Session 8 - Emerging topics in complement biology
The dynamics and functions of intracellular complement proteins have been increasingly described in immune and non-immune cells in recent years. However, studies investigating the presence and role of intracellular complement in neutrophils remain scarce. Here, we investigated the dynamics of complement protein uptake and production in neutrophils, focusing specifically on Complement 3 (C3). We utilized an in vitro time course culture model and used freshly-isolated human neutrophils from whole blood to examine the presence and dynamics of intracellular C3 protein levels by western blot and immunofluorescent imaging. Our analysis demonstrated that neutrophils contain large stores of C3 present throughout their cytoplasm immediately upon isolation from whole blood. Further, neutrophil C3 gene expression, measured by qPCR and RNA-seq, immediately following isolation was very low, indicating that the majority of protein C3 found at the time of isolation was taken up from the fluid phase while in the circulation. We next cultured neutrophils in either C3-sufficient or C3-deficient sera and found that neutrophils maintain a consistent level of intracellular C3 when cultured in C3-sufficient sera only. This, along with reports of T cells and macrophages increasing C3 gene transcription upon tissue diapedesis, led us to hypothesize that neutrophils may rely on uptake and shuttling of exogenous C3 until they extravasate into tissues at sites of inflammation. To test this hypothesis, we utilized an in vivo mouse model of LPS-induced acute lung injury. Using an RNAscope Assay probing for C3, we detected active C3 gene transcription in neutrophils throughout the inflamed lung, while neutrophils in the intravascular space and those attached to the luminal side of endothelium lacked significant C3 gene transcription. We further confirmed these findings in an in vivo mouse orthotopic lung transplant model and observed an identical phenomenon. Taken together, our results indicate that neutrophils rely on exogenous sources of complement C3 to supply their intracellular stores until such time that they extravasate into sites of inflammation, upon which neutrophils increase endogenous C3 production.
Opsonization and efferocytosis of dying cells are modulated by the presence of malondialdehyde epitopes.
Nikolina Papac-Milicevic1,; Frida C. Mohlin3; David Weismann1; Mirlinda Ademi1, Clara J Busch1; Daryna Katashynska1; Valentina Kovacic1; Florentina Porsch1; Barbara Bartolini Gritti1; Maria Gorna2; Lejla Alic1; Anna M. Blom3; Christoph J. Binder
1 Department of Laboratory Medicine, Medical University of Vienna, Austria.
2 Department of Chemistry, University of Warsaw, Poland.
3 Department of Translational Medicine, Lund University, Sweden.
Background: Increased oxidative stress leads to the formation of lipid peroxidation-derived structures that form oxidation-specific epitopes (OSEs) and act as damage-associated molecular patterns (DAMPs). Malondialdehyde (MDA)-adducts are prototypical examples of such OSEs and can be found on dying cells, oxidized lipoproteins, and a subset of extracellular vesicles. We and others have shown that MDA-epitopes are recognized by scavenger receptors, natural IgMs, factor H (FH), and FH-related proteins (FHR1, FHR3, FHR5). Once bound to MDA-epitopes, these proteins determine consequent innate immune responses. To elucidate the role of MDA-epitopes in the removal of dying cells and homeostasis maintenance, we investigated the mechanisms of their appearance and their roles in opsonization and efferocytosis.
Methods & Results: Using flow cytometry, we found that the presence of MDA-epitopes on dying cells correlates with loss of cellular membrane integrity, as measured by 7-Aminoactinomycin D positivity. MDA-epitopes were detected on the surfaces of dying cells generated by extrinsic or intrinsic apoptotic-, ferroptotic-, and necroptotic cellular death pathways. Furthermore, we demonstrated by ELISA that initiators of the classical and alternative complement pathways, C1q and C3b, also bind to MDA-epitopes. However, the extent of complement activation on MDA-decorated surfaces was controlled by the MDA-dependent recruitment of the complement regulators FH and C4b-binding protein (C4BP). Using flow cytometry, we further found that the opsonization of dying cells by IgMs, C1q, C4BP, C3b, and C5b-C9 complexes depends partially on the presence of MDA-epitopes. Finally, we observed that the efferocytosis rate was determined by the density of MDA-epitopes and the repertoire of opsonins.
Conclusion: Our data suggest that MDA-epitopes on dying cells with impaired membrane integrity guide opsonization by complement components and thereby modulate their recognition and clearance by phagocytic cells.
Background: C1q exerts pro-tumorigenic functions in the tumour microenvironment (TME), independently of complement activation (1). In malignant pleural mesothelioma (MPM), HA-bound C1q is able to increase tumor cell adhesion, migration and proliferation, but also to enhance the production of pro-inflammatory and pro-metastatic HA fragments due to HAS3 upregulation (2). An increasingly activated HA metabolism is associated with cancer progression. Here, we investigated HA-bound C1q contribution in HA degradation via modulation of hyaluronidases (HYAL1, HYAL2) and the involvement of globular C1q receptor/HABP1/p32 (gC1qR), as a receptor of both HA and C1q.
Methods: primary cell isolation, zymography, immunohistochemistry, Real-Time quantitative PCR (RT-qPCR), immunofluorescence, Western blot, surface biotinylation assay, flow cytometry, proximity ligation assay (PLA), RNA interference, bioinformatics analysis.
Results: After initial characterization of HYALs in MPM primary cells, we focused on HYAL2, since GEPIA bioinformatics analysis revealed an unfavorable prognostic index in MPM patients with higher HYAL2 mRNA levels. Upon seeding MPM cells onto HA-bound C1q, HYAL2 upregulation was highlighted by RT-qPCR, flow cytometry and Western blot. In the attempt to determine receptors involved in HA-C1q signaling, a striking membrane and intracellular co-localization between HYAL2 and gC1qR was found by immunofluorescence, surface biotinylation assay and PLA. RNA interference of C1QBP (gene for gC1qR) unveiled a promising regulatory effect of gC1qR on HYAL2 expression, determining an unexpected HYAL2 downregulation. Moreover, functional blockage of gC1qR by a specific antibody hampered HA-C1q signaling and impeded HYAL2 upregulation.
Conclusions: HA-C1q interplay can act as a tumor-promoting signaling complex by enhancing HYAL2 expression, suggesting a consequent higher rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments. Furthermore, we demonstrated a novel regulatory function of gC1qR in the TME due to its involvement in the modulation of HA metabolism. The regulation of HA metabolism is crucial in view of its key role in tumor progression and its connection with most of the hallmarks of cancer.
References: 1.Bullaet al., Nat commun 2016, doi:10.1038/ncomms10346; 2.Vidergar, Balduitet al., Cancers 2021, doi:10.3390/cancers13030416.
Background: Complement receptors CR3 and CR4 are members of the β2-integrin family. They are involved in complement-dependent phagocytosis and leukocyte adhesion, migration, and activation, thereby playing important roles in immune surveillance and inflammation. The direct involvement of β2-integrin receptors in autoimmune, inflammatory, and age-related diseases render them interesting drug targets. However, they often act as both immune activators and suppressors in a complex interplay that is not well understood. In addition, CR3 has been shown to interact with a vast number of ligands , with unknown biological implications. To address this unmet need, we are developing a platform of recombinant αI domains of β2-integrins and assays to test their interactions with endogenous and synthetic ligands. In this study, we employed this platform to assess newly developed tool compounds that may help to disentangle the promiscuous ligand binding profile of CR3 and its implication in pathologies.
Methods: We expressed recombinant ligand-binding domains (αI) of all β2-integrin members in E.coli. These αI-domains were used to investigate the interaction of C3-derived opsonins with members of the β2-integrin family in direct binding (SPR) and bead-based adhesion assays. Using phage display screening, we have identified tool compounds that bind to CR3 and are able to compete with the interaction of endogenous ligands.
Results: The αI-domains of all four β2-integrins could be uniformly expressed in E. coli, in both wildtype and high-affinity conformation. Recombinant CR3 αMI and CR4 αXI showed distinct binding profiles for C3-derived opsonins that were dependent on activation states and cofactors. Phage display screening against CR3 αMI identified cyclic peptides as CR3 ligands, which showed direct binding to the αMI domain, partially competed with opsonin recognition, and exerted modulating activities in functional adhesion assays.
Conclusion: The platform established here is suitable to characterize the interaction profile of β2-integrins with their ligands and develop tool compounds able to distinctly modulate integrin-ligand interactions. The availability of integrin/ligand-specific modulators will facilitate the elucidation of the β2-integrin receptor’s intricate functional interplay in health and disease and may enable the development of therapeutic integrin modulators that block specific -ligand interactions while maintaining immune defense functions.
Plenary 2: Luke O'Neill - Krebs Cycle Reprogrammed for Immunity and Inflammation
Session 9 - Complement and infection
Pasteurella multocida is a Gram-negative microbe that causes serious infectious diseases in cattle and other ruminants. In North America alone, 75% of annual cattle death can be traced back to this bacterial infection, underlining the huge economic impact of this pathogen on the farming industry. Recurring outbreaks of P. multocida worldwide and the limitations of current vaccines have thus prompted us to search for a novel antigen that can elicit a cross-protective and broad-spectrum response to all circulating bacterial strains. Through an extensive bioinformatic analysis, we identified a surface lipoprotein, termed PmSLP, that is present in more than 95% of bovine P. multocida isolates (Hooda et al., 2017). The prevalence and conservation of PmSLP suggests that the protein is important for bacterial physiology and pathogenesis. We also showed that using PmSLP as a vaccine antigen could protect animals from invasive P. multocida infections (Islam et al., 2023).
To further understand the role of PmSLP in bacterial immune evasion, we used PmSLP as a bait and identified bovine complement factor I (FI) as the mammalian binding partner. We were able to isolate stable protein complexes through size exclusion chromatography and demonstrated high affinity binding between PmSLP and FI via kinetic studies. We also solved the structure of PmSLP using X-ray crystallography and identified several key residues required for binding function. Moreover, functional characterization of FI in complex with PmSLP showed that the enzyme is proteolytically active against both C3b and C4b without needing its native co-factors, factor H and C4BP, respectively. This result suggests that PmSLP serves as a promiscuous co-factor of bovine FI, allowing the bacteria to switch off all three branches of the complement cascade. Finally, we carried out gain-of-function experiments where PmSLP was expressed on the surface of a serum-sensitive strain of Escherichia coli. Interestingly, the presence of PmSLP alone on the cell surface was sufficient to promote E. coli resistance to complement-meditated killing.
Taken together, this study highlights a previously unknown mechanism used by P. multocida to evade the complement system.
Background: Invasive aspergillosis is among the most common fungal infections in immunocompromised patients. With the increasing number of susceptible patients, the development of host-centric interventions is of paramount importance. Pulmonary hemorrhage is a characteristic clinical feature of invasive aspergillosis, and is associated with worse lung injury and a more lethal infection. However, the effects of free heme on the host during invasive aspergillosis are poorly understood. We hypothesized that a mechanism by which free heme leads to worse pulmonary injury is via activating the terminal complement pathway, driving inflammation and tissue injury.
Methods: C57BL/6 mice were neutrophil depleted and then infected with A. fumigatus. To characterize the dynamics of complement activation in the alveolar space, we measured levels of C3a and C5a in the bronchoalveolar lavage fluid during the infection. To test the effects of heme on the host, mice were administered exogenous hemin after infection, and C3a and C5a levels were measured by ELISA, and compared to mice challenged with vehicle control. As a secondary model of heme accumulation, we infected hemopexin -/- mice and measured complement levels. Lastly, we antagonized C5aR1 and measured fungal burden and lung injury on C57BL/6 compared to vehicle control.
Results: Complement is activated in the alveolar space of Aspergillus-infected mice with C5a levels peaking before C3a levels, and exogenous administration of hemin leads to higher levels of anaphylatoxins C3a and C5a in the alveolar space. Hemopexin-deficient mice had higher levels of alveolar C3a, and administration of exogenous hemopexin to hemopexin -/- mice led to a decrease of C3a in the alveolar space– suggesting that heme scavenging attenuates complement activation. Due to the early peak of alveolar C5a levels, and the putative inflammatory and potentially harmful role of C5aR1 signaling (as shown in other models of acute lung injury) we antagonized C5aR1, which led to a decrease in the lung fungal burden of infected female mice, but not in male mice.
Conclusion: Complement is induced in the alveolar space during invasive aspergillosis in mice. This study indicates that inhibiting C5a signaling results in better microbial clearance during invasive pulmonary aspergillosis in female mice.
The obligate intracellular parasite Plasmodium falciparum causes malaria tropica, the most severe form of malaria, which is particularly acute in the WHO African region. With more than 247 million cases and 619,000 deaths each year, malaria is still one of the deadliest infectious diseases in the world. Malaria infection is characterized by multiple symptoms, like fever, head- and body aches, and diarrhea. Severe complications further include anemia, shock, cerebral malaria, multiple organ failure, and ultimately death. During evolution, the parasite has developed various mechanisms to evade attack by human complement, such as binding of proteins of the Factor H (FH) family. In previous studies, we have shown that the Factor H-Related Protein 1 (FHR-1) competes with FH for binding sites and thereby prevents FH-mediated complement evasion (Reiss et al., 2018). Furthermore, FHR-1, when binding to malaria-lysed erythrocytes, activates neutrophils and thus contributes to inflammation. Remarkably, up to one third of the African population harbors a chromosomal deletion of the FHR-1 gene. To investigate a potential link between FHR-1 deficiencies and malaria severity, we investigated two cohorts of malaria tropica-infected African patients. We here show that patients with FHR-1 deficiencies are more likely to suffer from a mild malaria with less signs of anemia and lower levels of inflammation markers compared to FHR-1 expressing individuals. Our data underline the potent role of FHR-1 in inflammation, which likely enhances the immune response in Malaria infections and the risk of severe Malaria anemia.
Reiss, T., Rosa, T.F. de A., Blaesius, K., Bobbert, R.P., Zipfel, P.F., Skerka, C., Pradel, G., 2018. Cutting Edge: FHR-1 Binding Impairs Factor H–Mediated Complement Evasion by the Malaria Parasite Plasmodium falciparum. J. Immunol. 201, 3497–3502. https://doi.org/10.4049/jimmunol.1800662
In severe forms of COVID-19, the infection is associated with airway tissue damage and complications due to cellular infection and excessive inflammatory responses. A systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRM) with SARS-CoV-2 showed that the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid (N) and spike (S) proteins, respectively. MBL bound trimeric spike protein in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. After binding to S protein, MBL activated the lectin pathway of complement, potentially contributing to hyperinflammation and tissue damage1. PTX3 interacts with C1q, MBL and ficolins enhancing complement activation, and with factor H regulating complement response, while with Fcγ receptor modulating phagocytosis. In patients, the serum levels of complement proteins and PTX3 significantly increase during the infection period, correlating with severity2.
In this study, we asked the question whether these PRMs influence the interaction between phagocytes and SARS-CoV-2 infected cells, in term of phagocytosis and inflammation activation. We first observed that surface-exposed N proteins on the infected cells co-localize with PTX3 before being internalized by human bone marrow derived macrophages (MDM). By immunofluorescence, we observed the phagocytosis rate by MDMs of SARS-CoV-2 infected cells significantly increases with PTX3 or MBL treatment. In addition, in the MDMs with internalized infected cells, both N protein and dsRNA signals increase over time, suggesting a temporary viral replication activity in those MDMs. In parallel, cellular caspase-1 signal increases over time, showing inflammasome activation in both MDMs and infected epithelial cells. Lastly, we demonstrated complement protein deposition on SARS-CoV-2-infected epithelial cells. In conclusion, our study demonstrated that the interactions of PTX3 with N and MBL with S enhance the phagocytosis of SARS-CoV-2 infected epithelial cells, suggesting their involvement in promotion of antigen presentation and effective specific responses against SARS-CoV-2 infection.
Committee meeting - ECN
Lightning Poster Session 2
- 166 The complement system is dysregulated in long covid patients – Kirsten Baillie
- 132 Multiple pregnancy can induce preeclampsia-like changes in complement factor H-point mutation mice – Ying Tan
- 224 Specificity vs. versatility: Exploring the selectivity profile of the leech inhibitor gigastasin for serine proteases of the complement system and other pathways – Peter Ruthemann
- 168 Placenta specific complement activation in mice produces preeclampsia phenotype and adversely affects fetal liver function – Chandra Yallampalli
- 156 Activation of the complement system early on during brain death management – Laura Knijff
- 141 The activation of the lectin pathway serine proteases by fibrin and it’s subsequent effect in clot formation – David Eikrem
- 232 Effect of IL-1ß and C3a on complement expression and secretion on human osteoarthritic chondrocytes – Hannah Köck
- 18 A Phase 2, Open-Label Study Evaluating the Safety and Efficacy of Combination Pozelimab and Cemdisiran Therapy in Patients with Paroxysmal Nocturnal Hemoglobinuria Who Switch from Eculizumab – Petra Muus
- 50 Protective role of complement factor H against the development of preeclampsia – Roberta Bulla
- 72 Elucidation of novel exosite interactions in the activation and control of the classical pathway of complement – Ryan Garrigues
- 78 Characterisation of CSMD1 – a Complement Regulator Associated with Schizophrenia Risk – Robert Byrne
- 74 αβ T cells and complement meet again: TCR crosstalk with surface complement regulators – Daniel Chacon-Arguedas
Poster Sessions with cake / coffee
- 8 Humanization of the mouse anti-human complement C6 monoclonal antibody as a potential therapeutic for certain complement-mediated diseases
- 10 Structure/Function Analysis of collectin-K1 and collectin-L1
- 12 Prevention of terminal pathway activation improves survival in a lethal malarial infection mouse model associated with multiple organ failure.
- 14 Functional activity of CD35 (Complement Receptor 1; CR1) in the conversion of complement fragment iC3b to C3dg
- 16 Characterization and development of CSL040, a soluble truncated fragment of complement receptor 1
- 18 A Phase 2, Open-Label Study Evaluating the Safety and Efficacy of Combination Pozelimab and Cemdisiran Therapy in Patients with Paroxysmal Nocturnal Hemoglobinuria Who Switch from Eculizumab
- 26 Sustained Complement C1s Inhibition with Sutimlimab in Patients With Cold Agglutinin Disease Results in Continued Efficacy During Part B of the Randomized Placebo-Controlled Phase 3 CADENZA Study (NCT03347422)
- 34 Anti-tumor effect of resident macrophages bearing complement component C1q in targeted therapy with CD19- Chimeric antigen receptor (CAR)-T cell
- 36 Phase 1 clinical data for single-dose subcutaneous injection of RLYB116, a C5 blocking Affibody® molecule linked to an Albumod® albumin binding domain
- 42 APPARENT: A multicenter, randomized, double-blind, placebo-controlled Phase 3 study to assess the efficacy and safety of iptacopan in idiopathic (primary) immune complex-mediated membranoproliferative glomerulonephritis
- 44 Bi-specific antibodies targeting C1q and a tumor antigen activate the complement system independent of C1q-Fc interactions
- 46 The mycobacterial strain-dependent impact of the complement system in tuberculosis
- 50 Protective role of complement factor H against the development of preeclampsia
- 52 Human Factor H and Properdin Modulate SARS-CoV-2 Infection in a Complement-Independent Manner
- 54 Functional assessment of the alternative complement pathway in the first line treatment of B cell Non-Hodgkin Lymphoma
- 56 Complement biomarkers reflect the pathological status of neuromyelitis optica spectrum disorders
- 60 Pattern recognition plays a role in the initiation of the alternative complement pathway
- 72 Elucidation of novel exosite interactions in the activation and control of the classical pathway of complement
- 74 αβ T cells and complement meet again: TCR crosstalk with surface complement regulators
- 76 C1q-IgM interaction studies with recombinant globular C1q variants
- 78 Characterisation of CSMD1 – a Complement Regulator Associated with Schizophrenia Risk
- 80 C5b-9 and Bb factor levels as potential novel biomarkers in crescentic IgA nephropathy
- 84 Inhibition of SARS-CoV-2 spike interaction with angiotensin converting enzyme-2 (ACE2) by specific antibodies is enhanced by complement, determined with a novel flow cytometry assay
- 86 Assessing Complement Factor B Function: A Comparison of Hemolytic and Immunofixation Electrophoresis-based Assays
- 90 Complement activation during hypoxia-reoxygenation in human kidney epithelial cells
- 92 Treatment with warfarin but not Factor Xa inhibitors, is associated with dysregulated, noncanonical, complement activation
- 94 Citrullination of C1-inhibitor as a mechanism of impaired complement inhibition in rheumatoid arthritis
- 96 Association between acute stage biomarker levels, severity and long-term outcome in COVID-19
- 98 The complement C1s cleaves HMGB1, generating a potent anti-inflammatory product
- 100 LPS O1-antigen-mediated complement-resistance of Klebsiella pneumoniae clinical isolates
- 102 Role of Dengue NS1 and its Antibodies in Complement Activation in Hepatocytes
- 108 Design and in vitro Evaluation of Collectin-11 Antagonists for the Treatment of Ischemia-Reperfusion Injuries
- 112 Distinctive dosage requirements between C3 and Factor D (FD) in the activation of the alternative complement pathway
- 116 Differential complement iC3b levels in baseline plasma of individuals at clinical high risk for psychosis.
- 120 Differential complement activation in the blood of individuals at clinical high risk for psychosis compared with healthy controls
- 122 Autoantibodies targeting HMGB1 and the main C1s generated HMGB1 fragment in systemic lupus erythematosus: prevalence and characterization
- 124 A Role for P-selectin and Complement in the Pathological Sequelae of Germinal Matrix Hemorrhage
- 126 Complement Factor I Protein Concentration in Human Plasma and Ocular Tissue Samples Stratified by AMD Genotypes and Phenotypes
- 130 Low Gene Copy Numbers (GCN) of Complement C4 and C4A Deficiency Are Highly Significant Genetic Risk Factors for Idiopathic Inflammatory Myopathies and Its Major Subgroups
- 132 Multiple pregnancy can induce preeclampsia-like changes in complement factor H-point mutation mice
- 136 Complement C1q and von Willebrand Factor interaction in atherosclerosis of human carotid artery
- 138 Escherichia coli-induced platelet aggregation in human whole blood is partly dependent on complement C3b.
- 140 A novel selective leukocyte depletion human whole blood model reveals the specific roles of monocytes and granulocytes in the cytokine response to Escherichia coli
- 141 The activation of the lectin pathway serine proteases by fibrin and it’s subsequent effect in clot formation
- 142 Autoantibodies against complement C1q induced by Epstein Barr Virus antigen exacerbate renal disease in lupus-prone mice
- 144 FH desialylation as an acquired cause of Complement dysregulation in atypical Haemolytic Uraemic Syndrome
- 146 A transcriptome array-based approach to link SGLT-2 andtubulointerstitial synthesis of complement C5 in IgA nephropathy
- 150 Association between loss of immune checkpoint programmed cell death protein 1 and local synthesis of complement factor B in active ANCA-associated renal vasculitis
- 152 Assessing Function and Inhibition of a Synthetically Assembled C3 Convertase Using a Novel Functional Assay
- 154 Inhibition of terminal pathway activation by Diclofenac
- 156 Activation of the complement system early on during brain death management
- 158 Structure-guided design of derivatives of the complement inhibitor compstatin with improved species specificity profiles
- 160 CD59, C9-lock and horse C9 all inhibit MAC assembly through similar mechanisms.
- 162 Ficolin-2 high consumption is a hallmark of ischemic stroke with an etiology of large-artery atherosclerosis
- 164 Role of Nutraceuticals in ameliorating Inflammation in Alzheimer’s Disease
- 166 The complement system is dysregulated in long covid patients
- 168 Placenta specific complement activation in mice produces preeclampsia phenotype and adversely affects fetal liver function
- 170 Increased complement activation in recipients is associated with chronic rejection after lung transplantation.
- 172 Insulin secretion and blood glucose homeostasis in a CD59 double knockout mouse model
- 174 Factor H-related dimer equilibrium and kinetics revealed through novel specific ELISAs
- 176 A genome-wide association screening of complement activation from a general population study
- 178 Complement component C1q can predict patients’ response to cisplatin depending on tumour type: a step towards personalized medicine?
- 180 FUNCTIONAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST HUMAN COMPLEMENT C4 AND C4BP
- 182 Complement C7 and clusterin form a stable complex in circulation
- 186 Assessment of immunogenicity in mice and cross-species activity of an engineered complement regulator
- 192 Molecular imaging of renal C3d deposition to monitor lupus nephritis
- 198 Novel natural antibodies targeted against cardiolipin are associated with higher risk of rejection in kidney transplant recipients
- 206 Expression of complement proteins by M1 and M2 macrophages: Complement factor B induces inflammatory macrophage phenotype
- 210 Complement activation in patients with ANCA-associated vasculitis – focusing on complement activation markers in remission
- 212 Determinants resulting in high residual C5 activity in presence of Eculizumab or Coversin
- 222 Complement Activation in Cytokine Storms Associated with COVID-19, Sepsis, and Chimeric Antigen Receptor (CAR)-T Therapy
- 224 Specificity vs. versatility: Exploring the selectivity profile of the leech inhibitor gigastasin for serine proteases of the complement system and other pathways
- 230 Interaction of factor H and factor H-related proteins with S and N proteins of SARS-CoV-2
- 232 Effect of IL-1ß and C3a on complement expression and secretion on human osteoarthritic chondrocytes
- 238 Characterizations of structural and functional diversities of human complement C4A and C4B allotypes
- 242 Does c1q modulate myelin sheath development in zebrafish?
- 244 The role of the complement system in DCD liver transplantation
- 248 Trypanosoma brucei invariant surface glycoprotein 65 inhibits the alternative pathway of complement by accelerating C3b degradation
- 250 Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring
- 252 Towards a molecular understanding of a HIV-1:Complement interaction
- 254 Factor H-Capturing on HMEC-1 Cells with the Cyclic Peptide 5C6: Synthesis, Cell Surface Modification and Measurement of FH-Capturing from Human Blood Serum
- 258 The role of Complement C3aR in adolescent brain development
- 260 Are these cases C3 glomerulopathy? Or are they infection-related glomerulonephritis?
- 262 A novel CFHR3-1 hybrid protein provides insight into the pathogenic mechanisms leading to C3 glomerulopathy
Session 10 - Complement in neurodegenerative disease
Background: Genome-wide association studies (GWAS) in Alzheimer’s disease (AD) have highlighted the importance of the complement cascade in pathogenesis. Complement receptor 1 (CR1;CD35) is a top AD-associated GWAS hit. The long variant, CR1*2, is associated with increased AD risk. The roles of CR1 in brain and how variants influence AD risk are poorly understood. Our aim was to investigate the impact of the AD-associated CR1 length polymorphism on phagocytic function in induced pluripotent stem cell (iPSC)-derived microglia.
Methods: Donors were screened for erythrocyte expression of common CR1 variants, CR1*1 and CR1*2, using western blotting and junction PCR genotyping. Homozygote donors (CR1*1/CR1*1, CR*2/CR1*2) were identified, bled, peripheral blood mononuclear cells (PBMCs) isolated and reprogrammed to iPSCs using the CytoTune-iPS 2.0 Sendai Reprogramming kit. Clones were tested for virus clearance and expression of iPSC markers using immunofluorescence and qPCR prior to differentiation into microglia via embryoid bodies. Microglia precursors and iPSC-microglia were tested for marker expression using immunofluorescence, qPCR and flow-cytometry. Microglia expressing CR1*1 and CR1*2 variants were tested for phagocytosis of diverse targets (pHrodo-E.coli bioparticles, pHrodo-human synaptoneurosomes, Alexa-488-amyloid β fibrils), either unopsonised or opsonised with human serum or Factor I (FI)-depleted human serum to mediate C3 fragment deposition, verified by flow cytometry. Synaptoneurosomes were extracted from control or AD brain. Uptake of fluorescent targets was measured in real-time using live imaging in the OperaPhenix high content screening system with inbuilt Harmony software for analysis.
Results: iPSC lines were established by reprogramming CR1*1/CR1*2 homozygote donor PBMCs. Pluripotency was confirmed by demonstrating expression of pluripotency markers. iPSCs were differentiated into microglia and characterised for microglia-specific marker expression. CR1 was expressed in all iPSC-microglia lines for both CR1*1 and CR1*2 variants. iPSC-microglia were phagocytic and phagocytosis was enhanced by opsonisation of targets with human serum; target opsonisation with FI-depleted serum reduced this enhancing effect. Comparison of CR1*1 and CR1*2 expressing iPSC-microglia cells showed that expression of CR1*2, the AD risk variant, enhanced phagocytosis of opsonised targets.
Conclusions: CR1 is an important component of microglial phagocytic activity; expression of CR1*2 increased phagocytosis of opsonised targets, perhaps explaining its association with AD risk.
Alzheimer’s disease (AD), resulting in progressive cognitive decline, is the most prevalent form of dementia in the elderly. Neuroinflammation is now recognized as a contributor to the progression of late onset AD, and complement activation is associated with pathological hallmarks of AD. In animal models of AD, early components of the classical complement pathway are involved in aberrant synapse elimination. In previous studies, genetic ablation of C5aR1 or pharmacologic antagonism of C5aR1, resulted in less activation of microglia and astrocytes, preservation of neuronal complexity, reduction of cognitive loss and suppression of synapse engulfment by microglia, all implicating a role for C5a-C5aR1 signaling in disease progression. Gene expression data from adult microglia isolated from brain demonstrated that a genetic lack of C5aR1 prevented the polarization of microglia to a more inflammatory state while enhancing expression of genes involved in phagocytosis and lysosomal degradative enzymes and limiting synaptic pruning, all disease mitigating functions. Single cell (microglia)- and single nucleus-RNA seq now reveal differential expansion of specific cell clusters of astrocytes, as well as microglia, and predict pathways of cell communication involving oligodendrocyte precursor cells providing insight into the cellular and molecular mechanisms underlying the neuroprotection achieved by inhibiting C5a-C5aR1 signaling in adult mice. Specifically, some microglia and astrocyte clusters induced in the AD mouse models were C5aR1-independent, but others were almost completely suppressed by C5aR1 antagonism. Additional clusters were reduced in the AD mice, but restored by inhibition of C5aR1. Functional pathways that were suppressed by inhibition of C5aR1 included inflammatory, neurotoxic or synapse pruning, while those that were rescued by C5aR1 inhibition were neuroprotective or included regulation of inflammation. Importantly, these studies suggest that the specific inhibition of C5a-C5aR1 signaling is a promising strategy for suppressing loss of function in AD and other neurogenerative disorders in which neuroinflammation and synapse loss are evident.
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and imposes a severe social and economic burden on ageing populations. PD results from the progressive loss of dopaminergic neurons which is accompanied by a chronic neuroinflammatory response that propagates disease progression. Despite clear evidence for complement involvement in Alzheimer’s disease, the contribution of complement to PD neuropathology remains poorly defined. Using publically available data, we demonstrate that complement is widely upregulated in PD patient brains, at sites of dopaminergic neuron loss, and in peripheral blood. Similarly, activation of complement is observed in multiple preclinical PD models. Genetic deletion of key complement effectors at the level of C3, C5, and MAC highlighted a critical role for complement C5a receptors (C5aR1) in driving neurodegeneration in vivo in response to dopaminergic toxins. Fibrillar α-synuclein aggregates (PFF-synuclein), the predominant protein found in PD brain Lewy bodies, directly activated complement to generate C5a and markedly increased C5aR1 expression in human and mouse microglia. Oral administration of a C5aR1 antagonist significantly protected against behavioral motor deficits and nigrostriatal dopaminergic degeneration in acute (28-day 6-OHDA) and chronic (12-month PFF-synuclein) mouse models of PD. Notably, delaying drug administration until symptom onset prevented further motor functional decline and remained neuroprotective. Live visualisation of neuroinflammation using [18F]DPA-714 PET/CT-imaging, demonstrated that both prophylactic and therapeutic inhibition of C5aR1 blunted microglial activation in living mice. Mechanistically, cell-intrinsic microglial NLRP3 inflammasome activation by multiple stimuli was impaired in the genetic absence of C5aR1. Furthermore primary human microglia were unable to secrete IL-1β in response to α-synuclein fibrils in the presence of C5aR1 inhibitors. Targeting this complement-microglia-inflammasome axis with brain-permeable inhibitors could be a feasible approach to tame neuroinflammation, and slow neuronal death in people living with PD.
Background: Neuroinflammation is a critical component of Alzheimer’s Disease (AD). Dysregulation of complement leads to excessive inflammation, direct damage to self-cells and propagation of injury. This is likely of particular relevance in the brain where inflammation is poorly tolerated and brain cells are vulnerable to direct damage by complement. The membrane attack complex (MAC) is a highly pro-inflammatory product of complement activation, killing cells by lysis and/or causing sublytic damage, including initiating NLRP3 inflammasome activation, and provoking other damaging responses leading to death of vulnerable brain cells.
Methods: The role of MAC in AD was investigated in MAC-deficient animals and by using a newly developed anti-C7 monoclonal antibody (mAb) that efficiently inhibits formation of the MACin vitro andin vivo. Impact of C7 deficiency on brain complement dysregulation, synapse loss, amyloid load and cognitive decline was examined by comparing APPNL-G-F mice back-crossed to C7 deficiency (APPNL-G-FxC7) with unmodified APPNL-G-F mice. To assess the effect of therapeutic C7 blockade, unmodified APPNL-G-F mice were treated systemically (for four weeks) with anti-C7 mAb or control IgG and the same set of parameters of complement dysregulation, pathology and cognition measured.
Results: C7 deficiency in AppNL−G−F mice reduced levels of complement activation markers, reduced amyloid load and increased synapse density with a commensurate improvement in cognitive test performance. Systemic treatment of AppNL−G−F mice with a blocking anti-C7 mAb caused reduced brain levels of complement activation markers, reduced amyloid load and increased peri-plaque synapse density and cognitive performance when compared to controls.
Conclusions: We demonstrate that complement dysregulation occurs in brain in the AppNL−G−F mouse model of AD. C7 deficiency reduced brain complement dysregulation, reduced synapse loss and improved other pathological parameters and cognitive function. Systemic anti-C7 therapy reduced complement dysregulation and protected from synapse loss in the model. Current efforts to modify the anti-C7 mAb for brain delivery will enhance efficacy in the model. The findings highlight the potential for complement inhibition at the level of MAC as a therapy in AD.
Background: We previously described that non-GPI anchored CD59 splice isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2) are required for insulin secretion from pancreatic β-cells and that the expression of both IRIS-1 and 2 is significantly reduced in islets isolated from human type 2 diabetic (T2D) patients, as compared to healthy controls. While assessing the expression of IRIS-1 and IRIS-2 in various human tissues we found high RNA expression levels of these isoforms in the human brain, however, their protein expression or role in the brain was unknown.
Methods: Using confocal microscopy, noradrenaline ELISA, proximity ligation assay (PLA), and expression studies on primary human and mouse brain sections, and neuronal cell lines we assessed the localisation and function of IRIS-1 and IRIS-2.
Results: Both IRIS-1 and IRIS-2 exist at mRNA and protein levels in human and mouse brains, where they localize in astrocytes and neurons, but not in microglia. IRIS-1 and IRIS-2 are found within the cytosol of the neuroblastoma cell line (SH-SY5Y), and their expression significantly increases in neuroblastoma cells differentiated into mature neurons with retinoic acid. Knockdown of IRIS-1 and IRIS-2 in SH-SY5Y cells greatly reduces the SNARE complex formation (needed for synaptic vesicle exocytosis). As a result, cells with IRIS-1/2 knockdown display significantly reduced secretion of noradrenaline. We have also shown that neuronal IRIS-1 and IRIS-2 expression is reduced in patients with Alzheimer’s disease (AD) and that phosphorylated Tau expression (a key pathological feature of AD) is much higher in SH-SY5Y cells with IRIS-1/2 knockdown.
Conclusions: Our data prove the existence of non-GPI anchored CD59 splice isoforms IRIS-1 and IRIS-2 in human and mouse brains and indicate that these isoforms are required for synaptic vesicle exocytosis. Additionally, the knockdown of IRIS-1/2 in SH-SY5Y cells results in increased activity of cyclin-dependent kinase 5 (CDK5) which causes Tau protein hyperphosphorylation and the accumulation of phosphorylated Tau in these cells, accelerating AD pathology.
Background: Repetitive mild closed head injury (rmCHI) results in the development of cognitive deficits and may lead to neurodegenerative disease later in life. The underlying neuroimmune mechanisms linking rmCHI to cognitive decline are not well understood, and the role of the complement system in this context is unexplored.
Methods: We developed a mouse model of rmCHI and examined pathophysiological and cognitive outcomes in injured vs. non-injured animals in the context of complement inhibition. Flow cytometry was used to characterize local and peripheral immune cell recruitment after injury. Immunofluorescence microscopy was used to analyze complement involvement, as well as to characterize microglial morphology and activation status. A multi-omics approach including proteomics and RNAseq coupled with CyTOF was employed to further dissect the neuroimmune response and the role of complement in brain pathology and animal behaviour after rmCHI.
Results: Of various rmCHI paradigms investigated, a 12 hit model was found to be optimum in terms of measurable outcomes. Following 12 hit rmCHI over 24 days, animals exhibited worsened spatial learning and memory retention. Flow cytometry revealed increased infiltration of various innate and adaptive immune cells. Immunofluorescence staining showed elevated complement deposition in the hippocampus of injured animals compared to non-injured. Microglial ramification was reduced after 12-hits, which was also associated with decreased microglial cell volume. In a therapeutic approach, we treated animals with an injury site-targeted complement inhibitor, CR2Crry, which inhibits all activation pathways of complement at C3 cleavage. CR2Crry treatment of injured animals improved cognitive outcomes, which was associated with changes in the abundance of distinct microglial sub-populations as revealed by mass cytometry. RNAseq and proteomic analysis revealed major changes in neurodegenerative associated pathways after rmCHI, with many of these alterations moderated by complement inhibition.
Conclusion: We developed and characterized a closed head repetitive injury model and demonstrated a role for complement in cognitive decline and the upregulation of multiple neurodegenerative markers/pathways post rmCHI. Targeting the complement system as a therapeutic approach in repetitive brain injuries requires further investigation.
Tour of Brewery and tasting session (must have booked in advance)
ECR (Early Career Researchers) Pub Quiz (must have booked in advance)