler1, Timo Gemoll2, Anke Fähnrich3, Christian Karsten1
1ISEF, Lübeck, Germany. 2Section for Translational Surgical Oncology and Biomaterial Banks, Lübeck, Germany. 3Medical System Biology, Lübeck, Germany
Reasons for infertility in women and pregnancy complications can have various reasons, ranging from anatomical abnormalities to hormonal imbalances. However, approximately 16% of infertility-cases remain unexplained. Altered immunological effects may help address this gap. Research has shown that the maternal immune response against the semi-allogenic fetus is highly dynamic during pregnancy. A Th1-dominated response in the first trimester changes to a Th2-dominated during the second trimester. Another shift to a pro-inflammatory state initiates parturition. Alterations in immune cell ratios, especially Th1/Th2, have been shown to play a detrimental role in various pregnancy complications and infertility. Maternal decidual T cells, which have been shown in humans to exclusively express the anaphylatoxin receptor C5aR2 in absence of C5aR1, are part of a complex immune cell network within the pregnant uterus. This network is dominated in number by uterine NK (uNK) cells, which were also shown to solely express C5aR2 in absence of C5aR1 in human and mice.
Interestingly, a global deletion of C5aR2 in mice leads to increased resorptions, the murine analogue of spontaneous abortion.
To find the route of action of this global deletion, broad-band approaches like bulk-proteomics and spatial transcriptomics of pregnant and non-pregnant uteri from wildtype and C5aR2-deficient mice were utilized.
Previously published results from our lab showed increased mRNA levels of uNK, uDC and Th1-derived cytokines like IFN-𝛾, IL12 and IL-18, in C5aR2-deficient mice. Proteomics-interaction-terms revealed an increased upregulation of granzymes from non-pregnant to pregnant status, compared with witldtype mice. Differential expression and pathway enrichment analysis of spatial transcriptomics data showed down-regulation of GATA3, ID3 and TGF-𝛽 signaling, being involved in the differentiation of Th2, Treg and Th17 cells, respectively.
Together, these data suggest the involvement of C5aR2 in the differentiation of uterine CD4+ T cells. The subsequent establishment of a pregnancy-favoring Th1/Th2-ratio may influence numbers, maturation, and cytotoxicity of uNK cells. uNK cells and their secreted cytokines, in turn, are detrimental for uterine tissue remodeling, enabling the implantation of the blastocyst and, therefore, successful pregnancy. In the future, high-dimensional flow cytometry analysis will help to determine T cell ratios and maturation and cytotoxicity status of uNK cells.
P-138
A ‘sweet spot’ for complement receptor CD46 activity: CYT-1 regulates glycolytic and moonlighting functions of glycolytic enzymes during human CD4+ T cell activation
Natalia Kunz1,2, Gaelle Le Friec3,4, Jack A. Bibby5, Erin E. West5, Ayden Case5, Parul Singh5, Nicolas S. Merle5, Paul Lavender4, Majid Kazemian6, Christoph Hess7,8, Claudia Kemper5
1Laboratory of Molecular Immunology and Immunology Center, NHLBI, NIH, Bethesda, USA. 2Complement and Inflammation Research Section, NHLBI, NIH, Basel, Switzerland. 3UCB, Slough, United Kingdom. 4School of Immunology and Microbial Sciences, King’s College London, London, United Kingdom. 5Complement and Inflammation Research Section, NHLBI, NIH, Bethesda, USA. 6Department of Biochemistry and Computer Science, Purdue University, West Lafayette, USA. 7Department of Biomedicine, Immunobiology, University of Basel, Basel, Switzerland. 8Department of Medicine, University of Cambridge, Cambridge, United Kingdom
Autocrine engagement of the human-specific complement receptor CD46, driven by T cell receptor (TCR) activation on human CD4+ T cells, is critical for human Th1 induction: Processing of the intracellular CD46 domain CYT-1 by g-secretase mediates mTORC activation and metabolic adaptation required for cell growth, expansion and effector function. How, at the molecular level, CYT-1 enables this fundamental cellular adaptation remains unknown. Identifying the CYT-1 protein interactome in T cells via co-immunoprecipitation with antibodies recognizing cleaved-CYT-1 followed by protein mass spectrometry, we observed that CYT-1 associates with five of the ten metabolic enzymes mediating glycolysis.
Focusing on the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) we used ELISAs, Microscale Thermophoresis and Proximity Ligation Assays to confirm a direct interaction between GAPDH and CYT-1. Functionally, CYT-1 directly increased GAPDH’s classic enzymatic activity allowing it to meet the heightened energetic demand that T cell activation requires. Unexpectedly, CYT-1-augmented GAPDH glycolytic activity was accompanied by a concurrent increase in a non-canonical key moonlighting function of GAPDH: the ability of GAPDH to stabilize mRNAs selectively associated with metabolic states underlying Th1 differentiation. Accordingly, both classical and non-classical GAPDH functions were abrogated in in vitro-activated Jurkat and primary human CD4+ T cells with selective CYT-1-deficieny induced by CRISPR/Cas9 deletion. Consequently, CYT-1 deficient T cells exhibited reduced GAPDH enzymatic activity, reduced glycolytic flux and impaired cell proliferation. Employment of a xenogeneic Graft-versus-host disease model (GvHD) confirmed reduced T cell activation, activity and poor engraftment of CYT-1-deficient primary human CD4+ T cells in vivo, while engineered CYT-1 deficiency in Jurkat cells significantly reduced Jurkat cell expansion in a xenogeneic leukemia mouse model. CYT-1 binding to aldolase or enolase also augmented the glycolytic and moonlighting functions of these proteins, suggesting a more generalized effect of CYT-1 on CYT-1-intercating enzymes of the glycolytic pathway.
These data identify the CD46 CYT-1–glycolytic enzyme interaction as a direct facilitator of classic and non-classic glycolytic enzyme activities, solidify the notion that autocrine complement is a critical regulator of cell physiology, and suggest that the CYT-1-GAPDH axis may be a potential target in CD4+ T cell-associated disease states including autoimmunity and leukemia.
P-139
CD46 directly engages Sp1 to control gene transcription and HIV expression in human CD4+ T cells
Parul Singh1, Luopin Wang2, Pavitra Ramdas3, Gaelle Le Friec4, Natalia Kunz1, Prakriti Mudvari3, Ayden Case1,5, Nicolas Merle1, Jack Bibby1, Henrik Hasle6, Eli Boritz3, Majid Kazemian2, Claudia Kemper1
1NHLBI, NIH, Bethesda, USA. 2Perdue University, Indiana, USA. 3NIAID, NIH, Bethesda, USA. 4UCB, London, United Kingdom. 5Heart and Lung Research Institute, University of Cambridge, Cambridge, United Kingdom. 6Department of Pediatrics and Adolescent Medicine, Aarhus University Hospit, Aarhus, Denmark
CD46 is a ubiquitously expressed complement regulator, a cell entry receptor for several important pathogens, and a cell-intrinsically operating orchestrator of the human Th1 induction and contraction program via direct control of T cell gene programs largely associated with cell metabolism. Thus, CD46 signalling perturbations in T cells are associated with either hyper or hypo-Th1 activity and autoimmunity or recurrent infections, respectively. The molecular mechanism by which CD46 controls gene transcription is unknown. Utilizing protein chemistry, CUT&RUN, RNA-sequencing, a Jurkat T cell clone and primary CD4+ T cells in which the expression of the CYT-1 intracellular signaling domain of CD46 was ablated by CRISPR-Cas9 technology, in vitro human immunodeficiency virus (HIV) vector infections and assessment of T cells from people with HIV, we demonstrate that the CD46 signaling domain engages the transcription factor Sp1 in human CD4+ T cells to dynamically modulate Sp1–DNA interactions and control CD4+ T cells survival and HIV expression.
Specifically, CD46–Sp1-controlled gene programs allow survival and activation of naive CD4+ through expression of nutrient channels and concurrent suppression of genes encoding death-caspases. Together, these events enable initiation of IFN-γ production and Th1 induction. Somewhat surprisingly, the CYT-1–Sp1 interaction is not required to sustain Th1 memory.
Furthermore, based on extensive prior evidence supporting a role for Sp1 in the HIV life cycle, we explored the role of CD46 CYT-1 in regulating HIV transcription within infected cells. Indeed, the CD46–Sp1 axis restrained HIV transcription in in vitro-infected CD4+ T cells and differences in CD46 expression among CD4+ T cell subsets from people with HIV suggest roles for CD46 in shaping infected cell reservoirs in vivo. These findings provide important insights into human-specific aspects of complement and T cell biology and a needed tangible opening to define novel physiological and pathophysiological roles of CD46 across cells and diseases.
P-140
Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction
Yiyang Zhao, Min Chen
Department of Renal Medicine, Peking University First Hospital, Beijing 100034, China, Beijing, China
Background
Complement system-mediated metabolic disorders are considered an important contributor to the pathogenesis of diabetic kidney disease (DKD). However, the non-canonical roles of complement 5a receptor 2 (C5aR2) in metabolic regulation during DKD progression remain unclear.
Methods and Results
C5aR2 expression was significantly upregulated in the tubulointerstitium of patients with DKD and was correlated with disease severity and renal outcomes. In diabetic mouse models, genetic deletion of C5ar2 markedly aggravated the DKD phenotype. Combining lipidomic and transcriptomic analyses, we further found that C5aR2 deletion induced excessive lipid accumulation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in the kidneys, accompanied by a decrease in phosphatidylserine (PS) levels. Mechanistic studies using dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), and proximity ligation assays (PLA) demonstrated that C5aR2 activates the transcription factor c-FOS, which upregulates phosphatidylserine synthases (PSS) and promotes the interaction between PSS and mitochondrial fusion protein 2 (MFN2). This interaction facilitates the formation of mitochondria-associated ER membranes (MAMs), thereby sustaining PS biosynthesis and maintaining mitochondrial and ER function. Furthermore, treatment with the C5aR2-specific agonist P59 significantly restored PS levels and improved MAM integrity, ultimately exerting a protective effect in DKD.
Conclusion
Collectively, this study suggests that C5aR2 activation may represent a promising therapeutic strategy for DKD by preserving mitochondrial and ER function.
References
1. Li XX, Clark RJ, Woodruff TM. C5aR2 Activation Broadly Modulates the Signaling and Function of Primary Human Macrophages. J Immunol 205, 1102-1112 (2020).
P-141
The role of intracellular and cell-autonomous C3 in NF-κB priming in human macrophages
Charlotte Årseth1, Stine Kristensen1, Harald Husebye1, Terje Espevik1,2, Liv Ryan1, Tom Eirik Mollnes3,1,4,5, John D. Lambris6, Trude Helen Flo1,7
1Department of Clinical and Molecular Medicine, Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway. 2Clinic of Laboratory Medicine, St. Olavs Hospital, Trondheim, Norway. 3Department of Immunology, Oslo University Hospital, and K. G. Jebsen Inflammation Research Centre, University of Oslo, Oslo, Norway. 4Research Laboratory Nordland Hospital, Bodø, Norway. 5K. G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway. 6Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. 7Department of Infection, Clinic of Medicine, St. Olavs Hospital, Trondheim, Norway
In 2021, 1.6 million people died from Tuberculosis (TB). This makes Mycobacterium tuberculosis (Mtb), the causative agent of TB, one of our top infectious killers. The complement system is an integral part of the innate immune system that curbs such bacterial infections.
Although the complement system was previously thought to be hepatically derived and extracellular only, a cell-autonomous and intracellular complement system involved in metabolic and inflammatory pathways in several cell types has recently been identified. Limited research has been published on the role of complement in TB; thus it remains to be elucidated if the intracellular complement system plays a role in Mtb infection.
We have previously demonstrated that intracellular C5a signaling via a mitochondrial C5a receptor contributes to inflammasome activation upon sensing danger signals in monocytes and macrophages. We now want to explore the role of C3/C3aR in these cells, using Mtb as a model system for infection.
To investigate the role of intracellular C3 in human macrophages, we have used CRISPR-Cas9 knock-out (KO) THP- cells lacking C3 or the C3a receptor (C3aR). Following stimulation with LPS and Mtb, the C3 KO cells, but not the C3aR KO cells, showed a marked decrease in the release of several NF-κB-regulated pro-inflammatory cytokines, including TNF-α, IL-8, and IL-1β, compared to the wild-type cells. This effect was observed on both the mRNA and protein levels, indicating that C3 may play a C3aR-independent role in the NF-κB signaling pathway. Supporting this hypothesis, phosphorylation of the IKKα/β complex, an activation step required for nuclear translocation of NF-κB, was strongly reduced in stimulated C3 KO cells, but not in the C3aR KO cells. Other NF-κB pathway kinases, such as TAK1, also appear to be affected.
However, it remains to be determined whether full-length C3 or one of its split products is responsible for the observed effects. The exact step in the NF-κB pathway where C3 (or a split-product of C3) interacts is also unidentified. Overall, our data demonstrate that cell-autonomous C3 mediates inflammatory responses in human macrophages.
P-142
Exploring the role of intracellular C3 in activation of the NLRP3 inflammasome in human macrophages
Stine Kristensen1, Charlotte Årseth1, Liv Ryan1, Sindre Ullmann1, John D. Lambris2, Tom Eirik Mollnes3,4, Trude Helen Flo1, Harald Husebye1, Terje Espevik1
1Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. 2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. 3Department of Immunology, University of Oslo, Oslo, Norway. 4Research Laboratory, Nordland Hospital, Bodø, Norway
Background
In recent years, a cell-autonomous and intracellularly active complement system has been identified as an important regulator of basic metabolic pathways underlying the normal function of immune cells. We have previously demonstrated that human monocytes and macrophages harbour a cell-autonomous C5 activation system, in which C5a signals via a mitochondrial C5a receptor (mtC5aR1). Upon sensing of danger signals, mtC5aR1 signalling induces a metabolic shift in the cell, triggering activation of the NLRP3 inflammasome and thus cleavage of pro-IL-1β into active IL-1β by caspase-1. However, an intracellular role of complement component C3 in inflammatory responses in this cell type has not yet been established.
Methods
We have used confocal microscopy and antibodies targeting neo-epitopes revealed on C3a and C3bc following C3 cleavage to map the sub-cellular location of C3 activation products in human iPSC-derived macrophages (iPSC-DMs). Additionally, to investigate the role of intracellular C3 in human macrophages, we have used CRISPR-Cas9 gene edited THP- cells in which the gene encoding C3 or the C3a receptor (C3aR) have been knocked-out (KO). Immunoblotting, ELISA, and qPCR was used to measure the inflammatory response of these cells.
Results
We have identified intracellular stores of C3a and C3bc in both resting and LPS-stimulated iPSC-DMs, indicating that constitutive C3 activation occurs in this cell type. Interestingly, C3bc localised on vesicle-like structures, and we observed co-localisation of C3bc and NLRP3 on enlarged endosomes following LPS priming. Supporting a role of C3 in inflammasome activation, stimulation of the C3 KO THP- cells with LPS and nigericin to activate NLRP3 resulted in reduced cleavage of pro-caspase-1 and pro-IL-1β into their active forms, when compared to the WT. Furthermore, transcription of IL1B following LPS priming was decreased in the C3 KO cells when compared to the WT, indicating that C3 may also be involved in the priming step of NLRP3 activation. However, these effects were not observed in the C3aR KO cells, suggesting that this role of C3 is independent of signalling through C3aR.
Conclusion
Together, our results demonstrate the presence of an intracellular C3 activation system in human macrophages, which may regulate activation of the NLRP3 inflammasome.
P-143
Neutrophil Plasticity in Epidermolysis Bullosa Acquisita: Bridging Innate and Adaptive Immunity via C5aR2 Signaling
Dennis Niese1, Jovan Schanzenbacher1, Ralf Ludwig2,3, Christian Karsten1
1Institute for Systemic Inflammation Research (ISEF), Lübeck, Germany. 2Department of Dermatology, Allergy and Venerology, Lübeck, Germany. 3Lübeck Institute of Experimental Dermatology (LIED), Lübeck, Germany
Background:
Pemphigoid diseases like Epidermolysis Bullosa Acquisita (EBA) involve complement-mediated neutrophil recruitment to the dermal-epidermal junction. Our single-cell sequencing revealed elevated Semaphorin 4D (SEMA4D) expression in neutrophils stimulated with C5a compared to neutrophils deficient in C5aR2, suggesting functions beyond their classical roles. SEMA4D influences B cell maturation and autoantibody production, while C5aR2 appears critical in this process, highlighting complement-neutrophil interactions in mediating both innate and adaptive immunity. Neutrophils are essential for EBA development, with their effector functions driving tissue damage. This research aims to elucidate how neutrophils, through complement receptor signaling, contribute to autoantibody production and autoreactive lymphocyte responses in EBA pathogenesis. Additional evidence suggests neutrophils may directly impact adaptive immune responses through antigen presentation capabilities, challenging traditional understanding of immune cell compartmentalization.
Methods:
Using an immunization-induced EBA mouse model with C5aR2-deficient and wild-type mice, we characterize neutrophils at different disease timepoints in skin and bone marrow. Flow cytometry is used to identify neutrophil subsets expressing antigen-presenting markers (MHC-II, CD86, CD80) and adaptive capabilities. Single-cell RNA sequencing distinguishes bone marrow neutrophil subpopulations with unique expression signatures, while in vitro assays evaluated their capacity to process and present autoantigens.
Results:
Our preliminary investigations characterize neutrophil populations with antigen-presenting phenotypes in active EBA, comparing C5aR2-deficient and wild-type mice across disease development. We aim to identify neutrophil subsets expressing antigen presentation and adaptive function markers, determining their distribution during disease progression, and assessing their functional capacity to interact with adaptive immune components. These studies are revealing potential mechanisms by which neutrophils bridge innate and adaptive immunity in EBA pathogenesis, with particular focus on C5aR2-dependent pathways.
Conclusion:
Understanding neutrophil-mediated regulation of autoreactive lymphocytes could enable earlier detection and targeted therapies for EBA and related autoimmune disorders. This research provides insights into neutrophils' emerging role in adaptive immunity, potentially improving outcomes for autoimmune patients and revealing new therapeutic avenues targeting the complement-neutrophil axis.
P-144
Complement Membrane Attack Complex Alters the Proliferation of Neural Progenitor Cells During Late Embryonic Murine Neurogenesis.
Austin Read1, John Lee1, Jenny Fung1, Trent Woodruff2
1School of Biomedical Sciences, the University of Queensland, Brisbane, QLD, Australia. 2Queensland Brain Institute, the University of Queensland, Brisbane, QLD, Australia
Neurogenesis, the process by which neurons are formed from neural progenitor cells (NPCs), relies on the expansion and differentiation of NPCs. Our prior studies have demonstrated that several components of the immune complement system play key roles in regulating NPC proliferation during neurogenesis. Complement activation culminates in the formation of the cytolytic complement membrane attack complex (MAC), which forms membrane pores to eliminate pathogens. However, its role in neurodevelopmental processes remains unclear.
We investigated the presence and function of MAC during late embryonic neurogenesis in murine models. Using qRT-PCR, ELISA, and immunofluorescence, we confirmed MAC deposition in NPCs of the non-pathogenic murine neocortex. In C9-/- mice, which lack the terminal complement component C9, we observed a significant reduction in mitotic activity among apical NPCs. This decrease in mitosis correlated with an altered balance between proliferation and differentiation, reflected in the differential expression of Empty Spiracles Homeobox 1 (EMX1), associated with self-renewal, and TIS21, which drives differentiation. The disruption of this balance at embryonic day 15 (E15) led to notable changes in the cytoarchitecture of the developing neocortex.
Further investigation of MAC’s role in primary NPCs revealed that blocking the endogenous inhibitor CD59a and inducing MAC deposition with normal mouse serum increased MAPK pathway activation, cell cycle length, mitotic activity, and glucose metabolism, which are all critical indicators of cellular proliferation.
Our results suggest that MAC plays a crucial role in enhancing glycolytic metabolism via the MAPK pathway, thereby extending the cell cycle and promoting the proliferation of apical NPCs during neurogenesis. This study uncovers novel, non-redundant roles for complement components in the development of the mammalian neocortex, highlighting MAC as a key regulator of NPC proliferation and neurodevelopment.
P-145
Investigating complement receptor C3aR1 signalling in metabolic dysfunction in the R6/1 mouse model of Huntington’s Disease
Titaya Lerskiatiphanich1, Tanya McDonald1, Cedric Cui1, Jenny Fung1, Trent Woodruff1,2, John Lee1
1The University of Queensland, Brisbane, Australia. 2Queensland Brain Institute, Brisbane, Australia
Background: Huntington’s disease (HD) is a devastating neurodegenerative disorder characterised by progressive motor, cognitive, and psychiatric symptoms, combined with pronounced metabolic disturbances such as weight loss, lean mass decline, and glucose dysregulation. The molecular mechanisms driving these metabolic changes remain unclear. Emerging evidence suggests the complement receptor C3aR1 plays a role in metabolic regulation, particularly in glucose homeostasis and insulin secretion. TLQP-1, a neuropeptide reported to activate C3aR1, has shown potential in enhancing insulin secretion. In this study, we assessed the ability of TLQP-1 to restore metabolic function in the R6/1 transgenic mouse model of HD and explored the potential involvement of C3aR1 signalling in HD-associated metabolic dysfunction.
Methods: Metabolic phenotyping of R6/1 and wild-type (WT) littermate mice was performed using indirect calorimetry to measure energy expenditure, locomotor activity, and food intake. Glucose metabolism was examined using intraperitoneal glucose (ipGTT) and insulin (ipITT) tolerance tests. Circulating levels of metabolic hormones (insulin, leptin, adiponectin, GLP-) were measured using ELISA. Islets were isolated to evaluate insulin secretory responses to TLQP-1 stimulation.
Results: R6/1 mice exhibited progressive metabolic deterioration, characterised by significant weight loss, reduced lean mass, decreased food intake and altered hormonal changes. Despite preserved insulin sensitivity and normal islet morphology, R6/1 mice displayed impaired glucose tolerance and reduced glucose-stimulated insulin secretion compared to WT controls. Treatment with TLQP-1 significantly enhanced insulin secretion in R6/1 islets, restoring it to WT levels.
Conclusions: Our study demonstrates that R6/1 mice develop progressive metabolic disturbances, including weight loss, reduced lean mass and glucose intolerance, accompanied by impaired insulin secretion and dysregulated GLP- levels. The reduced insulin secretory capacity in R6/1 islets suggest that β-cell dysfunction drives metabolic abnormalities in HD. Importantly, TLQP-1 treatment restores islet function, implicating C3aR1 signalling in these secretory defects. These findings indicate that C3a-C3aR1 signalling plays an important regulatory role in HD systemic homeostasis. While further studies are needed to fully elucidate the mechanism, our results identify TLQP-1 and C3aR1 activation as plausible therapeutic targets for addressing HD-associated metabolic dysfunction.
P-146
Peptide-mediated modulation of T cell-expressed complement-like CD109boosts T cell anti-tumor responses
Nicolas Merle1, Jienyu Ding2, Martin Kolev1, Erin West1, Ira Phadke3, Jubayer Rahman1, Jack Bibby1, Bingyu Yan4, Kazemian Majid4, Behdad Afzali5, Se-In Son6, Ganesha Rai6, Rosa Nguyen3, Christian Biertuempfel2, Naoko Mizuno2, Claudia Kemper1
1Laboratory of Complement and Inflammation Research, NHLBI, NIH, Bethesda, MD, USA., Bethesda, USA. 2Laboratory of Structural Cell Biology, NHLBI, NIH, Bethesda, MD, USA., Bethesda, USA. 3Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA, Bethesda, USA. 4Departments of Biochemistry and Computer Science, Purdue University, West Lafayette, IN, USA., West Lafayette, USA. 5Immunoregulation Section, Kidney Diseases Branch, NIDDK, NIH, Bethesda, MD, USA., Bethesda, USA. 6National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA, Bethesda, USA
Background: Cell-intrinsic complement expression and function have emerged as critical regulators of tissue immunity and T cell biology. In particular, cell-intrinsic CD46 engagement and signaling on human CD4⁺ T cells is required for optimal Th1 response induction. Through transcriptomic profiling, we identified CD109 – a GPI-anchored, C3-like complement protein – as one of the top upregulated targets downstream of CD46 signal induction during T cell activation. However, the function of CD109 in T cells remains unexplored.
Methods: We investigated CD109 function in freshly isolated human CD4⁺ T cells using CD109-targeted CRISPR/Cas9 gene editing and used Cd109⁻/⁻ deficient mice to examine CD109’s role in vivo in an experimental autoimmune encephalomyelitis (EAE) model, with phenotypic analysis of immune populations from spleen, draining lymph nodes, brain, and spinal cord via restimulation assays and scRNA-seq. To identify CD109’s mode of action, we employed predictive modeling, protein chemistry, super-resolution microscopy (STED) and atomic force microscopy as well as the usage of an in-house generated CD109-targeting peptide in in vitro CAR T cell-mediated glioma tumor killing.
Results: CD109-deficient human and mouse CD4⁺ T cells exhibited hyperactive Th1 and Th17 (but not Th2) responses, with increased IFN-g and IL-17 production upon stimulation in vitro. Accordingly, Cd109⁻/⁻ CD4⁺ T cells exacerbated EAE pathology in a mouse model of MS. Mechanistically, CD109 directly interacted with (at minimum in cis) and inhibited the activity of a non-canonical co-stimulatory molecule expressed on T cells. A bespoke peptide interfering with the CD109/costimulatory interaction enhanced the CAR T cell-mediated tumor killing activity of human T cells in vitro. In vivo assessment of the CD109 peptide efficacy using human mesothelin-specific CAR T cells injected into NSG mice bearing human mesothelioma is currently ongoing (with IP application pending).
Conclusion: These findings identify the complement-like protein CD109 as a novel negative regulator of CD4⁺T cell co-stimulatory pathways that control the magnitude of Th1/Th17 inflammatory responses. Furthermore, pharmacological targeting of CD109 may enhance CAR T cell anti-tumor activity, which may open the door to new therapeutic avenues involving a currently little explored member of the complement family.
P-147
Intracellular C1r impacts tumor progression and immune microenvironment in renal cancer
Nicolas Merle1, Artemis Angelidou1, Alessandra Zarantonello1, Mikel Rezola Artero1, Margot Revel1, Idris Boudhabhay1, Marius Ueffing2,3, Christine Gaboriaud4, Lubka Roumenina1
1Centre de Recherche des Cordeliers, Institut National de la Santé et de la Recherche Médicale, Sorbonne Université, Université de Paris Cité, Inflammation, Complement and Cancer team, Paris, France, Paris, France. 2Institute for Ophthalmic Research, Department for Ophthalmology, Eberhard Karls University of Tübingen, 72076 Tübingen, Germany., Tubingen, Germany. 3German Center for Neurodegenerative Diseases (DZNE), 72076 Tübingen, Germany., Tubingen, Germany. 4Laboratoire de Cristallographie et Cristallogénèse des Protéines, Institut de Biologie Structurale Jean Pierre Ebel, CEA-CNRS-Université Joseph Fourier, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France., Grenoble, France
Background:
Metastasis and resistance to therapy are major drivers of cancer-related mortality. Complement component C1R is overexpressed in multiple tumor types and correlates with poor prognosis and lack of response to immunotherapy, particularly in clear cell renal cell carcinoma (ccRCC), the most common renal cancer. In several models, C1R knockout reduces proliferation, viability, and migration of cancer cells, suggesting critical cell-intrinsic functions. However, the underlying mechanisms remain poorly understood. We aimed to investigate whether and how C1r contributes to the hallmarks of cancer
Methods:
We used gene silencing, phenotype profiling, RNA-seq, and metabolomics in C1r-expressing ccRCC cell lines (A498, Caki-1) and fibroblasts (BJ) to study C1r function. Findings were corroborated using snRNA-seq data ccRCC patients, including cancer cells and cancer-associated fibroblasts (CAFs). Subcellular fractionation and multiplexed immunofluorescence were used to localize C1r, and co-immunoprecipitation with mass spectrometry identified candidate interactors. A 40-marker immunofluorescence panel was applied to ccRCC tumor sections (n=6), and a larger patient cohort was stained with an 8-plex panel targeting C1r, stromal and immune cell markers.
Results:
C1R expression was primarily detected in tumor cells and CAFs in ccRCC. Silencing C1R in vitro impaired proliferation, migration, viability, and sphere formation in both cancer cells and fibroblasts. This phenotype could not be rescued by adding purified C1r, indicating a non-canonical, intracellular role. C1r localized to organelles and nuclei in vitro and in situ. Proteomic analysis identified ~30 candidate interactors related to proliferation and migration. Transcriptomic and metabolic analyses of siC1R cells confirmed altered pathways involved in cell cycle, migration, energy and aminoacid metabolism. C1r levels also influenced immune signaling: C1R knockdown reduced inflammatory signatures in vitro, while its overexpression in tumors correlated with altered T cell infiltration in both RNA-seq and hyperplex imaging data. The relationship between C1r expression and immune cell phenotype is currently under investigation in a patient cohort
Conclusion:
Our findings reveal novel intracellular functions of C1r in tumor cells and CAFs, independent of the classical complement pathway. These results highlight C1r as a multifunctional, cell-autonomous regulator of tumor progression and immune contexture, and support its potential as a therapeutic target in cancers expressing C1r.
P-148
GC B cell autonomous complement activation rewires cell metabolism associated with delayed class switching and altered GC dynamics
Philip Einwohlt1, Sina Borus1, Anke Fähnrich2, Jörg Köhl1
1Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany. 2Medical Systems Biology Group, Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany
Background:
Anaphylatoxin receptor signaling is a key component in the humoral immune response. B cell activation, the formation of germinal centers (GC), and the class switch recombination (CSR) of immunoglobulins are critical in this process. Previously, we and others found that C5aR1-deficiency in B cells leads to impaired B cell activation and CSR recombination. To identify the mechanism by which C5aR1 controls these steps, we determined transcriptome activation of induced germinal center B cells (iGB) from wildtype and C5ar1-/- mice.
Methods:
We co-cultured naïve B cells from C57BL/6 wildtype and C5ar1-/- mice with fibroblasts expressing CD40 ligand and B cell-activating factor + IL-4, resulting in B cell expansion and differentiation into iGBs. During a 4-day co-culture, CD95+ iGB cells were analyzed for C5, C5a, C5aR1 and immunoglobulin expression via flow cytometry, subsequently sorted, and their gene expression profile was determined by single-cell RNA sequencing.
Results:
We found upregulation of C5 expression and C5a generation in iGB cells as compared with naïve B cells associated with intra- and extracellular C5aR1 expression. Gene expression profiling uncovered altered dynamics of zone-specific light and dark zone GC B cells, indicating that C5aR1 might control the dynamics of B cells in the GC. Further, we observed that the neutral amino acid transporter B(0) (SLC1A5), an L-glutamine transporter, was significantly lower expressed in C5ar1/than in WT iGBs. This decrease was associated with a markedly decreased expression of the oxidative phosphorylation (OXPHOS) pathway in iGBs from C5ar1-/- mice. A reduced glutamine concentration in the co-culture, or the inhibition of the OXPHOS pathway by oligomycin, inhibited the differentiation of iGB cells from naive B cells. Also, the shortage of glutamine reduced the frequency of iGBs that underwent IgM to IgG or IgE CSR, which is consistent with the results observed in iGB cells from C5aR1-deficient mice.
Conclusions:
The C5a/C5aR1 axis is critical for activation, differentiation, and CSR of B cells. Our findings suggest that autocrine C5a generation and C5aR1 activation controls this process by rewiring B cell metabolism, i.e. by upregulation of glutamine uptake in activated B cells, fueling the OXPHOS pathway and driving germinal center dynamics.
P-149
Factor H-Related Protein Dysregulation and Intracellular Localization in Osteoarthritis and Head & Neck Cancer
Daniela Schulz1, Andreas Moehwald2, Denis Vlasov1, Marianne Ehrnsperger3,4, Yvonne Reinders5, Sina Heimer1, Tobias Ettl1, Susanne Grässel2,4, Richard Bauer2,1, Nicole Schäfer2,4
1Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany. 2Department of Orthopaedic Surgery, Experimental Orthopaedics, Center for Medical Biotechnology (ZMB, Bio Park 1), University of Regensburg, Regensburg, Germany. 3Department of Trauma Surgery, University Medical Center Regensburg, Regensburg, Germany. 4Department of Orthopedic Surgery, University of Regensburg, Asklepios, Regensburg, Germany. 5Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany
Background:
Factor H (FH) and FH-Related (FHR) proteins are crucial complement regulators. Their roles may extend beyond extracellular functions, potentially influencing cellular processes in inflammatory diseases like Osteoarthritis (OA) and malignancies such as Head and Neck Cancer (HNC). However, comparative expression profiles across different disease contexts and their intracellular presence require detailed investigation.
Methods:
We quantified FH and FHRs (-2, -3, -4, -5) levels via ELISA in synovial fluid (SF) from OA patients (n=175), non-OA controls (n=15), and in serum from HNC patients (n=20) pre- and post-tumor removal versus controls. RNA sequencing and mendelian randomization (MR) analyses compared gene expression of CFH and CFHRs in OA vs non-OA tissues. To investigate subcellular protein distribution in HNC tumors, FH, FHR-1, FHR-2, FHR-3, and FHR-5 was assessed in cellular fractions (total lysate, membrane, cytoplasm, cytoskeleton, nucleus, chromatin), followed by protein identification and abundance measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Results:
Distinct protein profiles emerged significant elevated FHR-4 levels in OA-SF in male and female patients, while FHR-5 (males) and FH (females) also showed significant increased levels. Notably, FHR-2 and FHR-3 levels were unchanged in OA-SF compared to non-OA-SF. Conversely, sera from HNC patients exhibited significantly elevated FHR-2, FHR-3, FHR-4, and FHR-5 pre-operatively compared to healthy controls, which remained elevated post-surgery. Mass spectrometry analysis of subcellular fractions revealed widespread distribution. Remarkably, FH, FHR-1, FHR-2 and FHR-5 were found in the nucleus, indicating possible nuclear functions. MR analysis indicated that genetically predicted higher expression of CFH was associated with an increased risk of knee-OA.
Conclusion:
FH-family proteins display context-dependent dysregulation: specific elevations (notably FHR-4) typify the local OA joint environment, contrasting with broad systemic FHR increases in HNC. The confirmed intracellular, particularly nuclear, localization points towards non-canonical functions of FHRs. Together, these findings reveal disease-specific molecular signatures and potential genetic contributions involving FH/FHR pathways in OA and HNC pathogenesis, highlighting avenues for biomarker development and therapeutic targeting.
P-150
HMGB1 cleavage by C1s modulates the pro-inflammatory signal induced by TLR2
Jeanne Vigne1, Marie Lorvellec1, Nolwenn Haut1, Nicole M. Thielens1, Christine Gaboriaud1, Chantal Dumestre-Pérard1,2, Véronique Rossi1
1Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France. 2Laboratoire d'Immunologie, Pôle de Biologie, CHU Grenoble Alpes, Grenoble, France
Backgrounds
Extracellular HMGB1 protein acts as an alarmin, alerting the immune system to cell injury and inducing an inflammatory response. As a pro-inflammatory mediator, it interacts with immune components including innate immune receptors such as TLRs and the complement system (1). We recently observed that HMGB1 is cleaved in vitro by C1s into 3 N-terminal fragments and we are currently investigating their inflammatory properties (2). To this end, we sought to characterise the involvement of HMGB1 fragments generated by C1s cleavage in the interaction of HMGB1 with one of its receptors, TLR2.
Methods
HMGB1 and cleavage fragments (f1, f2 and f3) were produced and purified. Their ability to activate the NF-kB pathway via TLR2 interaction was evaluated using HEK Blue hTLR2 cells.
Results
The HMGB1/TLR2 axis, although described in numerous pathological contexts, is poorly characterised. Our study highlights the differential inflammatory potential of HMGB1 fragments generated by C1s cleavage. We confirm that the disulphide form of full-length HMGB1 is capable of binding to TLR2 and subsequently inducing pro-inflammatory signalling. The same response is observed with the longer C1s proteolytic fragment (f1) composed of the A and B boxes, without the C-tail. Interestingly, fragment f2, a shorter proteolytic product of f1, lacking the C-terminal part of the B box, exhibits greater pro-inflammatory activity via its interaction with TLR2. In contrast the shortest fragment f3, composed of a truncated part of the HMGB1 A box, does not show any pro-inflammatory signal. This last result fits well with the fact that the A box is known to be an antagonist of HMGB1 inflammatory function when isolated.
Conclusion
HMGB1 can play a pro-inflammatory role via its binding to TLR2. Our findings further suggest that the B box, which harbours the site responsible for the pro-inflammatory activity of HMGB1, is crucial for its interaction with TLR2. Cleavage of HMGB1 by C1s could therefore induce modulation of its pro-inflammatory activity via TLR2, providing a regulatory mechanism that may be relevant in diseases such as lupus nephritis, where the HMGB1/TLR2 axis is implicated.
References
1. Gaboriaud, Christine et al. “Complement System and Alarmin HMGB1 Crosstalk: For Better or Worse.” Frontiers in immunology vol. 13 869720. 28 Apr. 2022, doi:10.3389/fimmu.2022.869720 2. Lorvellec, Marie et al. “HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.” Frontiers in immunology vol. 14 1151731. 26 Apr. 2023, doi:10.3389/fimmu.2023.1151731
P-151
Inflammatory cytokines drive local C3 transcription to regulate metabolic reprogramming in kidney epithelial cells and subsequent fibrosis
Tilo Freiwald1, Md Tajmul2, Jorge-Luis Trujillo-Ochoa2, Daniel Chauss2, Dhaneshwar Kumar2, Baktiar Karim3, Julis Jager1, Yubing Guo1, Maja Gunkel1, Tobias Huber1, Thorsten Wiech1, Claudia Kemper4, Martin Kolev5, Majid Kazemian6, Didier Portilla7, Behdad Afzali2
1University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH), Bethesda, USA. 3National Cancer Institute (NCI) of the National Institutes of Health (NIH), Bethesda, USA. 4National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH), Betheda, USA. 5Apellis, Boston, USA. 6Purdue University, West Lafayette, USA. 7University of Virginia, Charlottsville, USA
Background Complement is classically viewed as a liverderived, extracellular defense cascade, but many components are also transcribed by parenchymal cells, where they regulate metabolism and injury responses. The kidneys, which are highly susceptible to complementmediated damage, commonly exhibit local complement deposition, yet the extent, distribution, and function of de novo complement produced by renal cells remain poorly defined. We asked whether tubular epithelial cell (TEC)-derived complement shapes the metabolic and fibrotic sequelae of acute kidney injury (AKI).
Methods
We analyzed existing and de novo bulk and single-cell high-throughput data from multiple acute kidney injury (AKI) models, followed by CUT&RUN, CRISPR editing, and regulon analysis to map transcription factor occupancy at complement loci. Mechanistic studies used proximal TEC specific C3 knockout mice, a cell permeable factor B (CFB) inhibitor, metabolic flux (Seahorse) profiling, confocal and histological imaging, and mass spectrometric mapping of C3. Human relevance was explored in transcriptomic cohorts of human disease.
Results Across AKI models, folicacid nephropathy (FAN) showed the strongest enrichment of complement genes. Singlenucleus RNAseq revealed injuryinduced Vcam1⁺ TECs coexpressing C3 and Factor B (Cfb), which signaled to infiltrating immune cells via C3a-C3AR1 and integrins. An inflammatory cytokine was the dominant upstream inducer: deletion of its receptor reduced C3 and Cfb transcripts in response to injury. Two downstream transcriptional regulators were confirmed as transactivators at both genes, as confirmed by CUT&RUN and CRISPR disruption, and their regulon activity correlated with C3/Cfb expression. Confocal imaging tracked C3 trafficking from ER/Golgi to endolysosomes and mitochondria, co-localizing with CFB. Conditional C3 deletion or intracellular CFB inhibition lowered glycolytic and oxidative phosphorylation and attenuated interstitial fibrosis in FAN mice. In human kidney datasets, C3 and CFB coexpression inversely correlated with eGFR.
Conclusion Inflammatorycytokine–driven transcription of C3 and factor B in proximal TECs couples intracellular complement activation to metabolic reprogramming and fibrogenesis after kidney injury. Targeting this intrinsic complement axis offers a therapeutic avenue beyond systemic complement blockade for inflammatory kidney diseases.
References
This has been submitted by the PI on behalf of an ECR
P-152
Spatial transcriptomics maps complementdriven celltocell interactions during acute kidney injury
Vikram Sabapathy1, Daniel Chauss2, Shuqiu Zheng1, Resmi Raju3, Jeremie Pina3, Rahul Sharma1, Mark Okusa1, Behdad Afzali2, Didier Portilla1
1University of Virginia, Charlottsville, USA. 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH), Bethesda, USA. 3Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH), Bethesda, USA
Background Understanding the mechanisms that drive (acute kidney injury (AKI) and subsequent tissue repair is crucial for developing novel therapeutic strategies to halt inflammation and prevent tissue scarring. Intracellular and locally secreted complement components are increasingly recognised as modulators of tissue injury and repair, yet the spatial niches in which they operate within the kidney remain undefined. Standard singlecell RNAseq detects extrahepatic complement transcription but loses positional context, obscuring microdomains where complement mediates crosstalk among parenchymal, stromal, and immune cells during acute kidney injury (AKI).
Methods Formalinfixed paraffinembedded kidney sections from mouse models of ischaemia–reperfusion injury (IRI), unilateral ureteral obstruction (UUO), and sham surgery underwent 10x Visium HD and Xenium insitu–hybridisation spatial transcriptomics. Data were preprocessed, normalised, and clustered, followed by differentialexpression, pathwayenrichment, cellneighbourhood, and ligand–receptor interaction analyses. Key transcripts and proteins were validated by RNAscope, RTqPCR, immunoblotting, and confocal microscopy.
Results Spatial maps revealed a loss of healthy S1/S2 proximal tubules and expansion of VCAM1⁺ injured tubules in both IRI and UUO compared with shams. Injured tubules showed robust upregulation of C3 and Cfb, while stromal cells and macrophage clusters selectively increased other complement components. Cellneighbourhood analysis identified discrete loci where C3high tubules and C3ar1high macrophages were spatially adjacent. Ligand-receptor modelling confirmed significant C3-C3AR1 and integrinmediated signalling within these niches. RNAscope and confocal imaging validated C3 protein localisation to injured tubules and C3AR1 to nearby macrophages.
Conclusion Highresolution spatial transcriptomics uncovers compartmentalised complement expression and signalling hotspots in AKI, linking injured proximal tubules with infiltrating macrophages through local C3 production. These findings provide a tissue atlas for targeting intrinsic complement pathways and will guide ongoing translation to human kidney biopsies.
References
This has been submitted by the PI on behalf of an ECR
P-153
Targeting complement anaphylatoxin C3a receptor to break immunotherapy resistance in colorectal cancer
Nadia St. Thomas, Brenton Puckett, Carsten Krieg, Silvia Guglietta
Medical University of South Carolina, Charleston, USA
Background. Microsatellite instability-high (MSI-H) colorectal cancer (CRC) is characterized by a high mutational burden and immune cell infiltration, rendering approximately 5% of metastatic cases responsive to immune checkpoint inhibitors (ICIs). In contrast, the majority of CRCs are microsatellite stable (MSS), exhibit low immunogenicity, and are unresponsive to ICI therapy. Despite this paradigm, some MSI-H tumors develop resistance to ICI, and emerging data suggest that a subset of MSS tumors display robust inflammatory signatures that may support immune-based interventions. We recently identified that down-regulation of the complement anaphylatoxin receptor C3aR correlates with increased innate and adaptive immune cell infiltration in CRC. However, the mechanisms through which C3aR loss fosters immunologically “hot” tumors and their relevance to ICI responsiveness remain poorly understood.
Methods and Results. Using an APCMin/+/C3aR-/- mouse model, we show that tumors, although MSS, exhibit a heightened immune signature and respond to anti-PD-1 therapy. High-dimensional profiling of the tumor microenvironment revealed increased frequencies of polyfunctional NK cells, macrophages, and dendritic cells following ICI. Bone marrow chimera experiments indicated that C3aR deficiency in non-immune stromal or epithelial cells was the primary driver of the pro-inflammatory tumor microenvironment. To further investigate tumor-intrinsic effects, we established APCMin/+/C3aR-/- tumor cell lines and transplanted them into wild-type hosts. Remarkably, tumors lacking C3aR in cancer cells alone were sufficient to create an ICI-responsive microenvironment, with the most significant responses observed upon combination treatment with anti-CTLA-4 and anti-PD-1 antibodies.
Conclusions. Our findings demonstrate that loss of C3aR in CRC—particularly within tumor cells—can reprogram the tumor microenvironment to support anti-tumor immunity and enhance responsiveness to ICI therapy, independent of mutational burden. These insights uncover a novel mechanism to convert immunologically “cold” MSS CRC into “hot,” ICI-responsive tumors and may inform future strategies to overcome therapeutic resistance in CRC patients.
References
This work was supported by ACS-IRG (to C.K.), American Cancer Society RSG-24-1255025-01-IBCD and R01 CA258882 to S.G.
P-154
Beta-Cell Intrinsic C3 Regulates Pancreatic Islet Responses to Metabolic Stress in vivo
Vaishnavi Dandavate, Ben King, Anna Blom
Lund University, Malmö, Sweden
Background: Recently, the complement system, particularly C3, has been linked to the regulation of several metabolic processes. Elevated C3 levels are correlated to a higher risk of Type 2 diabetes (T2D) onset associated with insulin resistance, high BMI, body fat percentage, waist circumference and triglyceride concentrations1. This suggests that circulating C3 may contribute to the onset of obesity, T2D, and associated cardiovascular diseases. However, in contrast to these detrimental effects, intrinsic C3 expression in pancreatic beta-cells has been shown to protect against stress-induced apoptosis in vitro2. This paradox highlights the need to investigate the role of C3 in beta-cells in vivo.
Methods: To investigate the role of beta-cell-intrinsic C3, we generated a beta-cell-specific C3 knockout (beta-C3-KO) by crossing C3fl/fl tdTomato reporter mice with Tg(Ins2-cre)23Herr mice, which express Cre recombinase under the rat Ins2 promoter. Male beta-C3-KO and control mice were placed on either a low-fat diet (LFD) or high-fat diet (HFD) for 20 weeks, and pancreatic islets were analyzed using RNA sequencing.
Results: As expected, male mice on HFD gained more weight than LFD, with no significant physiological differences between genotypes on either diet. Though, RNA-Sequencing results exhibited significant gene expression differences between control and beta-C3-KO islets under LFD, but not in HFD conditions. Notably, ~60% of differentially expressed genes in LFD:control vs. LFD:beta-C3-KO overlapped with those in LFD:control vs. HFD:control, indicating that the absence of C3 in beta-cells induces a transcriptional profile resembling that of HFD exposure. Among the affected pathways, PI3K-Akt signaling emerged as a top hit, highlighting C3’s potential role in regulating beta-cell responses to metabolic stress.
Conclusion: Our findings suggest that beta-cell-intrinsic C3 plays a protective role in maintaining metabolic homeostasis, preventing transcriptional changes characteristic to HFD exposure. This emphasizes the need for further exploration of C3 as a potential target for modulating beta-cell function in metabolic disorders.
References
1) Engström, G. et al. Complement C3 Is a Risk Factor for the Development of Diabetes: A Population-Based Cohort Study.Diabetes (2005). 2) King, B. C. et al. Complement Component C3 Is Highly Expressed in Human Pancreatic Islets and Prevents β Cell Death via ATG16L1 Interaction and Autophagy Regulation.Cell Metabolism (2019).
P-155
A Versatile Screening Platform to Guide Applications for the FH-Recruiting Peptide 5C6 on Non-Self Surfaces
Jannes Felsch, Ekaterina Umnyakova, Shugirshan Suthagar, Simona Jacquemai, Haijie Zhao, Alexander Lander, Said Rabbani, Daniel Ricklin
University of Basel, Basel, Switzerland
Introduction
When non-self surfaces encounter biological fluids, complement-mediated responses may seriously impact patient health and transplant/biomaterial function. As the alternative pathway is driving such processes, surface recruitment of the regulator factor H (FH) serves as elegant approach to prevent complement attacks directly on vulnerable surfaces. While the potency of FH-recruitment via the 5C6 peptide has been demonstrated,1 its broader evaluation on relevant surfaces was limited by the availability of suitable coating strategies. Here, we introduce a click-chemistry-based surface modification platform that enables biocompatible coating of endothelial cells via metabolic engineering and the rapid preparation of affinity chromatography columns, among other applications.
Methods
5C6 was prepared by peptide synthesis and functionalized with alkyne handles. Human (HMEC-1) and porcine (PIEC) endothelial cells were incubated in medium containing azido-sugars. Glycocalyx modification was verified using a clickable dye, followed by flow cytometry and fluorescence microscopy. FH-recruitment onto 5C6-coated cells was assessed using labelled FH and endogenous FH from serum. The surface distribution of FH was visualized using fluorescently-labelled anti-FH antibodies. Sequence-scrambled peptide coating and unmodified cells were used as controls. To prepare 5C6 columns, azide-agarose was clicked to 5C6 and packed into fritted syringes; binding of intact FH from serum was evaluated by SDS-PAGE, western blot, and microscale thermophoresis.
Results
Efficient 5C6-coating of endothelial cells could be achieved without affecting cell viability and metabolic activity. Coating with active peptide provided strong recruitment of FH over the entire surface and reduced C3b-opsonization, whereas no comparable effect was observed for the controls. 5C6-based affinity columns verified that the peptide selectively binds FH from serum and enabled the purification of intact FH for further assays.
Conclusions
By establishing a surface modification platform for FH-recruiting peptides, we confirmed the high efficacy and selectivity of 5C6-coatings to recruit FH from serum and prevent complement attacks on biomedical surfaces, including living cells. Due to its broad adaptability to different peptides and surfaces, the system serves not only as a screening assay for 5C6 analogues but could be applied to other coating strategies for improving transplantation outcomes. Moreover, it enables new assay formats and facilitates the purification of bioactive FH.
References
1. C. Bechtler et al., Complement-regulatory biomaterial coatings: Activity and selectivity profile of the factor H-binding peptide 5C6. Acta Biomater. 2023; 155: 123-138
P-156
C5aR2 Fuels Autoimmune Skin Disease in Murine Epidermolysis Bullosa Acquisita
Jovan Schanzenbacher, Jörg Köhl, Christian M. Karsten
Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany
Background: Epidermolysis bullosa acquisita (EBA) is a blistering autoimmune skin disease caused by IgG autoantibodies (aAbs) targeting type VII collagen (COL7). In experimental EBA, passive transfer of anti-COL7 IgG aAbs mimics the effector phase, while active immunization with the von-Willebrand-factor-A-like domain 2 (vWFA2) of COL7 covers both, the afferent and effector phases. C5aR1 activation is crucial in EBA pathogenesis, as C5ar1/ mice are completely protected in both models. C5ar2/ mice are partially protected in passive EBA. The role of C5aR2 in the afferent phase remains elusive. Here, we determined C5aR2 function in active EBA.
Methods: WT and C5ar2/ mice were immunized with vWFA2, and disease progression, aAb production, and immune cell phenotypes were analyzed over 12 weeks. Since IgG aAb-producing B cells are key players in the afferent phase, we used an in vitro B cell–fibroblast co-culture system mimicking the germinal center (GC) B cell response to assess the impact of C5aR2-signaling on key markers of GC B cells, their activation, differentiation, and class switching.
Results: C5ar2/ mice were nearly fully protected in active EBA, showing markedly reduced skin lesions. Serum analyses revealed a disease-attenuating aAb profile in C5ar2/ mice, correlating with reduced inflammatory marker expression in the draining lymph nodes (dLNs), spleen, and bone marrow (BM). In vitro, survival marker expression was altered and activation, GC B cell differentiation, and IgG class switching in C5ar2/ GC B cells was impaired.
Conclusions: Our findings reveal a previously unrecognized pro-inflammatory role of C5aR2 in the early stages of autoimmune skin inflammation, underscoring its complex function in immune regulation. While C5aR2-deficient mice were only partially protected in passive EBA, they were almost completely protected in active EBA. This suggests that C5aR2 operates at two distinct disease stages: initiating autoimmunity by priming GC B cells for pathogenic IgG aAb production and exacerbating tissue damage in the effector phase by promoting neutrophil activation. The disease-initiating function is associated with alteration of key survival factors in GC B cells, critical for their development and multiple immune responses. Our findings highlight C5aR2 as a potential novel therapeutic target to treat autoimmune skin blistering in EBA.
P-157
Divergent Effects of Early Adult Microglial versus Global C1q Deletion on Amyloid Pathology and Synaptic Engulfment in an Alzheimer's Disease Mouse Model
Tiffany Petrisko, Shu-Hui Chu, Blossom Zhang, Andrea Tenner
University of California, Irvine, Irvine, USA
The complement system contributes to enhanced inflammation and cognitive decline in Alzheimer’s disease (AD). Previous reports showed that constitutive genetic deletion of C1q reduces glial activity and attenuates neuronal loss in AD mouse models. It is now known that microglia are the primary producers of C1q within the CNS. Therefore the aggressive Arctic (Arc) AD mouse model was used to determine if early adult microglial and/or global deletion of C1q would reduce engulfment of synapses, glial reactivity and amyloid plaque pathology.
Briefly, C1qaFL/FL and Arc C1qaFL/FL mice with and without the RosaCreERT2 transgene were treated with tamoxifen at 11-wks of age to induce global C1q deletion (designated iC1qKO). C1qaFL/FLCX3CR1CreERET2 (designated C1qΔMG) and ArcC1qΔMG mice, previously shown to delete microglial C1q by 8-12 weeks of age in the absence of tamoxifen, were also generated. All mice were aged to 10m of age, at time at which behavior loss is beginning to be detected in the Arc mice. Hippocampi were collected and confocal and immunofluorescence microscopy (IF) was used to quantify Vglut1 engulfed by microglia, lysosome-associated engulfment of amyloid by glia, glial activation (Iba1/GFAP), C3 and C5aR1 expression, as well as amyloid pathology.
Engulfment of Vglut1 within microglial lysosomes was significantly reduced in hippocampal CA1 of ArcC1qΔMG mice and in both the CA1 and CA3 subregions in ArciC1qKO mice compared to C1q-sufficient Arc controls. Expression of C3 was significantly reduced in both ArcC1qΔMG (37%) and ArciC1qKO mice (48%), although no changes in C5aR1 expression nor astrocyte (GFAP) or microglia (Iba1) reactivity was observed. While early adult microglial deletion of C1q failed to reduce amyloid levels, the amount of CD68 colocalized amyloid within both microglial (48%) and astrocytes (43%) was reduced. In contrast, early adult global deletion of C1q significantly reduced amyloid plaque burden levels by both IF (33%) and MSD (41%) assay for insoluble amyloid but did not alter lysosomal associated amyloid.
These findings support the role of C1q in synapse engulfment and astrocyte C3 induction . The data are also consistent with the hypothesis that peripheral C1q can contribute to amyloid plaque load, either directly or by modulating peripheral inflammation.
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Opposing effects of animal models of Type 1 and type 2 diabetes on factor D expression and systemic alternative pathway activity
Lucie Colineau1, Olga Kolodziej1, Daniel Ajona2, Ruben Pio2, Anna Blom1, Ben King1
1Lund University, Malmö, Sweden. 2CIMA Universidad de Navarra, Pamplona, Spain
Background: Alternative pathway (AP) production of C3a is demonstrated to increase insulin secretion and adipocyte function, therefore affecting systemic metabolism. While most complement proteins are hepatocyte-derived, factor D (FD), the rate-limiting factor of the AP, is an adipokine predominantly produced in adipose tissue. We therefore investigated the effects of both type 1 and type 2 diabetic mouse models (T1D and T2D) on systemic expression of complement proteins, with focus on the AP.
Methods: T2D models included use of high-fat diet induced obesity, and hyperphagic db/db mice. T1D models included streptozotocin induction of beta-cell apoptosis, and the spontaneously diabetic Akita mouse model. Complement gene expression was assessed by qPCR, and protein levels by Western blot and ELISA of serum samples. Serum AP activity was assessed by flow cytometry of C3 deposition on zymosan beads. Obesity-induced FD deficiency was treated with recombinant FD injections in db/db mice.
Results: We confirmed previous observations that FD is downregulated in obesity models, with strong negative correlations between serum FD and body weight, blood glucose, and serum insulin. However, liver expression of C3 was also significantly downregulated in animals on high fat diet. In contrast, in both T1D models, characterized by insulin deficiency and weight loss, FD expression in adipose tissue was powerfully upregulated and hepatic C3 expression was significantly increased, in the absence of inflammatory markers or acute phase response. This was not purely due to weight loss, as no upregulation was seen in mice subjected to calorie restriction alone. As a result, serum AP activity was significantly upregulated in T1D models. Consequences of local complement upregulation in T1D in tissues relevant to diabetic complications, such as kidney, are also being investigated, as well as the treatment of FD-deficient obese db/db mice with recombinant FD.
Conclusions: Downregulation of complement in mouse models of obesity may downregulate both negative inflammatory and positive homeostatic roles of complement activation. However, complement is upregulated in T1D models, where destruction of beta-cells prevents positive homeostatic roles. The relative roles of targeting homeostatic compared to pro-inflammatory roles of complement should therefore be assessed separately in T1D and T2D.
P-159
Cell-autonomous complement C3 activity in prostate epithelial cells suppresses malignant transformation through tonic restrain of the MYC-tumorigenesis program
Natalia Kunz1, Sumeyra Kartal2, Jack A. Bibby1, Nicolas S. Merle1, Hidekazu Yamamoto3,4, Jubayer M. Rahman1, Adam F. Maleki1,5, Luopin Wang6, Md Tajmul7, Jianping Ma7, Martin V. Kolev8, Fatima Karzai2, Nikhil Pramod2, Jonas Loetscher9, Tamara Kellerhals9, Maria L. Balmer9, Christoph Hess9,10, Behdad Afzali7, Ash Chandra11, Jeffrey R. Brender12, Murali K. Cherukuri12, Prokar Dasgupta3,4, Majid Kazemian6, Adam G. Sowalsky2, Erin E. West1, Claudia Kemper1
1Complement and Inflammation Research Section, NHLBI, NIH, Bethesda, USA. 2Laboratory of Genitourinary Cancer Pathogenesis, NCI, NIH, Bethesda, USA. 3Department of Urology, Guy’s & St. Thomas’ NHS Foundation Trust,, London, United Kingdom. 4School of Immunology & Microbial Sciences, King’s College London, London, United Kingdom. 5Translational Neuroradiology Section, NINDS, NIH, Bethesda, USA. 6Departments of Biochemistry and Computer Science, Purdue University, West Lafayette, USA. 7Immunoregulation Section, Kidney Diseases Branch, NIDDK, NIH, Bethesda, USA. 8Discovery, Apellis Pharmaceuticals, Waltham, USA. 9Department of Biomedicine, Immunobiology, University of Basel, Basel, Switzerland. 10Department of Medicine, University of Cambridge, Cambridge, United Kingdom. 11Department of Histopathology, Guy’s & St. Thomas’ NHS Foundation Trust, London, United Kingdom. 12Radiation Biology Branch, Center for Cancer Research, NCI, NIH, Bethesda, USA
Prostate cancer (PC) remains among the leading causes of cancer-related deaths with a better understanding of the mechanisms underlying its pathogenesis urgently needed. The synthesis of complement component C3 by tumor-infiltrating immune cells in the tumor microenvironment (TME) facilitates tumor progression via immunosuppression and angiogenesis support, denoting TME C3 as a therapeutic target. However, the role of C3 expressed by tumor cells themselves during oncogenesis remain inadequately characterized.
Using data from three clinical PC patient cohorts in combination with controlled ex vivo modulation of C3 in benign and malignant human prostate epithelial cells (PECs), molecular and RNA-sequencing analyses, a xenograft in vivo PC mouse model, a novel mouse PC model based on prostate-specific C3 ablation and mouse prostate magnetic resonance imaging (MRI), we found that PEC-intrinsic C3 is, unexpectedly, required to maintain normal cell homeostasis. C3 transcript and C3 protein levels were significantly reduced in malignantly transformed regions when compared to benign sections of human PC tissues. Aligning with this, C3 expression ablation in human benign PECs caused uncontrolled proliferation while provision of intracellular C3a to the aggressive PC cell line DU145 reduced in vitro proliferation and tumor burden in vivo in a xenograft cancer model. Moreover, aged Pbsn-Cre/C3Fl/Fl mice lacking cell-intrinsic C3 specifically in PECs exhibited increased prostate weights with MRI-assessment revealing changes in prostate lobe morphologies consistent with prostate inflammation and possibly neoplastic growth.
Mechanistically, steady-state, endogenous C3 expression, C3a generation, and C3aR receptor engagement in PECs maintains normal cell turnover through restraint of a PI3Kd-dependent MYC program upstream of ribosomal biogenesis. Collectively, these findings ascribe a non-traditional, tumor-suppressor function to prostate cell-intrinsic C3 and advocate for dissecting the context-specific roles of complement in malignancies to better inform on complement-targeted cancer therapies.
P-160
Investigating the Role of Neuronally-expressed C5aR1 in Motor Neuron Disease
Grace Risby-Jones, Trent M Woodruff, Jenny N Fung, John D Lee
School of Biomedical Sciences, University of Queensland, St Lucia, Australia
Motor Neuron Disease (MND) is characterised by the progressive degeneration of both upper and lower motor neurons within the central nervous system. Increasing evidence suggests that innate immunity, including the complement system, plays a significant role in MND pathogenesis. Studies have shown elevated complement activation fragments in MND patients and animal models, with C5a and its receptor, C5aR1, increasing as the disease progresses. Genetic deletion or pharmacological inhibition of C5aR1 using PMX205 has been shown to improve motor function, slow disease progression, and extend survival in MND models. Interestingly, we found that C5aR1 expression is detected on motor neurons across multiple species. However, the specific role of neuronal C5aR1 in disease pathology remains poorly understood. Evidence suggests that C5aR1 activation in cortical neurons may contribute to apoptosis and impair mitochondrial energy production, but studies directly investigating motor neurons are limited. Understanding these mechanisms may be critical for translating complement therapies to slow neurodegeneration and improve outcomes for individuals living with MND. This study aims to evaluate the role of C5aR1 in energy metabolism linked to cell death pathways in motor neurons. The NSC-34 motor neuron cell line, primary motor neuron cultures from SOD1G93A mice, and human iPSC-derived motor neurons from MND patients will be used. Motor neurons will be exposed to stressors including glutamate, hydrogen peroxide and aggregates of MND-associated proteins (TDP-3, SOD1, C9orf72), with C5a added as a co-treatment to assess its impact on cell death and mitochondrial function. Cell viability and apoptosis will be assessed alongside energy metabolism and mitochondrial membrane potential assays. The results from this study will provide valuable insights into how C5aR1 activation impacts apoptotic pathways and energy metabolism in motor neurons under stress conditions relevant to MND. Specific targeting of C5aR1 in motor neurons may represent a novel therapeutic approach for MND by directly modulating cell death and mitochondrial function.
P-161
A detrimental role for complement C5aR1 activation in Huntington’s disease
Jianina Marallag1, Jenny Fung1, John Lee1, Trent Woodruff2
1SBMS, University of Queensland, Brisbane, Australia. 2SBMS, University of Queensland, Brisbane, Australia
Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative disease, which causes progressive and irreversible loss of striatal neurons leading to cognitive and motor decline. Accumulating evidence indicates a link between neuroinflammation in accelerating the progression of HD. We have previously demonstrated that complement C5a-C5aR1 signaling is an important driver of motor neuron degeneration in motor neuron disease. Hence, the current study aimed to determine the role of C5aR1 in motor decline of HD using the selective and orally active C5aR1 antagonist PMX205 in the R6/1 transgenic mouse model of HD. The effect of PMX205 on motor deficits in R6/1 mice was determined using body weight, motor score and hind-limb grip strength. Inflammation was also measured in these animals by determining the degree of immune cell infiltration and cytokine expression in the spinal cord and skeletal muscles using immunohistochemistry and quantitative PCR at defined disease stages. R6/1 mice treated with PMX205 had significantly improved motor scores and hind-limb grip strength when compared with vehicle treated R6/1 mice. These improvements in the PMX205 treated group were associated with reductions in the infiltration of macrophages and pro-inflammatory cytokines in spinal cord and skeletal muscles. R6/1 mice crossed to C5aR1-/- mice also showed significant improvements in neuropathology, confirming a key role for C5a-C5aR1 signaling in disease development. These results confirm that C5aR1 activation produces a detrimental response in R6/1 mice by driving neuroinflammation and motor decline. The C5a – C5aR1 signaling axis may thus be a potential therapeutic target to slow disease progression in people living with HD.
P-162
Complement C3a receptor signalling mitigates motor neuron degeneration by modulating neurotoxic astrocytes
Alana R. Julianisya, Jenny N. T. Fung, Titaya Lerskiatiphanich, Trent M. Woodruff, John D. Lee
School of Biomedical Sciences, University of Queensland, Brisbane, Australia
Motor neuron disease (MND) is a fatal neurodegenerative disorder characterised by the progressive loss of upper and lower motor neurons. Dysregulation of the complement system, along with reactive glial activation is a hallmark of MND (1). Activation of the complement cascade through all pathways leads to the generation of C3a, a bioactive peptide that modulates inflammation via its receptor, C3aR. Our data suggests a neuroprotective role for C3aR in MND. Genetic ablation of C3aR in the SOD1G93A mouse model of MND resulted in accelerated disease progression, reduced survival, and impaired muscle strength. Furthermore, C3aR deficient SOD1G93A mice exhibited elevated levels of inflammatory cytokines and an upregulation of markers for astrocytes compared to C3aR-expressing SOD1G93A mice. Immunohistochemical analysis demonstrated astrocytic-specific expression of C3aR during early to mid-symptomatic stages in SOD1G93A mice. Given the proposed involvement of neurotoxic astrocytes in MND pathogenesis (2), we next explored the functional role of C3a in astrocyte activation. Primary and immortalised mouse astrocytes were treated with the immune stimulator lipopolysaccharide or MND-associated TDP-3Q331K protein aggregates, in the presence or absence of recombinant mouse C3a. qRT-PCR analysis showed that C3a treatment suppressed the expression of neurotoxic astrocyte genes in immune-primed astrocytes. Furthermore, quantification of neurotoxic astrocyte markers in the spinal cord of WT, C3aR-/-, SOD1G93A, and SOD1G93A x C3aR-/- mice revealed that C3aR deletion in SOD1G93A mice was associated with accelerated and widespread induction of neurotoxic astrocyte, supporting an in-vivo role for C3aR in modulating astrocyte reactivity. Finally, bulk RNA sequencing of spinal cord tissue from SOD1G93A x C3aR-/- mice identified key neuroactive ligand-receptor pathways implicated in disease progression. Together, our results demonstrate a protective role for the complement C3a-C3aR axis in MND, potentially through the suppression of neurotoxic astrocyte induction, and highlight C3aR activation as a potential therapeutic strategy to mitigate astrocyte-mediated neurodegeneration.
References
(1) Lee, J.D., et al., Dysregulation of the complement cascade in the hSOD1G93A transgenic mouse model of amyotrophic lateral sclerosis. J Neuroinflammation, 2013. (2) Guttenplan, K.A., et al., Knockout of reactive astrocyte activating factors slows disease progression in an ALS mouse model. Nature Communications, 2020.
P-163
CD93: A New Complement-Linked Target for Immunomodulation and wound healing in SCI
Melina Haritopoulou-Sinanidou, Mia I. W. Shapland, Hong Wa Lao, Laura F. Grice, Marc Ruitenberg
The University of Queensland, Brisbane, Australia
Background: CD93, also known as C1qRp, is a transmembrane protein historically implicated in endothelial cell function and angiogenesis. Emerging evidence now also supports a broader role for CD93 in immune regulation and ECM remodeling. Interestingly, CD93 has been associated with both protective and pathological responses, with this duality seemingly dependent on the disease context. We identified CD93 as being upregulated in traumatic spinal cord injury (SCI), a condition marked by chronic dysregulated inflammation and scarring, but its role in this pathology remains poorly understood.
Methods: We performed single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining to map CD93 expression at various time points (up to 42 days) after contusive SCI in mice. We used cell marker genes and/or antibody approaches, respectively, to identify the various cell types expressing CD93.
Results and Conclusion: We found that CD93 is robustly expressed at the site of SCI in association with both blood vessels and myeloid cells, including subsets of neutrophils, monocytes and/or monocyte-derived macrophages, and microglia. These findings provide a basis to further explore a functional role for CD93 in relation to neuroinflammation and spinal cord wound healing after injury.
References
This work is supported by a Project Grant (WFL-AU-14/25) and fellowship (to LFG; WFL-AU-04/22) from the Wings for Life Spinal Cord Research Foundation, and SpinalCure Australia.
P-164
Macrophage-Derived Complement Factor H Restrains Kidney Inflammation and Fibrosis in Immune Complex-Mediated Disease.
Jessy Alexander
University at Buffalo, Buffalo, USA
Complement factor H (FH) is a key regulator of the alternative pathway, limiting complement activation by negatively controlling C3 consumption. Deficiency or dysfunction of FH is strongly associated with kidney disease, where monocytes and macrophages are critical contributors. Notably, FH is expressed by these myeloid cells and regulates cell-intrinsic complement activity to maintain local immune homeostasis. Given the heterogeneity of macrophages and their context-dependent roles, we investigated the specific contribution of macrophage-derived FH in kidney pathology and fibrosis. We generated FloxFH (FHfl/fl) mice and bred them with CMVcre, CX3CR1cre, and Csfr1cre strains to produce conditional FH knockout (cFHKO) mice: systemic FH deficiency (FHfl/flCMVcre), and macrophage-specific FH deletion (FHfl/flCX3CR1cre and FHfl/flCsfr1cre). Mice were subjected to chronic serum sickness (CSS) using horse spleen apoferritin to induce immune complex-mediated kidney injury. FHfl/flCMVcre mice displayed significant glomerular immune complex deposition compared to controls. Strikingly, macrophage-specific cFHKO mice (FHfl/fCX3CR1cre and FHfl/flCsfr1cre) developed kidney dysfunction and immune complex accumulation, with FHfl/flCX3CR1cre mice recapitulating features seen in global FH deficiency. Furthermore, elevated renal expression of TGF-β and laminin indicated enhanced fibrotic responses. These findings establish, for the first time, that macrophage-derived FH plays a pivotal role in modulating complement activation, controlling inflammation, and preventing fibrosis in immune complex-driven kidney disease.
P-165
Effects of Cannabinoid receptor-1 Antagonist MRI-1867 on C5ar1 Signaling in Attenuating Lung Remodeling in Mice Induced with Experimental Arthritis
Haydn E. Rich1, Aleksey Y. Domozhirov2, Yi Dong Li2, Stacey L. Mueller-Ortiz2, Hunter G. Devaughn1, Lavanya Gunamalai3, Sunil K. Tripathi4, Nandakumar Natarajan5, Kai-Lieh Huang6, Madeline Jensen6, Hari Krishna Yalamanchili7, Tingting Weng-Mills8, Marie-Francoise Doursout9, Eric J. Wagner6, Malliga R. Iyer10, Pooja Shivshankar1,2
1Center for Metabolic and Degenerative Diseases, Institute of Molecular Medicine, UTHealth-McGovern Medical School, Houston, USA. 2Hans J. Müller-Eberhard and Irma Gigli Center for Immunology and Autoimmune Diseases, Institute of Molecular Medicine, UTHealth-McGovern Medical School, Houston, USA. 3Center for Cardiovascular Genetics, Institute of Molecular Medicine, UTHealth-McGovern Medical School, Houston, USA. 4Department of Chemistry, Georgia State University, Atlanta, USA. 5Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, USA. 6Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, USA. 7Department of Pediatrics Neurology, Baylor College of Medicine, Houston, USA. 8Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, USA. 9Department of Anesthesiology, University of Texas Health Science Center at Houston, Houston, USA. 10Section on Medical Chemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, USA
Rheumatoid arthritis-associated interstitial lung disease is an extra-articular manifestation of rheumatoid arthritis (RA) with significantly increased mortality. Research in recent years has investigated the role of complement dysregulation in disease pathogenesis and progression, particularly examining anaphylatoxin C5a. As standard disease-modifying treatments for RA may exacerbate existing ILD, we previously examined the protective activity of novel cannabinoid receptor antagonist MRI-1867 in mice with collagen-induced arthritis (CIA) (1). Here, we hypothesized that the protective effect of MRI-1867 may involve direct interaction and sequestration of C5aR1 signaling.
We performed in-silico studies to understand the binding and interaction of MRI-1867 with human and mouse C5aR1 and C5aR2 using AutoDock 4.2.6. Discovery Visualizer 3.1 Studio tools. Our results demonstrated the best binding conformation of MRI-1867 with human C5aR1, wherein MRI-1867 undergoes hydrogen bonding with Trp213. MRI-1867 also formed hydrogen bonds with key residues of human C5aR1, with TRP213, LEU209, and GLY162 most represented. More importantly, residue ARG175 in both human and mouse C5aR1 commonly binds with MRI-1867, with mouse C5ar1 forming two hydrogen bonds at ARG175. NCBI blast shows widespread similarities between human and mouse C5aR1, including nucleotide position of ARG175 and structural similarity pertinent to MRI-1867 binding.
We further studied MRI-1867 interaction with C5aR1 using b-arrestin-2 recruitment assay using HTLA cells (a HEK293 cell line stably expressing tTA (tetracycline transactivator)-dependent luciferase reporter and β-arrestin2-TEV-fusion gene) transfected with TANGO-C5aR1. 24h post-transfection, cells were transferred to flat-bottomed 96-well plates (~25,000 cells per well) and stimulated with C5a (100nM) with or without MRI-1867 (40nM) in 10mM HEPES buffer for 3 hours and luminescence was measured using BioTek Cytation 5 Cell Imaging Multimode Reader. Relative luminescence was obtained by subtracting base values collected prior to adding Bright-Glo solution for all experimental groups tested. We observed significantly increased luminescence in MRI-1867+C5a group, suggesting MRI-1867-mediated internalization of C5aR1 via enhanced b-arrestin-2 recruitment. In vivo, MRI-1867 treated WT-CIA mice showed significant downregulation of tissue remodeling mediators, including Notch4, Jcad, and Qars expression and the phenotype was comparable to double C5a-receptor knockouts (C5ar1/C5ar2-DKOs), suggesting that MRI-1867 may block both C5a binding receptors and therefore attenuate CIA-induced lung remodeling.
Rich HE, et al. Eur. J. Immunol. 2024 Sep 2-6; 54: 2470300. https://doi.org/10.1002/eji.202470300 This work was supported by the NIH to P.S. (R01-AI158694) and the National Institute on Alcohol Abuse and Alcoholism to M.R.I (ZIA AA000360-03). We acknowledge former chair Dr. Rick Wetsel for financial support (R01-AI025011) and Chair funds.
P-166
TiO2 nanoparticles act as an Alternative Pathway-specific C3 inhibitor
David Eikrem1, Jacob Whittaker2, Mats Sandgren2, Karin Fromell1, Gulaim Seisenbaeva2, Kristina Nilsson-Ekdahl1,3, Bo Nilsson1, Vadim Kessler2
1Uppsala University, Uppsala, Sweden. 2Swedish University of Agricultural Sciences, Uppsala, Sweden. 3Linnaeus University, Kalmar, Sweden
Background
The human complement system is bombarded with human-made foreign particles on a daily basis. Titanium oxide nanoparticles are one such foreign material which have now found their way into several aspects of everyday life, including within implanted surgical and dental devices, foods and cosmetics. Despite their apparent benefits, very little insight is available into the mechanisms behind their beneficial action, with safety information remaining quite limited. Coupling structural biology with biochemical techniques, we uncover the direct binding of small (3 nm) TiO₂ nanoparticles to the surface of C3, leading to structural restrictions that hinder its activation and subsequent complement cascade progression.
Methods
A suite of biophysical assays were used (DLS, nanoDSF) to first confirm that a meaningful interaction occurs between the two species over a range of environments and time. Native C3 and TiO2 nanoparticles were then incubated before being applied to a TEM grid for cryo-EM analysis. The effect of the TiO2 nanoparticles on the alternative pathway was assessed through the cleavage of factor B determined by capillary electrophoresis and rabbit red blood cell haemolysis.
Results
Careful sample alignment and reconstruction of the best 2D classes produced a density map which showed the direct interaction between C3 and a single nanoparticle at the MG7 domain. This site is reported by others to bind tailored nanobodies and appears to be important for the native motion experienced by C3. Importantly, our biochemical results show that the formation of the C3 convertase is inhibited as the TiO2 nanoparticles block the binding site necessary for further complement cascade events to proceed highlighted by deterred factor B cleavage and inhibited haemolysis.
Conclusion
We have uncovered the first protein-nanoparticle interaction responsible for the systemic downregulation of C3 and subsequent complement byproducts. Our results provide the foundation for the design and engineering of oxide nanoparticles as bioactive nanomaterials capable of immunomodulation. We hypothesise that our new findings may potentially be used in the future for improved biocompatibility of organ implants.
References
Gadeberg, T. A. F.; Jorgensen, M. H.; Olesen, H. G.; Lorentzen, J.; Harwood, S. L.; AlmeiGadeberg and Jørgensen et al. Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring. Nat Struct Mol Biol 2025, 32 (5), 884-895. DOI: 10.1038/s41594-024-01467-4.
P-167
CIMED: A High-Serum Concentration Diagnostic Platform for Complement Pathway Activity and Drug Response Monitoring in Patients
Sadam Yaseen1, Peter Garred2, Youssif Ali3, Wilhelm Schwaeble3
1Compvide Inc, Seattle, USA. 2University of Copenhagen, Copenhagen, Denmark. 3University of Cambridge, Cambridge, United Kingdom
Background
Accurate assessment of complement activity in patients undergoing complement inhibitor therapy is critical for guiding treatment decisions and supporting drug development. Conventional complement assays typically require substantial serum dilution (1–6%), which disrupts native protein interactions and destabilizes drug–target complexes. This not only produces false-positive and false-negative results in clinical monitoring but also reduces the physiological relevance of drug screening and IC₅₀ determination during preclinical development. To address these limitations, we present CIMED—a novel, high-serum (40–50%) assay platform that preserves in vivo protein-binding dynamics and enables precise monitoring of complement activity and drug efficacy.
Methods
CIMED includes specialized buffer systems for the classical (CP), lectin (LP) and alternative (AP) pathways, allowing robust complement activation in minimally diluted human serum. ELISA-based assays were performed using pathway-specific surfaces, followed by detection of complement activation products (C3, C4, C5b-9) in the presence or absence of known inhibitors.
Results
The CIMED CP test yielded enhanced C4 deposition in 50% serum and quantifiable IC₅₀ values with C1s inhibition. For LP, the CIMED test enhanced Ficolin-3/2 binding and produced clean, Futhan-sensitive activation profiles, again at 50% serum. The AP test enabled strong, AP-specific C3 activation in 20–40% serum with complete inhibition of CP/LP, confirmed by the absence of C4 deposition. Iptacopan (LNP023) inhibition was dose-dependent and reproducible, demonstrating CIMED’s suitability for pharmacodynamic profiling.
Conclusion
CIMED offers a transformative solution for complement diagnostics and therapeutic evaluation by maintaining near-physiological serum conditions. This enables:
More accurate clinical monitoring of complement inhibitor efficacy
Robust IC₅₀-based profiling for drug development
Potential translation to point-of-care formats using capillary blood (e.g., finger-stick samples)
By overcoming core limitations of traditional assays, CIMED supports personalized medicine and advances in complement-targeted therapy.
References
Zelek WM et al. Immunobiology. 2018;223(12):744–749. Seelen M et al J Immunol Methods. 2005 Jan;296(1-2):187-98
P-168
Point-of-Care Assessment of sC5b-9: A Reliable Quantitative Tool for Rapid Detection of Complement Activation
Tam Cung1,2,3, Søren Pischke1,2,3, Bettina Mülleder4, Loek Willems5, Camilla Schjalm1, Max Sonnleitner4, Erik J.M. Toonen5, Tom Eirik Mollnes1,6, Andreas Barratt-Due1,3
1Department of Immunology, Oslo University Hospital and University of Oslo, Oslo, Norway. 2Department of Research and Development, Division of Emergencies and Critical Care, Oslo University Hospital, Oslo, Norway. 3Department of Anesthesia and Intensive Care Medicine, Oslo University Hospital, Oslo, Norway. 4GENSPEED Biotech GmbH, Rainbach im Mühlkreis, Austria. 5Research and Development Department, Hycult Biotechnology, Uden, Netherlands. 6Research Laboratory, Nordland Hospital, Bodø, Norway
Background
Rapid identification of complement activation in critically ill patients remains a diagnostic challenge, hindering the prompt recognition and management of complement-driven diseases. The growing use of complement inhibitors underscores the urgent need for rapid, reliable tools to detect complement activation and guide early treatment. We hypothesized that rapid point-of-care (PoC) quantification of the complement activation product soluble C5b-9 (sC5b-9) can provide diagnostic accuracy comparable to the gold-standard enzyme-linked immunosorbent assay (ELISA).
Methods
Plasma-EDTA samples were obtained at admission and on day 2-4 from 38 critically ill intensive care unit (ICU) patients with suspected complement activation. Fifteen age- and sex-matched healthy individuals served as negative controls, while three patients undergoing cardiopulmonary bypass were included as positive controls. Plasma sC5b-9 was measured using a newly developed PoC assay, built on the Genspeed microELISA platform incorporating highly specific antibodies provided by Hycult. PoC assay results were benchmarked against our in-house sC5b-9 ELISA and the Hycult sC5b-9 ELISA.
Results
Patients with critical conditions such as cardiac arrest, subarachnoid hemorrhage, systemic inflammation or sepsis were included, with an ICU mortality rate of 31.7%. Seven patients died before day 2-4 sampling. PoC assay time was 45 minutes and revealed increased sC5b-9 levels in positive controls (median 8035, IQR 2951-8598 mAU/ml) and low levels in healthy controls (median 1286, IQR 1032–1903 mAU/ml). sC5b-9 levels were significantly elevated in patients, irrespective of underlying diagnosis, compared to healthy controls - both at admission (median 3572, IQR 1946–6504 mAU/ml; p < 0.0001) and on day 2-4 (median 3554, IQR 2130-5081 mAU/ml; p < 0.0001). Non-parametric Spearman analyses demonstrated strong correlations between sC5b-9 quantification by PoC, the in-house ELISA (r ≈ 0.82, 95% CI 0.74–0.88), and the Hycult ELISA (r ≈ 0.87, 95% CI 0.80–0.91).
Conclusion
The novel PoC assay enables rapid bedside quantification of sC5b-9 with accuracy comparable to gold-standard ELISAs, allowing early detection of complement activation. Complement activation was evident across a spectrum of clinical diagnoses, emphasizing the broad potential for bedside testing. PoC sC5b-9 assays may facilitate earlier, targeted complement inhibition therapy in a range of critical conditions.
P-169
Evolutionary Insights into the Complement System: Functional Diversification and Ancestral Mechanisms in Bony Fish
Miki Nakao, Akhil Kizhakkumpat, Takahiro Nagasawa, Tomonori Somamoto
Kyushu University, Fukuoka, Japan
Bony fishes represent one of the most primitive vertebrate groups possessing adaptive immunity, utilizing immunoglobulins and T-cell receptors as antigen receptors. They are also the earliest lineage known to harbor the classical pathway (CP) of the complement system, developed alongside the emergence of antibodies. A notable feature of the bony fish complement system is the extensive gene duplication of complement components, leading to functional diversification among isoforms.
This study focused on elucidating the functional differences among isoforms of complement component C3 and factor B (Bf) in C3-convertase formation in the common carp (Cyprinus carpio). We found that a non-typical C3 isoform (C3-S), which contains a non-catalytic thioester, and a novel Bf isoform (Bf-3) can form a C3-convertase independently of factor D (Df). In contrast, other combinations of isoforms (C3-H1/C3-S and Bf-1/Bf-3) required Df for convertase activity. Phylogenetically, the Df gene appears to have emerged in bony fishes. In more basal vertebrates—such as cartilaginous fishes and jawless agnathans—as well as in invertebrates, both C3 and Bf are present as components of the alternative complement pathway (AP), but no ortholog of Df, the protease that activates Bf into Bb, has been identified. Our discovery of Df-independent C3-convertase activity in carp may reflect an ancestral mechanism of AP activation.
Additionally, depletion of Df from normal carp serum abolished hemolytic activity mediated by the antibody-dependent CP, while still allowing C3b-deposition on target cells (i.e., antibody-sensitized sheep erythrocytes, or EA). These C3b-tagged EA were efficiently lysed by normal carp serum under EGTA/Mg²⁺ conditions, which permit only AP activation. This suggests that the bony fish CP may not independently generate C5-convertase, instead serving primarily to tag pathogens for further AP-mediated attack.
Collectively, these findings imply that the CP in bony fishes may have immature effector functions, underscoring the evolutionary significance of the AP in the early complement system of vertebrates.
References
Technical assistance was provided by Dr. Makiko Nakahara and Mr. Kentaro Arase.
P-170
Classical and Lectin Pathway Activation in ANCA-Associated Vasculitis with Kidney Involvement: Prognostic Value and Comparison with Alternative Pathway Components
Laura Lucientes Continente1, Alejandro Chozas Hernández2, Evelyn Cobos Paladines3, Lina León Machado3, Gema Fernández-Juarez3, Elena Goicoechea de Jorge2
1Universidad Complutense de Madrid, Madrid, Spain. 2Center for Biological Research Margarita Salas, Madrid, Spain. 3Department of Nephrology, Hospital Universitario La Paz, Instituto de Investigación de la Paz (IdIPAZ), Madrid, Spain
Background
Dysregulation of the complement alternative pathway (AP) is a known major contributor to ANCA-associated vasculitis (AAV), impacting prognosis and kidney disease severity [1]. Recent findings also suggest activation of the classical (CP) and/or lectin (LP) pathways in AAV, as indicated by plasmatic C4d levels correlating with disease activity [2]. Our study aimed to further investigate CP and LP activation in a well-characterized Spanish cohort of AAV patients with kidney involvement [1]. We sought to evaluate their potential as biomarkers for prognosis, disease activity, and kidney disease severity, comparing them to other complement parameters.
Methods
We measured levels of C1s/C1-INH and MASP-1/C1-INH complexes via ELISA as early markers of CP and LP activation in AAV and control plasma samples. We also analysed levels of C3, C4, sC5b-9, FB, properdin, FH, and FHRs. Pearson's and Spearman's rho correlations, along with ROC analysis, were performed on all components.
Results
Consistent with previous findings for AP components, samples from patients with active disease showed significantly higher levels of C1s/C1-INH and MASP-1/C1-INH compared to controls. Interestingly, only C1s/C1-INH levels significantly decreased in longitudinal samples from patients achieving remission. ROC analysis of measurements taken at diagnosis revealed that FH levels were the most effective in discriminating between patients who would achieve remission and those who would not, outperforming even proteinuria and serum creatinine levels. Kidney disease severity, as assessed by the Berden classification and CD163 urinary levels, was best determined by the plasma FH/FHR-1 ratio and urinary C5b-9 levels. In longitudinal studies, urinary sCD163 levels proved to be the best discriminator between active and remission samples, followed by plasma FH, eGFR, serum creatinine, and plasma C3 levels.
Conclusion
Despite clear evidence of CP and LP activation in active AAV, levels of C1s/C1-INH and MASP-1/C1-INH complexes did not correlate with disease prognosis, activity, or kidney damage severity, unlike AP components. This suggests that while all complement pathways may be activated in AAV, the degree of AP dysregulation is the primary driver of disease outcome and therefore represents the most promising therapeutic target.
References
(1) Lucientes-Continente et al. 2024. Kidney International, 105(1), 177–188. (2) Juto et al. 2025. Arthritis Res Ther. 5;27(1):49.
P-171
Rescue Therapy with Pegcetacoplan in a Patient with C3 Glomerulopathy: A Case Report
Hormaz Dastoor1, Emad Khater1, Suhail Al Salam2, Stephen Holt1
1SEHA - Kidney Care, SEHA Abu Dhabi Health Services Ltd (a PureHealth company), AbuDhabi, UAE. 2Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE
Background: C3 glomerulopathy (C3G) is a rare, complement-mediated renal disease characterised by predominant glomerular C3 deposition due to dysregulation of the alternative complement pathway. The condition often leads to progressive kidney damage, with up to 50% of adult patients developing kidney failure within 10 years. Conventional immunosuppressive therapies such as corticosteroids and mycophenolate mofetil offer limited efficacy. Pegcetacoplan, a novel C3 inhibitor, has demonstrated promising results in recent clinical trials. However, these trials excluded patients with advanced kidney dysfunction (eGFR <30 mL/min/1.73 m²), leaving a critical gap in evidence for this high-risk population.
Methods: We report the clinical course of a 50-year-old male diagnosed with biopsy-proven C3G, presenting with nephrotic-range proteinuria, acute kidney injury (serum creatinine 240 μmol/L), and hypocomplementemia. Initial treatment included corticosteroids, mycophenolate mofetil, and renin-angiotensin system inhibitors, resulting in partial biochemical and histological improvement. Repeat biopsy showed reduced C3 staining, with mild residual IgG and C1q deposition. However, kidney function declined significantly over the subsequent months, with eGFR dropping to 13 mL/min/1.73 m² and proteinuria worsening to 5 g/g. The patient was initiated on pegcetacoplan following failure of conventional therapy.
Results: Pegcetacoplan led to marked renal recovery. Within six weeks, serum creatinine decreased to 196 μmol/L, eGFR improved to 34 mL/min/1.73 m², and proteinuria declined to 0.86–1.6 g/g. By two months, creatinine further improved to 146 μmol/L and eGFR to 47 mL/min/1.73 m². Complement levels normalised (C3: 4.45 g/L; C4: 0.42 g/L). The therapy was well tolerated, with no infectious or systemic complications reported. Notably, these improvements occurred despite the withdrawal of SGLT2 inhibitors, suggesting the clinical benefit was directly attributable to complement inhibition.
Conclusion: This case provides real-world evidence supporting the efficacy and safety of pegcetacoplan in a patient with advanced C3G and severely reduced kidney function. The observed reversal of renal decline underscores the potential for complement-targeted therapy in late-stage disease. These findings advocate for broader inclusion criteria in future trials and further studies to validate pegcetacoplan’s role in patients with eGFR <30 mL/min/1.73 m².
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