Session 5 - Complement in metabolic disorders
Background: Non-alcoholic fatty liver disease (NAFLD) is a growing threat to global public health, affecting approximately 30% of adults and becoming the leading reason for liver transplantation. Nevertheless, no approved specific treatment is currently available for NAFLD. Accumulating evidence suggests an important role of the complement system in causation of NAFLD. As the axial component of the complement system, C3 is predominantly produced in the liver, the metabolic ‘engine’ of the body; however, its role in metabolism remains little known.
Methods: In this study, we have carried out a comprehensive characterisation of C3 spatial expression in normal and NAFLD human liver using single cell and bulk RNA sequencing data. These findings were validated in free fatty acid-induced steatotic human hepatocytes and a high fat diet-induced mouse NAFLD model.
Results: We found that C3 was expressed in a zonal manner, with abundant expression in both periportal and pericentral hepatocytes. Cellular deconvolution of NAFLD liver showed a disruption of hepatic zonal structure, with a depletion of pericentral hepatocytes while periportal hepatocytes were expanded. This alteration of hepatic zonal distribution was disease severity dependent with involvement of liver progenitor activation. RNA sequencing data from 408 liver biopsies confirmed significant alterations of complement C3 expression in response to NAFLD development. C3 was upregulated in all forms of NAFLD and positively correlated with liver fat load in patients with steatohepatitis. C3 expression was increased in steatotic human hepatocytes and hepatic C3 expression was higher in a mouse NAFLD model. C3 knockout protected against high fat diet induced hepatic lipid droplet accumulation. In the hepatic response to a high fat diet, C3 knockout repressed hepatic de novo lipogenesis and triglyceride synthesis through downregulating liver lipogenic regulators.
Conclusion: We demonstrate the impact of lipid accumulation on C3 expression in hepatocytes and show that C3 knockout impacts hepatic lipid handling. Overall, our study provides new insights into the role of C3 in NAFLD pathogenesis and suggests its potential as a therapeutic target for the treatment of NAFLD.
Objectives: CD59 is known as a membrane regulator of the complement system and inhibits the final step of membrane attack complex (MAC) formation to protect host cells from MAC-mediated injury. Noncanonical function was suggested for CD59 in relation to insulin secretion. Here we investigate its possible noncanonical function in developing insulin resistance and non-alcoholic fatty liver disease (NAFLD).
Methods: The study population was comprised of male C57BL/6 mice, aged 2 months, divided into 4 groups: CD59+/+ high fat diet (HFD), CD59-/- HFD, CD59+/+ standard chow diet (SD) and CD59-/- SD. Mice had unrestricted access for 16 weeks to a pelleted HFD [45%kcal Fat Diet (21% MF, 2% SBO)] or a SD. Intraperitoneal glucose tolerance test (IPGTT), insulin tolerance test (ITT), pyruvate assays, and insulin, glucagon, and blood chemistry measurements were performed every 4 weeks during the HFD. Western blots of the insulin signaling pathway were performed. PET-MRI was performed. Tissues of all mice were extracted at the end of the experiment for further investigations.
Results: CD59-/- HFD mice gained significantly less weight than CD59+/+ HFD mice. In metabolic cages, we found no significant differences in food intake, activity, or spirometry between the 4 groups, suggesting that weight gain did not result from these factors. IPGTT, ITT, and Western Blot of the insulin signaling pathway showed that absence of CD59 improved glucose intolerance and significantly reduced insulin resistance. Pyruvate assay indicated that CD59+/+ HFD mice had augmented gluconeogenesis compared to CD59-/- HFD mice. Moreover, the HFD resulted in fatty liver in the CD59+/+ HFD group but not the CD59-/- HFD group. In CD59+/+ HFD mice we observed weight increase, lipid droplets in the liver, elevated cholesterol, lipids and SGPT in blood serum, and more infiltration of macrophages in liver and muscle tissues.
Conclusion: A novel unpredicted tole for CD59 was found in insulin resistance, glucose intolerance and NAFLD.
Complement component 3 and C5 fragments C3a and C5a are anaphylatoxins involved in promoting cellular responses important in immune response and host defense. Its receptors (C3a/C5a receptors, C3aR and C5aR) are distributed on the plasma membrane; however, intracellular localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and complement activation in mitochondrial dysfunction in retinal pigment epithelial (RPE) cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. Focusing on plasma membrane C5aR, C5a stimulation was found to control fission/fusion state of mitochondria, leading to fusion when presented to control cells, and excess fission when presented to oxidatively stressed cells. While overall C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial calcium uptake, and inhibited mitochondrial respiration; mitochondria from control cells did not respond to C3a. Our findings suggest that oxidative stress increases C5aR-dependent fission as well as mtC3aR transfer, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.
Background: In type 1 and type 2 diabetes, pro-inflammatory cytokines are produced in pancreatic islets and contribute to loss of function and death of insulin-secreting beta-cells. Although traditionally seen as a secreted protein, we have reported the presence of C3 within the cytosol, where it interacts with cytosolic partners such as ATG16L1, in regulation of beta-cell autophagy. We now investigated pro-survival functions of C3 in beta-cells, focusing on cytosolic C3.
Methods: We used primary rodent and human pancreatic islets, and CRISPR/Cas9 gene-edited clonal beta cells to investigate the roles of C3 expression, and cytosolic C3 in particular, in beta-cell survival under cytokine exposure. We also generated a beta-cell specific C3 knockout mouse to study beta-cell function and survival in vivo.
Results: Loss of C3 expression results in increased levels of apoptosis in beta-cells exposed to the diabetogenic cytokine IL-1beta. Although both C3 and factor B expression are significantly increased in diabetic pancreatic islets from humans and rodent models, we found no evidence for alternative pathway activation in beta-cell survival; addition of exogenous C3 or C3a did not rescue C3-knockout cells from increased apoptosis, and C3aR KO also had no effect on survival. We therefore investigated non-canonical functions of C3. By protein microarray and proximity ligation assay, we identified an interaction between C3 and Fyn-Related Kinase (FRK), which mediates cytokine-related apoptosis in beta-cells. Consistent with the known function of FRK, clonal beta-cells lacking C3 expression, but those with cytosolic C3 expression, had increased levels of PTEN and pro-apoptotic phospho-cJun, and decreased levels of phosphorylated AKT, which mediates beta-cell survival. Finally, beta-cell specific C3-KO mice, which have normal levels of serum C3, were more sensitive to diabetes induction by streptozotocin, consistent with published results from beta-cell specific FRK- or PTEN-KO mice, and consistent with a protective role of beta-cell intrinsic C3.
Conclusions: Cytosolic C3 interacts with and inhibits the activity of FRK in beta-cells, thereby limiting cytokine-induced cytotoxicity. C3 upregulation in diabetic islets may therefore have a role in preserving beta-cell function. The findings suggest that cytosolic C3 may represent a therapeutic target for preserving beta-cell function and improving outcomes in diabetes.
Introduction to Hans Müller-Eberhard award
Hans Müller-Eberhard Award Lecture: Complement and lupus – lessons from the past and new insights for the future
Session 6 - Complement in kidney disease
Background: IgA nephropathy (IgAN) is the commonest primary glomerular nephritis in the world, with glomerular IgA and C3 deposition, inducing mesangial cell proliferation and inflammatory injury. Complement factor H (CFH), a key regulator of alternative pathways, was reported as an important factor for the development and progression of IgAN, mainly focused on the complement inhibition function of circulating factor H. Recent studies reported non-canonical function of CFH in both retinal pigment epithelium and kidney endothelial cells[1, 2]. Here, we investigate the potential role of mesangial cells derived CFH in IgAN.
Methods: CFH expression at mRNA and protein levels were firstly detected in primary human glomerular mesangial cells (pHGMC) treated with or without IgA1-containing immune complexes derived from IgAN patients (IgAN-IgA1-IC). Then, inflammatory factors secretion, cell proliferation and complement activation were evaluated under modified expression of CFH in pHGMC, including siRNA targeting CFH and overexpression of CFH.
Results: Expression of CFH at mRNA and protein levels were observed in pHGMC, and IgAN-IgA1-IC decreased CFH expression. In vitro, we found C3c and C5b-9 deposition and increased IL-6 secretion in pHGMC after IgAN-IgA1-IC treatment. Moreover, we found that inhibited production of CFH using siRNA in pHGMC led to increased secretion of IL-6 (P<0.001), deposition of C3c (P<0.001) and C5b-9 (P<0.001), and decreased cell proliferation (P=0.001), while overexpression of CFH in pHGMC decreased IL-6 secretion, C3c and C5b-9 deposition, and promoted cell proliferation.
Conclusion: Glomerular mesangial cells derived CFH contributes to renal complement activation, as well as mesangial cell proliferation and inflammation in IgA nephropathy.
1. Mahajan S, Jacob A, Kelkar A, et al: Local complement factor H protects kidney endothelial cell structure and function. Kidney Int 2021, 100(4):824-836.
2. Armento A, Schmidt TL, Sonntag I, et al: CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-kappaB Pathway. Int J Mol Sci 2021, 22(16).
IgA Nephropathy (IgAN) is the most common form of primary glomerulonephritis world-wide. Patients develop chronic kidney disease and up to 30% advance to end stage renal failure (ESRF). IgAN pathophysiology remains poorly understood. However, the lectin and alternative pathways of the complement system have been implicated. Indeed, altered Factor H (FH) and FH related protein 1 (FHR1) levels have been shown to associate with disease severity. Given the immune predisposition in IgAN, we decided to assay for the presence of autoantibodies to complement proteins (FH, FB, etc) and for altered expression levels of FHR proteins.
EDTA plasma samples were obtained from healthy blood donors (~200) via the Newcastle blood donor service and NRCTC. Over 500 biopsy-proven IgAN patient plasma samples were obtained from the Glomerulonephritis DNA Bank (UKGDB). Patients were classified as either non-progressors (with stable serum creatinine levels) or progressors (where serum creatinine doubled or patients reached ESRF) within 10 years of diagnosis. The standardised anti-FH autoantibody ELISA protocol was modified to test for anti-complement protein autoantibodies, and in house assays developed to measure FH and FHRs.
Approximately, 5% of IgAN samples were positive for anti-FH autoantibodies (> 100RU) and 1% had detectable Factor B autoantibodies but no significant anti-C3b reactivity was detected in the IgAN samples. Strikingly, mean FHR4 serum concentration was 2.3-fold greater in a cross section of the IgAN population (2.205±1.393µg/mL) compared to controls (1.248±0.639µg/mL), p<0.0001. Interestingly, samples from patients deemed to be progressors, had significantly higher levels of FHR4, compared to non-progressors (p=0.0009). As well as this, FH/FHR4 molar ratios were significantly decreased compared to healthy samples (progressors (p=0.0076) and non-progressors (p=0.0089)). FHR4 levels did not correlate with age and gender.
Our research into the reasons why the alternative pathway may be dysregulated in IgAN has identified the presence of anti-complement protein autoantibodies (although generally at a relatively low titre) and a suggestion of skews in the FH/FHR4 ratio. Strikingly, FHR4 levels were significantly increased in progressors compared to non-progressors, suggesting FHR4 may provide some utility as a potential biomarker when determining patient risk status, although expansion and replication of this work is needed.
C3 glomerulopathy (C3G) is caused by a dysregulation of the complement system leading to C3 deposition and formation of glomerular deposits. Several C3G patients harbor mutations or copy number variations in the human Factor H (FH) and/or Factor H-Related (FHRs) genes. Therefore, FH and FHRs are emerging immune targets for inhibition of the complement cascade, as well as markers to monitor patients on complement regulatory drugs to test their efficiency. Here, we focused our study on FHR2, known to inhibit in vitro formation of the terminal complement complex. We identified new variants for the FHR2 gene in a cohort of C3G patients and performed detailed functional studies on the novel variant FHR2L46, which has the Pro at position 46 replaced by Leu. Patients with FHR2L46 variant presented increased FHR2 plasma level, as compared to controls and displayed FHR2 deposits in glomeruli. We generated a recombinant FHR2L46mutant protein to gain insight into the effect of this novel FHR2 variant on complement regulation. As the amino acid exchange occurred in the first short consensus repeat (SCR1), we first tested if the Leu at position 46 altered FHR2 homodimerization and heterodimerization of FHR2 with FHR1 and FHR5. We observed that FHR2L46 binds significantly less to FHR2 and FHR1 but more to FHR5. Furthermore, FHR246L acquired the capacity to bind to cell surfaces by interacting with glycosaminoglycans heparin and malondialdehyde (MDA)-modified amino group (MAA) epitopes. FHR2L46 also bound substantially more to necrotic cells compared to wild-type FHR2 (FHR2WT). In contrast, no difference was observed between FHR246L and FHR2 WT binding to C3 and C5. Taken together, the present study identified a novel FHR246L variant in a C3G patient and suggests that the FHR2L46 mutant forms stable oligomers with FHR5 and enhances complement activation.
Background: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small vessel vasculitis affecting multiple organ systems, including the kidney. The activation of the complement system contributes essentially to its pathogenesis by autoantibody-antigen recognition directed against host cells in ANCA-associated renal vasculitis. We herein provide evidence for intrarenal synthesis of complement C3 localized to distinct vascular compartments in ANCA-associated renal vasculitis that associated with distinct inflammatory signaling pathways.
Methods: A total number of 43 kidney biopsies with ANCA-associated renal vasculitis were retrospectively included and evaluated for presence/absence of C3 deposits localized to distinct vascular compartments in association with clinicopathological biopsy findings. In addition, intrarenal C3 mRNA expression levels specifically from microdissected tubulointerstitial and glomerular compartments were extracted from transcriptome datasets.
Results: C3 deposits were present in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules in ANCA-associated renal vasculitis. Most C3 deposits are localized to the glomerular tuft overlapping with peritubular capillaries. The presence of C3 deposits in the glomerular tuft correlated with impaired kidney function and overall short-term survival. Intrarenal complement C3 deposits were not associated with consumption of respective serum levels, supporting the concept of intrarenal C3 synthesis. Finally, intrarenal synthesis of complement C3 was linked to distinct inflammatory signaling pathways in the kidney that is especially relevant in ANCA-associated renal vasculitis.
Conclusion: Considering recent advances in AAV therapy with the emergence of new therapeutics that inhibit complement activation, we here provide novel insights into intrarenal complement synthesis and associated inflammatory signaling pathways in ANCA-associated renal vasculitis.
Committee meeting - ICS
Lightning Poster Session 1
- 129 Complement propagates visual system pathology following traumatic brain injury – Khalil Mallah
- 51 Activation of complement in a mouse model of severe COVID-19 ” – Peter Szachowicz
- 231 Expression and Function of Complement Components in Head and Neck Cancer: Analysis Across Various Model Systems – Nicole Schäfer
- 41 Assessing the Feasibility of Genetically Modified Porcine Red Blood Cells as an Alternative to Human Red Blood Cells for Transfusion – Hee Jung Kang
- 39 Humoral complementomics – exploration of non-invasive complement biomarkers as predictors of renal cancer progression – Mikel Rezola Artero
- 245 Sez6L2 is a brain-resident complement inhibitor that is necessary for proper brain development –Julia Granato
- 75 The molecular basis for C1s inhibition by Lyme disease spirochetes – Brandon Garcia
- 213 Complement C3 deficiency enhances the effector function of CD8+ T cells and inhibits tumour growth – Pradipta Pal
- 215 The role of the complement system in the induction of antibodies directed against post-translationally modified proteins – Stef van der Meulen
- 49 The Binding and Kinetics of Normal versus Pathogenic C3 Convertase Autoantibodies – Christopher Culek
- 223 Beta cell-derived C3 is protective in a mouse model of diet-induced obesity and diabetes – Lucie Colineau
- 149 SARS-CoV-2 nucleocapsid protein is not responsible for the activation of complement lectin pathway – Andrea Kocsis
- 190 Characterization of the interaction of mannose-binding lectin with variant SARS-CoV-2 spike proteins – Adrian Sutta
Poster Sessions with cake / coffee
- 5 Synovial Complement Activation and Imbalance after Anterior Cruciate Ligament Injury or Meniscus Tear as a Risk Factor in the Development of Post-Traumatic Osteoarthritis
- 7 A nanobody-based complement inhibitor targeting complement component 2 reduces hemolysis in a complement humanized mouse model of autoimmune hemolytic anemia
- 9 Structural evolution of a complement evasion determinant shapes Lyme borreliae host tropism
- 13 Differential contributions of C5b-9 and C5a/C5aR pathways to microvascular and macrovascular thrombosis in complement-mediated thrombotic microangiopathy patients
- 15 Defining the Impact of Factor H, Factor H-related 1, and Factor H-related 5 on C3b Deposition on Mouse Mesangial Cells
- 17 A Phase 2, Randomized Trial Evaluating the Safety and Efficacy of Pozelimab and Cemdisiran in Patients with Paroxysmal Nocturnal Hemoglobinuria
- 19 Patient-Reported Outcomes from a Phase 2, Randomised Trial Evaluating the Safety and Efficacy of Pozelimab and Cemdisiran in Patients with Paroxysmal Nocturnal Haemoglobinuria
- 21 Exploring the impact of social media on mental health: A qualitative study
- 27 Elucidating the molecular mechanisms mediating the tumor suppressive actions of complement inhibitor CSMD1 in gliomas
- 29 Humoral Innate Immunity and Acute Phase Proteins in COVID-19
- 35 Deciphering complement system-dependent cellular pathways in human rheumatoid arthritis synovial tissues using large single-cell computational omics
- 37 Intracellular complement in Atypical Hemolytic-Uremic Syndrome.
- 39 Humoral complementomics – exploration of non-invasive complement biomarkers as predictors of renal cancer progression
- 41 Assessing the Feasibility of Genetically Modified Porcine Red Blood Cells as an Alternative to Human Red Blood Cells for Transfusion
- 43 Evaluation of iptacopan in atypical hemolytic uremic syndrome: Design and rationale of the Phase 3 open-label multicenter APPELHUS study
- 45 Expression of complement regulators on red blood cell progenitors duringin vitro and in vivo erythropoiesis
- 47 Plasma factor D cross-sectionally associates with low-grade inflammation, endothelial dysfunction and cardiovascular disease, but not with intima-media thickness or ankle-brachial index: The Maastricht Study.
- 49 The Binding and Kinetics of Normal versus Pathogenic C3 Convertase Autoantibodies
- 51 Activation of complement in a mouse model of severe COVID-19 “
- 53 Increased infiltration of CD4+ T cell in the complement deficient lymphedema model
- 55 Association between autoantibodies and complement activation in systemic lupus erythematosus
- 61 Association between novel lipoprotein particles and age-related macular degeneration
- 71 Acinetobacter baumannii clinical isolates evade complement-mediated lysis by inhibiting the complement cascade and improperly depositing MAC.
- 73 Dengue virus NS1-epitope specific monoclonal antibodies inhibit NS1-mediated complement evasion
- 75 The molecular basis for C1s inhibition by Lyme disease spirochetes
- 79 The Functional Consequence of two CFI Ultra Rare Variants in Complement-Mediated Diseases: Insights from In Vitro Splicing Assay
- 81 Clinical and Histologic Correlations in C3 Glomerulopathy
- 85 Association of a new variant of complement regulator FHR2 with C3 glomerulopathy
- 87 Activation of complement pathways and formation of membrane attack complex contribute to the development of severe COVID-19
- 91 Complement genetics: implementation of a custom made panel for genetic testing of complement-associated kidney patients
- 93 Modeling complement activation on human glomerular microvascular endothelial cells
- 95 Complement-associated prothrombotic state is caused by MAC-induced lysis of cells
- 97 Decreased Expression of CR1 on Peripheral White Blood Cells During Acute Dengue Infection
- 99 Targeted complement inhibition using engineered bispecific antibodies that bind local antigens and endogenous complement regulators as a novel therapeutic approach
- 105 Elucidating the role of anaphylatoxins and their receptors in osteoarthritis regarding cartilage calcification
- 107 Ex situ porcine liver machine perfusion activates the complement system and increases cytokines independent of pre-induced liver injury
- 111 Proteolytic activity of secreted proteases from pathogenic leptospires and effects on phagocytosis by murine macrophages
- 117 Complement receptor 1 long homologous repeats (A, B, and C) as a novel tool to study immune-adherence functionality of human erythrocytes
- 119 Characterising complement in amyloid-beta plaques
- 121 Cell-based assay for the measurement of complement convertase activity.
- 127 Complement drives chronic inflammation and progressive hydrocephalus in murine neonatal germinal matrix hemorrhage
- 129 Complement propagates visual system pathology following traumatic brain injury
- 131 Dual Inhibition of the Complement System and Toll-like Receptors Prevents Systemic and Local Kidney Inflammation in Mice Experiencing Brain Death
- 133 Complement system activation is mediated by COVID-19 severity, inducing endothelial cell injury and cell permeability
- 135 External quality assurance program for diagnostic complement laboratories: Evaluation of the past six year’s results
- 137 CA19-9 versus an inflammatory profile in blood samples from pancreatic cancer patients
- 139 Antibodies Targeting Human Complement Receptor C3aR
- 145 C1q Dependent Synaptic Pruning in Sepsis-Associated Encephalopathy (SAE)
- 147 Antibodies against Streptococcus pneumoniae pneumolysin correlate with modified HDL levels and complement activation markers in peripheral arterial disease patients
- 149 SARS-CoV-2 nucleocapsid protein is not responsible for the activation of complement lectin pathway
- 151 Complement system remains unaltered in an acute schizophrenia
- 153 Visualizing complement activation and regulation in the tumor microenvironment
- 157 From parasite to therapeutic: Expression, functional characterization, and therapeutic potential of the leech-derived complement inhibitor gigastasin
- 159 Functional characterization of two novel Complement Factor B variants in patients with primary Immune-Complex-mediated MPGN
- 161 VIS954 is a Potent Anti-C5aR1 Antibody for the Treatment of ANCA-Associated Vasculitis
- 167 Immune Evasion Potential Associated with Infection Type of Staphylococcus aureus
- 171 The dominant form of the C1 inhibitor R444C variant causing type II hereditary angioedema is its covalent adduct with serum albumin explaining elevated levels
- 173 Roles of complement dysregulation and membrane attack complex formation in developmental synapse loss
- 175 Transposon mutagenesis: a molecular switch in Acinetobacter baumannii for rapid adaptation to bacteriophages and the complement system
- 177 Factor H-related proteins bind to extracellular matrix components and affect complement activation
- 181 Complement C7 and clusterin form a stable complex in circulation
- 185 CPV-104, an improved recombinant variant of human complement factor H produced in moss, is a prime candidate for clinical application in complement mediated diseases.
- 190 Characterization of the interaction of mannose-binding lectin with variant SARS-CoV-2 spike proteins
- 195 Measurements of complement regulators in peritoneal dialysis patients
- 197 Keeping properdin in check: serum component(s) inhibit(s) the binding of properdin
- 199 A role for complement in sickle cell disease
- 201 Leptospira interrogans leptolysin displays proteolytic activity against complement proteins
- 203 Annexin A2: A positive regulator of the Alternative complement pathway in the kidney
- 205 The complex role of complement in hepatocellular carcinoma
- 209 Pregnancy-related Thrombotic Microangiopathy has a spectrum of underlying causes that affect vascular endothelium
- 211 Platelet supernatants modulate the inflammatory response in thrombocytopenic blood ex vivo.
- 213 Complement C3 deficiency enhances the effector function of CD8+ T cells and inhibits tumour growth
- 215 The role of the complement system in the induction of antibodies directed against post-translationally modified proteins
- 217 An ecotin ortholog turns out to be a potent classical pathway inhibitor and might provide information on the relative efficacy of C1r versus C1s inhibition
- 219 Therapy induced senescence in cancer cell lines affects complement activation and complement regulatory proteins expression
- 221 SYNERGY-1: A Phase 1, first-in-human, randomized, double-blind, placebo-controlled safety, tolerability, immunogenicity, PK and PD study of KP104 in escalating single and multiple doses
- 223 Beta cell-derived C3 is protective in a mouse model of diet-induced obesity and diabetes
- 227 Nomacopan as an anti-shock drug for pre-hospital treatment of traumatic hemorrhage through switching the injury phenotype to survival phenotype
- 229 Effects of combined complement complement and CD14 inhibition on Escherichia coli-induced thromboinflammation in human whole blood in the presence and absence of antibiotics
- 231 Expression and Function of Complement Components in Head and Neck Cancer: Analysis Across Various Model Systems
- 235 Crystallographic and SAXS structure of the immune evasive factor GAPDH from Leptospira interrogans and interaction with human C5a
- 245 Sez6L2 is a brain-resident complement inhibitor that is necessary for proper brain development
- 251 Deciphering protease activity in human plasma from systemic lupus erythematosus patients
- 253 Unraveling Predisposing and Protective Functional Consequences of CFB Variants in Atypical Hemolytic Uremic Syndrome
- 259 “Unveiling Novel Crosstalk: C1q/MASP-3 Complexes Activate Pro-factor D, Linking Diverse Biological Pathways”
Session 7 - Mechanisms of activation and control
The membrane attack complex (MAC) is a large macromolecular immune pore that punches holes in target cells. While a potent weapon of the innate immune defense, MAC pores can also damage human cells if not properly controlled. Here we use cryoEM to understand the molecular basis for how MAC pore formation is controlled in human cells during an immune response. By solving the structure of a soluble regulated form of MAC called sMAC, we explain how blood-based chaperones scavenge and clear potentially harmful complement activation by-products. Most recently we have created a membrane model system that incorporates a synthetic GPI-anchored cellular receptor (CD59) that inhibits MAC. Using cryoEM, we show how CD59 captures and deflects pore-forming beta-hairpins of complement proteins, rerouting their membrane trajectory. Moreover, we have discovered how the membrane environment influences the role of CD59 in complement regulation and in host-pathogen interactions. Our results open new lines of investigation into the importance of lipids in immune homeostasis that may be relevant for therapies that regulate complement.
Factor H (FH) is an abundant plasma glycoprotein that restricts alternative pathway activation in the fluid phase and on host surfaces. FH is also exploited for therapeutic purposes, e.g., the cyclic peptide 5C6 was developed to recruit FH via its central domains to biomaterial and cell surfaces that require protection from complement attack. The central CCP domain segment of FH is thought to orient the regulator’s functional termini to facilitate self vs. nonself discrimination. However, structural details about the centre of FH have been scarce until recently when a crystal structure of FH CCPs 8-14 had been presented at a resolution of 2.2 Å. Whereas CCPs 8-9 are elongated and show few inter-domain contacts, CCPs 10-14 form a complete loop that is induced by intricate contacts of CCP14, which is wedged into a cleft formed by CCPs 10 and 11.
Intriguingly, structure-activity relationship studies (SAR) of 5C6 identified FH CCPs 10-14 as the essential binding region for the peptide, suggesting that 5C6 binds to a discontinuous domain platform. To cross-validate the unprecedented central domain arrangement of FH and target interaction mode of 5C6, we solved the co-crystal structure of 5C6 peptide and FH CCPs 8-14 by crystallography at a resolution of 2.87 Å.
In agreement with SAR studies, 5C6 is moulded into a small niche that arises at the ternary contact interface between FH CCP domains 10, 11 and 14 and interacts with all three domains. The peptide is in closest contact with CCP10, followed by CCP11 and CCP14. All 5C6 residues that were experimentally shown to be required for FH binding indeed form contacts in the 5C6:FH8-14 structure, predominantly via the cyclic core and exocyclic C-terminus of 5C6.
The structural complex of FH8-14 with 5C6 validates the unprecedented domain arrangement of CCP domain 10, 11 and 14 in FH. At the same time, the structure provides a rationale for the highly specific interaction of a small peptide with a presumably elongated and flexible target such as FH and an important basis for additional SAR studies to facilitate the rational design of enhanced 5C6 analogues for biomedical applications.
Background: Immunoglobulins type-M (IgM) are the first antibody isoforms that are produced by vertebrates in response to a range of antigens from viruses to tumor cells, making them promising therapeutic targets. In serum, IgMs are found in two oligomeric forms, pentamers (5 promoters joined by J chain) and hexamers (6 promoters without J chain). These assemblies make a rigid inner core formed by the constant fragment (Fc), while the antigen-binding regions (Fab) are positioned at the extremities and adopt a very flexible conformation. The antigen-bound IgMs are known to strongly activate the complement system, in particular the classical pathway, by binding to it’s first recognition molecule, C1q. Despite their essential role, the mechanism of the complement activation by the different oligomeric forms of IgMs are only starting to be elucidated in fine details through newly developed methods in protein engineering, biophysics and structural biology (Chouquet et al., 2021; Sharp et al., 2019).
Methods: Fab-truncated forms of IgMs containing only the Fc-core were produced in HEK293F cells. The purified samples were characterised by coupling size exclusion chromatography with mass photometry and negative-stain transmission electron microscopy to finely elucidate their oligomeric distributions. The binding of Fc samples to C1q and their potency in complement activation were subsequently measured using Bio-Layer Interferometry (BLI) and in-house ELISA-like assay, respectively.
Results and Conclusion: The oligomeric distribution in the Fc samples was either heterogeneous or homogeneous depending on the presence of the J chain, similar to recombinant full-length IgMs studied before (Chouquet et al., 2021). Unexpectedly, both forms exhibited the ability to bind to C1q although with lower affinity than full IgMs and to activate the classical pathway in BLI and ELISA functional assays. Notably, the level of complement activation was correlated to the oligomer ratios in the different samples, confirming that Fc-core hexamers have a stronger potential to trigger complement than pentamers, as observed in full IgMs. In conclusion, the findings from BLI and in vitrocomplement activation studies challenge the idea that, in the absence of antigen and Fab domains, the IgM-Fc (either pentameric or hexameric) is incapable of activating the complement by C1q binding.
Anti-C1q autoantibodies are present in several autoimmune diseases and are known to associate with nephritis in systemic lupus erythematosus (SLE). However, these antibodies have also been detected in up to 10% of healthy individuals. Importantly, analysis of sera suggests that the anti-C1q autoantibodies in both healthy subjects and SLE patients are selective for ligand-bound, solid-phase, C1q, and do not bind to fluid-phase C1q. This is underscored by the observation that in patients with anti-C1q, C1q levels tend to be in the normal range and the autoantibodies are thus not depleting.
To study the nature of human C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and cloned 9 monoclonal antibodies (mAb) from 4 individual healthy donors. The mAb were produced recombinantly and characterized in biochemical and cellular assays.
The isolated clones were of IgG isotype, contained a highly mutated variable domain and showed high affinity to the Collagen-Like Region (CLR) of C1q. Binding of anti-C1q mAb was observed only to solid-phase C1q that was bound on a range of natural ligands such as IgG, IgM, CRP, antibody-opsonized cells and necrotic cells. The binding to solid-phase C1q was not inhibited by fluid-phase C1q. Electron microscopy confirmed that multiple anti-C1q mAb can bind to a single solid-phase C1q molecule. In competition experiments, we observed 2 separate groups of human anti-C1q mAb indicating that at least 2 epitopes are targeted. Both epitopes are also targeted by anti-C1q from SLE sera, implying that the cloned anti-C1q mAb are representative for anti-C1q autoantibodies found in SLE. The presence of anti-C1q on C1q-containing immune complexes did not enhance complement activation. However, the presence of anti-C1q on C1q-opsonized immune complexes greatly enhanced triggering of cellular Fc receptors, a process that is normally impaired when C1q is bound to the immune complex. Indeed we observed increased phagocytosis of C1q-opsonized beads and bacteria after addition of anti-C1q mAb.
Thus, anti-C1q autoantibodies are specific for solid-phase C1q, do not enhance complement activation, but importantly enhance Fc-receptor triggering which may contribute to the immunopathology of autoimmune diseases.
The classical complement cascade can be activated via antigen-bound antibodies (IgG or IgM) in response to pathogens during infection or to autoantigens in autoimmune diseases. Whereas IgM circulates as a pre-formed pentamer or hexamer, IgG exists as monomers which have the ability to oligomerize via non-covalent Fc-interactions upon surface binding. Furthermore, it is known that at least two IgGs are required for C1 binding, with recent studies having shown that IgG1 mutants with enhanced hexameric oligomerization potential are able to activate the complement system more efficiently. However, structural data reveal that it is not necessary to bind all 6 C1q arms to initiate the complement cascade, but that already binding of 4 or 5 C1q arms is sufficient. These observations reveal a symmetry mismatch between C1 and the activating hexameric Abs complex, which has not been adequately explained.
Here we use DNA nanotechnology to produce specific nanostructures in order to template nanometre precise chemically conjugated antigens for antibody binding. We can bind these self-assembled DNA platforms to cell-mimetic lipid membranes, and assess complement activation in a controlled environment. This system enabled us to determine the effect of valency on complement activation, without the requirement of mutated antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography (cryoET). Our data revealed that pre-formed antibody complexes activated complement to a greater extent than the same number of antibodies left un-patterned on the surface of liposomal cell mimetics. Furthermore, biophysical analysis showed that, for similar C1 binding, increased valency caused an increased activation of the protease domains of C1 as measured via C4 cleavage and MAC pore formation.
Together, these data provide insights into how nanopatterning antigen-antibody complexes influences activation of the C1 complex, and suggests routes to modulate complement activation by antibody engineering. Furthermore, this is the first time DNA nanotechnology has been used to study the activation of the complement system.
Background: According to the state-of-art, the alternative pathway (AP) is initiated by spontaneous proteolysis of C3 to C3b in the fluid phase. Continuous formation of C3(H2O) has been suggested to be the nidus of this reaction. However, we have previously shown that fluid phase C3(H2O) is a poor initiator of the AP compared to the in vivo continuously formed C3b (1). As an alternative AP activation pathway, we have in this study investigated the ability of native C3 to specifically bind and form an AP convertase on various biosurfaces.
Methods: Adsorbed native C3, C3b and preformed C3(H2O), e.g., methylamine treated C3 (C3(met)) was investigated on various biosurfaces, and the conformation and convertase activity of the adsorbed C3 was evaluated using methods such as ELISA, flow cytometry, and quartz crystal microbalance with dissipation monitoring (QCM-D).
Results: Purified C3, C3b and C3(H2O), all bound to activated platelets, but it turned out that native C3 bound more efficiently to platelets compared to C3b and C3(H2O). Similarly, the generation of C3a in the presence of factor B, factor D and properdin was most efficient with platelet-bound native C3, compared to C3b and C3(H2O). Native C3 also bound to apoptotic cells. Competitive binding of C3 and Annexin V indicated that they compete for the same binding site, i.e., phosphatidyl serine, which is exposed on the cells during apoptosis. As assessed by QCM-D, C3 bound to all types of biomaterial surfaces tested, likely without covalent binding via the thioester. However, C3 only formed C3 convertases on surfaces with certain properties, e.g., rigid polymer surfaces, hydrophobic polystyrene surfaces, and negatively charged liposomes containing cholesterol. The convertase formation was correlated to an preceding conformational change of C3 to C3(H2O) on the specific surfaces and required properdin for the activation complex assembly.
Conclusion: These results propose an alternative targeted activation mechanism of C3 that leads to opsonization of altered self and foreign material surfaces, and formation of initial C3 convertases.
(1) Fromell K, Adler A, et al. Assessment of the Role of C3(H2O) in the Alternative Pathway. Front Immunol. 2020;11:530