Teaching Day Agenda
The morning lectures are open to all ICW delegates.
Welcome and Introduction (Room G.56)
The (canonical) complement cascade and amplification loop. (Room G.56)
Complement and disease (Room G.56)
Intracellular roles of complement (Room G.56)
Complement therapeutics. (Room G.56)
Using murine models to understand human disease and develop therapeutic strategies (Room G.56)
Diagnostics (Room G.56)
Round-up before afternoon session (Room G.56)
Neuroinflammation / degeneration (Room 2.16 – 2nd floor)
Genetics and disease (Room 1.17 – 1st floor)
Infectious disease (Room 2.14 – 2nd floor)
Complement and cancer (Room 2.15 – 2nd floor)
Non-canonical roles of complement (Room 1.18 – 1st floor)
Neuroinflammation / degeneration (Room 2.16 – 2nd floor)
Genetics and disease (Room 1.17 – 1st floor)
Infectious disease (Room 2.14 – 2nd floor)
Complement and cancer (Room 2.15 – 2nd floor)
Non-canonical roles of complement (Room 1.18 – 1st floor)
We are hosting a welcome reception, open to all delegates on the evening of the 31st August at The Baltic, located along Newcastle/Gateshead’s famous quayside.
We will have some local music, canapes and drink, so hope to you all there.
The event will start at 7pm, finishing at 10pm
Baltic Centre for Contemporary Art
South Shore Road,
Information to getting to the venue can be found here.
Visualizing Immunity and Inflammation in Tissues Using Dynamic and Multiplex Static Imaging – Ron Germain
Session 1 – Bridging to cellular immunity
Background: Previously, we observed a reduced switch from antigen-specific IgM to IgG autoantibodies in C5ar1-/- mice in a murine model of autoimmune skin disease, associated with strong upregulation of C5aR1 in Germinal Center B cells (GCB). Here, we used an in-vitro model of GCB induction to define the pathways downstream of the C5a/C5ar1 axis controlling the switch from IgM to IgG production in induced GCBs (iGCB).
Methods: We co-cultured 3T3 cells overexpressing CD40-ligand and B-cell-activating factor (BAFF) (40LB cells) (Haniuda and Kitamura 2019) with naïve B cells from either C57BL/6 WT or C5ar1-/- mice in the presence of IL-4 for 4 or 6 days. At both time points, we determined the expression of the GCB markers GL7 and CD95 (Fas), IL-4-receptor, IgG1 and IgM production as well as CD138+ plasma cell (PC) differentiation. Further, we assessed expression of C5 and C5aR1 as well as C5a generation in 40LB, naïve B cells and iGCBs.
Results: We observed strong intracellular expression of C5, C5a and C5aR1 in 40LB cells. A small fraction of naïve B cells from WT or C5ar1-/- mice expressed C5 but not C5a or C5aR1. After 4d of co-culture CD95 but not GL7 expression was reduced in iGCBs from C5ar1-/- as compared to WT mice. In iGCBs from WT mice, 65% expressed C5 and 50 % C5a at d4, which was significantly lower in iGCBs from C5ar1-/- mice. Strikingly, IL-4 receptor expression was downregulated in iGCBs from C5ar1-/- mice at d4, which was associated with a reduced IgM to IgG1 switch at d4 and d6 as compared with iGCBs from WT mice. Finally, we found induction of PCs from iGCBS at d6, the frequency of which was significantly lower in cells from C5ar1-/- as compared to those from WT mice.
Conclusion: Our findings suggest a model in which autocrine (in iGCBs) and/or paracrine (in 40LB cells) production of C5 and generation of C5a activates C5aR1 in iGCBs to drive their differentiation and expression of IL-4 receptor, which is crucial for the switch from IgM to IgG1 antibody production. Later, C5a/C5aR1 axis activation controls the differentiation of PCs from iGCBs.
Complement C5a has been shown to be critically involved in many autoimmune diseases through activation of its cognate receptors, C5aR1 and C5aR2, expressed on effector cells. While the role of C5aR1 in autoimmune disease is well understood, the function of C5aR2 remains elusive. Initially thought to be a decoy receptor that negatively regulates the functions of C5aR1, C5aR2 has recently been shown to exert both pro- and anti-inflammatory properties independent of C5aR1. Recent experiments by our group using C5aR2 reporter mice showed that B cells express C5aR2 but not C5aR1 in the steady state. We also observed an altered maturation of B cells in C5ar2–/– mice. These findings prompted us to further investigate the role of C5aR2 in B cells in the context of early autoimmunity. First, using flow cytometry to assess the role of C5aR2 for B cells under homeostatic conditions, we found that naive splenic B cells from C5ar2–/– mice showed a drastically increased expression of the decay accelerating factor (DAF, CD55) compared to B cells from wild-type and C5ar1–/– mice. Since the regulation of DAF expression has been shown to be critical for a functional germinal centre response, we hypothesised that the increased DAF expression in B cells of C5ar2–/– mice might affect their differentiation. To investigate this, we used an in vitro culture system in which we co-cultured splenic B cells with 40LB fibroblast feeder cells that exogenously express CD40‑ligand (CD40L) and B cell activating factor (BAFF), which promote B cell proliferation and survival and mimic the germinal center response. Flow cytometric analysis of the co-cultured B cells revealed attenuated activation, differentiation and class switching of B cells from C5ar2–/– mice compared to the wild-type. These results also suggest a potential role for C5aR2 in autoimmunity, which we will investigate in future experiments. Taken together, our findings highlight a potential role of C5aR2 as a novel checkpoint for B cell development, possibly by regulating DAF expression during B cell maturation.
Background: Complement factor H (FH) is a critical complement regulator and its absence results in kidney disease. FH deficiency renders C57BL6 mice susceptible to immune complex mediated glomerulonephritis. Significant macrophage infiltration occurs in this setting and understandably, depletion of macrophages reduced the disease pathology. Macrophages participate in defense, inflammation, restoration and fibrosis of the kidney.
Gap in knowledge: Macrophages express intracellular FH, but its role in these cells remains incompletely understood. The present study steps into this gap in our understanding and investigates the role of FH in macrophages.
Methods and Results: Wild type (WT) and FH knockout (KO) bone marrow derived macrophages were isolated, cultured and analyzed to understand the impact of FH deficiency. In FH deficient macrophages, nanostring gene expression analysis using mouse myeloid panel and stringent analysis revealed that of the 754 genes on the array, 73 genes were increased and 20 genes were decreased with at least 1.5 fold change and p<0.05. The significantly increased genes include the inflammatory genes TNF, Nlrp3, IL1B and IL6. In line with the increased mRNA expression of VEGF, ATF3, Egr3, Myc, Csf1R genes, macrophage trafficking and migration were increased compared to WT macrophages. Phagocytosis was reduced in FH deficient macrophages, and oxidative metabolism was decreased by 40%. Since transient receptor potential (TRP) channels modulate macrophage functions such as phagocytosis and cytokine generation, we studied the effect of FH on these channels. FH modulated the mRNA expression of the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in macrophages. In line with increase in mRNA expression of TRPM7, patch clamp assessment showed that TRPM7 current was significantly higher in FH deficient macrophages compared to wildtype macrophages. The TRPM7 current was blocked by FTY720, a specific TRPM7 inhibitor. FTY720 also significantly attenuated the FH regulated proliferation of macrophages.
Conclusion: For the first time our results show that FH is of functional relevance in macrophages, and establishes a critical link between intrinsic FH and macrophage effector functions through TRPM7, which could serve as a potential therapeutic target in FH modulated disease settings.
Background: Complement and B cells are implicated in the pathophysiology of rheumatoid arthritis, an autoimmune disease characterized by synovial inflammation. Loss of complement regulator CD55 leads to delayed onset of inflammatory arthritis in the collagen-induced mouse model of arthritis (CIA).1 Marginal zone (MZ) B cells, an innate-like B cell population, serve as first responders to blood-borne pathogens by rapidly differentiating into antibody-producing plasmablasts, and are important initiators of CIA.2 Whether and how CD55 regulates MZ B cells in CIA pathogenesis is unknown.
Methods: We examined naïve and sorted MZ B cell populations in 3-5 week-old and 10-12 week-old CD55-/- and compared to wildtype (WT) C57BL/6 mice using flow cytometry, immunofluorescence, bromodeoxyuridine (BrdU) incorporation, and bulk RNAseq. Differential gene expression analysis was performed, followed by Gene Sequence Enrichment Analysis (GSEA).
Results: We found that loss of CD55 decreased the number of MZ B cells in 10-12 week-old, but not 3-5 week-old mice. The precursors to MZ B cells were unchanged at both endpoints, indicating that loss of MZ B cells was not due to failure of MZ B cell differentiation. En vivo BrdU incorporation showed no difference between CD55-/- and WT, indicating MZ B cell differences are not due to loss of proliferative capacity. Differential gene expression analysis showed decreased expression of genes involved in preventing apoptosis in MZ B cells of CD55-/- compared to WT mice (HSP90b1, STIP1, AHSA1, and FKBP4). GSEA showed decreased expression of cell cycle/cell survival pathways in CD55-/- cells relative to WT. Flow cytometry revealed that CD55-/- MZ B cells had corresponding increases in activated caspase-3 and caspase-7, as well as increases in cellular reactive oxygen species relative to WT cells.
Conclusion: Loss of CD55 led to increased reactive oxygen species and activation of caspases in MZ B cell populations, which may prevent the normal expansion of the MZ B cell subset over time. These studies suggest a role for the complement regulator CD55 in MZ B cell homeostasis and survival. Future experiments are aimed at defining whether loss of CD55 impacts MZ B cell survival through complement-dependent or independent mechanisms.
Session 2 – Preclinical models in kidney diseases
Complement mediated atypical haemolytic uraemic syndrome (c-aHUS) arises from impaired regulation or gain-of-function changes in the alternative pathway (AP). Eculizumab is the gold standard therapy for c-aHUS but there is room for improvement in patient care. The unique mouse model of aHUS based on a single D1115N amino acid substitution in C3, (C3N/N, PMID: 30714990) provides a testbed to evaluate targeted complement inhibition and we aimed to establish new effective therapies in c-aHUS.
Therapeutic blockade of the AP using anti-properdin monoclonal antibodies (mAb H4, 14E1), CR2-FH, homodimeric minimal-FH (HDM-FH – protein and gene therapy) or FH1-5, and terminal pathway (TP) blockade with an anti-C7 mAb or an oral C5aR antagonist (ACT-1014-6470) was carried out using previously published dosing strategies. A prophylactic (from weaning) or rescue strategy (after 2 days of haem at 25 ery/ul) was used as appropriate. Upon study completion, animals were culled, tissue collected, and analysed for histological evidence of renal disease, fibrin deposition and glomerular complement deposition. Plasma was analysed for renal impairment (BUN), haemolysis indices (Haemoglobin, reticulocyte and platelet count) and complement levels.
No C3N/N mice succumbed to aHUS during prophylactic use of the oral C5aR1 antagonist (up to 80 days) which mirrored genetic deletion of C5aR1 (as previously reported1). Similarly, anti-C7 mAb therapy rapidly reduced evidence of renal damage and no mice succumbed to disease. Anti-properdin mAb 14E1 (similar to genetic deletion of CfP2) and HDM-FH (protein) were also highly effective in restoring renal function but CR2-FH (even when given daily) and FH1-5 largely failed to prevent acute TMA and renal damage in C3N/N mice. Therapeutic use of the oral C5aR antagonist and prophylactic gene therapy using HDM-FH is ongoing with no mice succumbing to TMA thus far.
Our data suggest that therapeutic targeting of either arm of the TP and disruption of the C5 convertase can significantly reduce renal damage and prevent c-aHUS (in the context of a C3 gain-of-function change) providing novel insight into disease mechanisms as well as illuminating potentially new disease maintenance and treatment strategies.
References: Conference abstracts by 1. Smith-Jackson et al and 2. Kamala et al, see 10.1016/j.molimm.2022.07.003
Atypical haemolytic uraemic syndrome(aHUS) is rare kidney disease, caused by thrombotic microangiopathy (TMA). Mutations in complement regulators and activators can ‘trigger’ TMA but exact mechanisms remain unclear. Recently, we showed that a C3 gain-of-function point mutation (C3D1115N) when in homozygous (or C3N/N) led to TMA and aHUS in mice. However, as patients seldom have homozygous mutations, we sought to investigate whether aHUS could be ‘precipitated’ in C3+/N (heterozygous) mice following various stimuli, that would mimic infections long suspected to be linked to aHUS in patients.
C3+/N mice were injected intraperitoneally with O111:B4 lipopolysaccharide (LPS) at various concentrations (0.1, 0.2 or 0.5 mg/kg) and were monitored (weight and urine analysis) until day (D)3. In additional studies, mice received 0.2 mg/kg LPS on D0 and D7, mice were tracked until D10. Alternatively, C3+/Nand wild type mice received an intra nasal dose of influenza strain X31 (7.8×105 plaque forming units) and monitored until D14. In our final approach, a Freund’s adjuvant free method of nephrotoxic serum (NTS) nephritis was performed. Briefly, mice received two priming doses of sheep IgG on D0 and D7, before receiving 25, 50, 100 or 200µl of NTS on D14, mice were monitored to D17. In all cases, serum was analysed at cull for evidence of renal disease and complement activation split products. Kidneys were assessed for TMA by histological approaches.
While there was evidence that C3+/N mice where more susceptible to these agents, with increased proteinuria/weight loss being common, no mice succumbed to TMA or exhibited significant haematuria. Furthermore, while altered B and T cell populations were found and greater levels of C3 breakdown products detectable in the serum and kidney of the C3+/N mice, this did not translate into aHUS.
These data confirm that excess complement activation systemically, through viral infection or immune complex is not sufficient to precipitate aHUS in C3+/N mice. These data indicate the mouse is potentially more robust than man with respect to development of aHUS and CRRY may play a role in this situation. Although further investigation is needed to clarify this finding.
Background: Mannose-binding lectin-associated serine protease-1 (MASP-1) and MASP-3, transcribed form the common Masp1 gene, play critical roles in the lectin and alternative complement pathway activation, respectively. We previously reported that Masp1-knockout (deficient for both MASP-1 and MASP-3) lupus-prone MRL/lpr mice showed significantly reduced glomerulonephritis and albuminuria compared to their wild-type littermates. The data suggested that MASP-1 and/or MASP-3 play an important role in the development of lupus-like glomerulonephritis in these mice via activation of the lectin and/or alternative pathways. Here, we aimed to clarify the role of MASP-1 in the development of renal disease in lupus by analyzing disease expression in MRL/lpr mice monospecifically deficient for MASP-1.
Methods: MASP-1-deficient mice were generated by the CRISPR/Cas9-mediated genome editing in the C57BL/6 background and then backcrossed to the MRL/lpr background for eight generations. Sera and urine were collected biweekly from groups of MASP-1-deficient and wild-type MRL/lpr mice starting at 12 weeks of age. Serum C3, anti-dsDNA IgG antibody and urinary albumin excretion levels were measured by ELISA. Circulating immune-complex levels were measured by ELISA utilizing anti-C3 and anti-mouse IgG antibodies. Mice were sacrificed at 24 weeks of age and kidneys were collected for histopathological analysis.
Results: MASP-1-deficient MRL/lpr mice showed no significant differences in serum C3, anti-dsDNA IgG antibody and circulating immune-complex levels compared to their wild-type littermates. Wild-type MRL/lpr mice developed albuminuria as early as 12 weeks of age, whereas MASP-1-deficient MRL/lpr mice developed albuminuria around 16 weeks of age. Wild-type MRL/lpr mice started to die as early as 16 weeks of age, whereas no MASP-1-deficient MRL/lprmice died until 20 weeks of age (p = 0.019 at 19 weeks of age). However, histopathological analysis of the kidneys collected at 24 weeks of age showed no significant differences in C3 deposition levels and in the development of lupus-like glomerulonephritis including proliferative, necrotic and crescentic changes between the two groups.
Conclusion: Our results suggest that MASP-1 accelerates the development of albuminuria and mortality in MRL/lpr mice, most likely via activation of the lectin pathway.
Leptospirosis is a neglected zoonosis affecting approximately 1 million people each year worldwide and causing near 5% deaths. Most patients are asymptomatic or have only mild clinical manifestations. However, some patients develop severe chronic and end-stage kidney disease responsible, which may be fatal. We are investigating the role of C3 in the chronic leptospirosis by using C57BL/6 wild-type (WT) and C57BL/6 C3-knockout (C3KO) male mice infected with 108 L. interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) or PBS (negative control) after 15, 30, 60, 90 and 180 d.p.i.. We observed the following results: i) all infected mice survived. However, more leptospires were observed in the kidney of LIC-C3KO mice up to 90 d.p.i., when compared to LIC-WT mice, by immunohistochemistry analysis; ii) LIC-infected mice lost more body weight during the first 5 days of infection when compared to PBS, independent of the presence of C3; iii) no significant differences in the spleen/total body weight ratio were observed between LIC-WT and LIC-C3KO after 15 d.p.i. or 30 d.p.i.; iv) LIC-C3KO mice presented higher scores of histopathological alterations in the liver and kidney, when compared to LIC-WT mice at 30 d.p.i up to 180 d.p.i.; v)) higher percentage of fibrosis was observed in the kidney from LIC-C3KO mice when compared to LIC-WT at 30 d.p.i., after Red Sirius staining, which reveals the presence of collagen I and III in the tissue. In conclusion, C3 did not affect the survival of LIC-infected C57Black/6 mice but it was important for the in vivo killing of leptospires. Leptospira remains for longer time in the liver and kidney of LIC-C3KO mice when compared do WT in the same conditions. In addition, the lack of this protein may trigger renal fibrosis in the infected mice at 30 d.p.i. suggesting that the presence of local C3 may control tissue repair after damage by pathogens.
Committee meeting ECCO
Session 3 – C5a and receptors in health and disease
Background: C5a regulates the differentiation and function of dendritic cell (DC) subsets from bone marrow-derived precursors through C5aR1 activation. Here, we determined the impact of the C5a/C5aR1 axis on FMS-like tyrosine kinase 3 ligand (Flt3L)-induced in vivo generation and function of splenic conventional DC (cDC) subsets.
Methods: Flt3L expressing B16 melanoma cells were subcutaneously injected into wildtype and C5ar1-/- mice. After 10 days spleens were harvested. cDC1 and cDC2 subsets were FACS-sorted, incubated with LPS-free ovalbumin (3 or 10 ug/ml) ± TLR ligands (LPS, PAM3) and co-cultured for 72h with ovalbumin-specific TCR transgenic OT-II CFSE-labelled CD4 T cells. Then we determined the generation of effector memory (TEM) and effector T cells (TEFF) and their differentiation towards Th1 (IFN-y) and Th17 (IL-17A) effector lineages.
Results: Flt3L induced mobilization of cDC1 and cDC2 cells in the spleen was independent of C5aR1 expression. Ovalbumin stimulation ex vivo resulted in strong TEFF and less TEM generation by cDC2, which was markedly reduced in cDC2 from C5ar1-/- mice at low ovalbumin concentration. Surprisingly, TEFF and TEM generation was absent using cDC1 cells. Additional LPS/PAM3 stimulation of cDC2 cells from either wildtype or C5ar1-/- mice increased the dominant TEFF cell differentiation. In contrast, TEM and TEFF induction was similar using cDC1 cells from wildtype or C5aR1-deficient mice. Strikingly, we found a strong and dominant IFN-y production upon wildtype cDC2 cell stimulation with OVA ± TLR ligands and OT-II cell co-culture, which was significantly reduced using C5ar1-/- cDC2 cells.
Conclusion: Our findings suggest that cDC2 but not cDC1 cells are critical for antigen-driven T cell proliferation and TEFF differentiation in the absence of TLR ligands. Further, C5aR1 activation seems to be crucial for this effect at low but not at high doses of antigen. Also, while C5aR1 activation did not affect TEFF or TEM cell differentiation induced by cDC1 or cDC2 following OVA ± TLR ligand stimulation, it controlled the dominant Th1 induction mediated by cDC2 cells. Thus, we identified a novel role for C5aR1 in antigen-driven TEFF differentiation by splenic cDC2 cells and subsequent differentiation into Th1 cells in response to pattern recognition receptor activation.
Mycobacterium avium (Mav) is one of the most successful pathogens infecting billions of people with daily exposure to water and soil. Mav belongs to the group of non-tuberculous mycobacteria (NTM) which affects the elderly and immunocompromised individuals. With the evasion and alteration of host inflammatory response, Mav persistence leads to severe pathogenicity resulting in interstitial chronic lung disease. Given that the C5a peptide is pivotal in chronic inflammatory and infectious diseases, we studied the role of C5a receptor 1 (C5ar1) signaling response in the in vivo Mav pathogenicity. We infected C5ar1-deficient mice (C5ar1KOC57B6/J), and the wildtype littermates (WTC57B6/J) with the aerosol exposure of a clinical strain ofMav(5×108 CFU/ml) and examined the immunopathologic changes up to 90 days post-infection (dpi). The Ashcroft scores obtained using Masson’s trichrome staining revealed significant fibrogenic burden withMav granulomas in the 30-dpi Mav-WT lung tissues that increased through 60 and 90-dpi than their C5ar1-deficient counterparts. Significantly increased inflammatory cytokines expressions including Il-1β and Tnfα were observed in the Mav- WT lungs at 60 and 90-dpi, which also corroborated with significantly increased proinflammatory helper T cell (Th1) specific marker Ifnγ, and its regulator T-Box transcription factor 21, Tbx21expressions in the BAL fluid cells. These markers were significantly low in the Mav-C5ar1KO mice correlating with low Ifnγ levels measured by ELISA. Importantly, marked Tbx21/T-bet immunofluorescent staining indicated Th1-specific immune cell subset in the Mav-WT lungs. Finally, we confirmed the role of C5ar1-dependent Th1 response in Mav-induced fibrogenic changes using splenocytes isolated from WT and C5ar1KO mice. In the absence of C5ar1 signaling, Mav-infected splenocytes demonstrated an impaired helper T cell response with a significant decrease in the Ifnγ and Tbx21 transcript levels, and decreased CD4+ /CCR5+-proinflammatory Th1 cell subsets were measured flow-cytometrically. Therefore, our data indicated a C5ar1-dependent proinflammatory Th1 response during the fibrotic tissue remodeling in Mav-infected mouse lungs.
Streptococcus pneumoniae (Spn) is one of the leading bacterial pathogens causing mortality among adults. Complement activation plays a pivotal role in the clearance of encapsulated Spn and the pathogenesis of ARDS. The subsequent release of the anaphylatoxin C5a promotes the recruitment and activation of inflammatory cells including neutrophils after C5a recognition by its two homologous receptors, C5aR1 and C5aR2. The blockade of C5a attenuates pro-inflammatory responses and rescues lethality in various experimental models of sepsis and inflammation. However, there is insufficient evidence whether C5a neutralization exerts protective or harmful effects during Spn infection.
We established a novel homozygous C5aR1/2-/- double-knockout mouse strain using CRISPR/Cas9 guided gene editing to assess the direct role of C5aR1 and C5aR2 during Spn infection. To further profile alveolar cell populations at the single cell level, we performed TOTAL-seq on FACSorted live CD45+ BALF cells using ~200 oligonucleotide-conjugated antibodies enabling simultaneous detection of surface protein markers and RNA.
The C5a-induced influx of neutrophils to the airways was completely abrogated in C5aR1/2-/- mice compared to C57BL/6J wild-type (WT) mice. In striking contrast, C5aR1/2-/- mice showed a stronger inflammatory response in the alveolar spaces after Spn infection, as indicated by enhanced neutrophil infiltration, increased cytokine/chemokine secretion, and exacerbated lung vascular permeability. Interestingly, the neutrophils recruited in C5aR1/2-/- mice released more neutrophil extracellular traps (NETs) but exhibited impaired bactericidal function as suggested by higher CFUs in BALF. Single cell proteotranscriptomics revealed 8 distinct subclusters of alveolar macrophages and 7 subclusters of neutrophils after Spn infection – which displayed heterogeneous levels of C5aR1 protein and C5aR2 RNA expression in WT mice. Consistently, neutrophil clusters were more abundant in C5aR1/2-/- mice but macrophage counts were reduced. Furthermore, C5aR1/2-/- neutrophils developed signs of exhaustion resembling human septic neutrophils with elevated expression of PD-L1 and ICAM-1, and decreased expression of CXCR2 and CD62L. These findings would explain the reduced capacity of neutrophils to control bacterial growth in C5aR1/2-/- mice, creating a potent chemotactic local environment for a persistent neutrophil influx and therefore, resulting in greater lung damage.
Concomitant inhibition of the C5a-C5aR1/2 axes could drive neutrophil exhaustion and lung injury after Spn infection.
Background: The anaphylatoxin receptors play a critical role in the pathogenesis of various autoimmune diseases. In this context, the enigmatic second receptor for the anaphylatoxin C5a, C5aR2, is often overlooked. In this study, we aimed to elucidate the impact of C5aR2 on the pathogenesis of epidermolysis bullosa acquisita (EBA), a prototypical autoimmune skin disease characterized by neutrophil-dependent skin blistering in which complement activation is a prerequisite.
Methods: We used mice with a global or a LysM-specific deficiency of C5ar2 in a passive model of EBA. In this model, mice were subcutaneously injected with antibodies against type VII collagen (COL7) to induce neutrophil-dependent blistering of the skin. In addition, we performed in vitro assays to assess how targeted deletion of C5ar2 affects C5a-induced activation and effector functions of neutrophils. In these experiments, neutrophils from wild-type and C5ar1–/–mice served as controls. We employed single-cell RNA-sequencing to identify differentially expressed genes and elucidate the molecular mechanisms underlying C5a-induced activation in neutrophils.
Results: Mice lacking C5aR2 globally or specifically in LysM-positive cells were found to have a significantly ameliorated disease phenotype compared with wild-type mice. The relative C5a-mediated activation and effector functions of neutrophils in these mice were correspondingly decreased. This reduced responsiveness to C5a stimulation was associated with a lower number of differentially expressed genes and a changed transcriptome profile compared with neutrophils from wild-type mice. Notably, neutrophils lacking C5ar1 expression did not respond to C5a stimulation, which was reflected in an unchanged transcriptome profile.
Conclusion: These results suggest that the often-neglected C5aR2 is a critical contributor to neutrophil-driven autoimmune diseases such as EBA. While our in vitro assays clearly indicate a dependence of C5aR2 on C5aR1, our single-cell RNA-sequencing results shed light on how signaling through C5aR2 affects gene expression. However, to fully understand the signaling cascade downstream of C5aR2 and its dependence on C5aR1, further research is needed. This will also help determine the potential efficacy of targeting C5aR2 to treat neutrophil-driven diseases such as EBA.
Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a major cause of death in RA patients with 40% mortality rate within 72 months from the time of clinical diagnosis. RA-ILD is manifested with usual and non-specific interstitial pneumonia and lung fibrosis. The role of complement activation is poorly understood in the pathogenesis of RA-ILD. We hypothesized that complement anaphylatoxin C5a via its receptor C5ar1, may contribute to the pathogenesis of RA-ILD. To unravel the molecular mechanisms of C5ar1-signaling, we induced collagen-induced arthritis in C5ar1 deficient-C57B6/J (C5ar1KO) mice and their wildtype littermates (WT). A set of arthritic mice were challenged with intradermal C5a (2.5mg/50μl) at 24-hour intervals for 27 days. All mice were euthanized on day 28 and lung tissues were collected for histologic and molecular analyses. Masson’s trichrome staining revealed pleural hypertrophy and fibrogenic foci in the WT arthritic lungs. C5a-challenged WT arthritic mice revealed enhanced hyaluronan production and presence of myofibroblast activation marker, α-SMA as compared to the C5a-challenged C5ar1KO group. We further approached our hypothesis with Poly(A)-ClickSeq RNA sequencing analysis and the PolyA-miner algorithms to study alternative polyadenylation (APA) as a post-transcriptional mechanism of gene regulation involving 3’-UTR sequences. Distinct 3’-UTR landscapes in proinflammatory genes result in transcript isoforms lacking regulatory miRNA binding sites during chronic inflammation. Our data indicated increased 3’-UTR lengthening profile with 477 transcripts in arthritic C5ar1KO mice compared to their WT counterparts. This included the presence of cleavage and polyadenylation specificity subunit 5 (Cpsf5/ Nudt21), a key regulator of pulmonary fibrosis. C5a-pulsed WT mice showed 1240 3’-UTR-shortened genes including Notch4 and RNA-binding protein Zfp871, compared to their C5ar1KO counterparts. C5a-pulsed arthritic WT mice demonstrated 972 disease-specific 3’-UTR-shortened genes relating C5ar1 signaling with endosomal trafficking, histone modifications, cellular senescence, and fibrogenesis. Intriguingly, significant upregulation of miRNA-29a and miRNA-30 was observed in C5a-arthritic mice, given that miRNA-29a is involved in inhibiting histone modification and fibrogenesis; and miRNA-30 targets cellular senescence. Thus, our data offer a novel mechanism of C5ar1-dependent 3’-UTR shortening in profibrogenic modulators that attenuates regulatory targeting by miR29a and miR-30 during arthritic lung remodeling.
In ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization and assessed its role if diseases featuring tissue ischemia in patients.
We have employed genetic mouse models to substantiate this. Indeed, the presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model, a mouse model of ischemia-induced revascularization. Ischemia-driven angiogenesis was significantly improved in C5aR1-/- mice, but not in C5-/- mice suggesting a specific role of C5aR1. Experiments using supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. WE characterized this secretion ot be a specific sub-alpha-granule specific event featuring a specific signaling cascade. Finally, we applied the C5aR1 inhibitor PMX-205 in vivo in the hindlimb ischemia model. This drug induced a phenotype of improved revascularization in mice.
Furthermore, we assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in coronary artery disease and found a correlation between specific but not all markers of platelet activation and C5aR1 expression on platelets. Furthermore, we have assessed the role of C5aR1 in peripheral artery disease (PAD) patients. We have matched a cohort with proven high-grade arterial stenoses of the leg or groin arteries but no ischemic walking pain with patients with PAD and typical symptoms. We hypothesized that lack of symptoms might be due to a better capacity for collateral artery growth in asymptomatic PAD patients. Strikingly, the C5aR1 expression on platelets and CXCL4 correlated with pain-free walking distance Thus, the C5aR1-CXCL4 axis seems to play a role in adaption to diseases featuring tissue ischemia also in cardiovascular patients.
We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by release of antiangiogenic CXCL4. Importantly, we can present evidence for the significance of this mechanism in cardiovascular patients suffering from diseases featuring tissue ischemia.
SVAR Prize: Awardees announced by SVAR representatives
Session 4 – Complement in cancer
Background: Loss of function STK11 mutations occur in 15-20% of non-small cell lung cancer (NSCLC) and are associated with poor survival and resistance to immune checkpoint inhibitors (ICI). We observed in human STK11-mutant NSCLC upregulation of C3, Factor D, Factor H, and CD55. We previously observed that in the tumor microenvironment (TME), neutrophils (PMN) acquire complement-dependent T cell inhibitory function. Less is known about the roles of tumor-derived complement in tumor progression. We hypothesized that tumor-derived complement will promote growth of STK11-mutant tumors in vivo.
Methods: CMT167 (syngeneic NSCLC) tumor cells with Stk11 deletion with and without C3 deletion were generated by CRISPR-Cas9. To evaluate the role of tumor-derived versus systemic complement in tumor progression, we compared growth of subcutaneous (s.c.) administered Stk11KOC3WT and Stk11C3KO tumor in WT versus C3-/- mice. To test the interaction of tumor-derived C3 and T cell immunity in regulating tumor growth, we compared tumor growth in WT versus nude mice. Finally, we evaluated whether PMN depletion with anti-Ly6G or Cxcr2 inhibition would overcome resistance of Stk11KO tumors to anti-PD-1 treatment.
Results: We observed high infiltration of PMN and low CD8 T cell in Stk11KO tumor in vivo. Systemic C3 deficiency resulted in modest delay of growth of Stk11WT tumors, but had no effect on growth of Stk11KO tumors. Deletion of C3 in CMT167-Stk11KO (CMT167 Stk11/C3KO) resulted in dramatic inhibition of tumor growth in immune competent mice, but had no effect in nude mice. While anti-PD1 treatment alone had modest to no effect in the growth of Stk11KO tumors, the addition of anti-Ly6G or Cxcr2 inhibitor resulted in improved control of tumor growth.
Conclusions: Tumor-derived C3 drives Stk11KO tumor growth in mice. Growth of Stk11KO tumor was strikingly dependent on tumor-derived C3 in immunocompetent mice but dispensable in nude mice. These results support a role for tumor-derived C3 suppressing T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in STK11-mutant NSCLC.
Background: Normal cells are equipped with numerous membrane-bound complement inhibitors that protect them from misguided complement attack. However, soluble complement inhibitors are rarely produced in locations other than the liver. Previously, we reported the expression of complement factor I (FI) in non-small cell lung cancer (NSCLC) cell lines.
Methods: FI expression in cancer biopsies from lung adenocarcinoma and squamous cell carcinoma patients was assessed by immunohistochemical staining and associated with clinicopathological characteristics and clinical outcomes. To approach the question of whether the expression of FI by tumor cells was aimed to protect tumor cells from host innate immunity, the deposition of C4d – the end degradation product of FI-supported inactivation of active complement component C4b was analyzed in the same tissue. To elucidate the role of FI in lung cancer cell physiology, three human non-small lung cancer cell lines naturally expressing FI were engineered with CRISPR/Cas9 technology and the transcriptome of FI-deficient and FI-sufficient clones was compared in each cell line. These cells were also compared in in vitro colony-formation assay.
Results: FI immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, differentiation grade, stage, T cell infiltrates or PD-L1 expression, but was associated with progression-free survival (PFS) and disease-specific survival (DSS). Multivariate Cox analysis of high vs. low expression of FI revealed HR 0.55, 95% CI 0.33-0.95, p=0.031 for PFS and HR 0.35, 95% CI 0.15-0.78, p=0.011 for DSS. Importantly, only negligible C4d staining was found in cancer tissue. RNA sequencing in FI knockout and wild-type NSCLC cells revealed differentially expressed genes of potential importance for intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. In vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium.
Conclusions: We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.
This project was supported by National Science Centre (Poland) grant no. 2014/14/E/NZ6/00182
Background: The complement system can exert pro- or anti-tumorigenic functions based on different tumour types and contexts, suggesting novel perspectives for therapeutic targeting in selected subgroups of patients (1). We aimed at developing an innovative prognostic tool based on the in situ quantification of several complement components for the prediction of malignant pleural mesothelioma (MPM) patient outcome, introducing the definition of “Complement Score”.
Methods: Bioinformatics analysis via GEPIA and UALCAN (TCGA-MESO); immunohistochemistry on MPM tissue microarrays (TMAs,n= 88) and whole tissue sections (n=17); clinical information collection and statistics analysis.
Results: Bioinformatics analysis of genes encoding for complement activation components (n= 27) and regulators/receptors (n= 29) allowed the selection of four promising markers in MPM: the mRNA expression of C1S, SERPING1, CFB and CFI resulted to be positively correlated with patient overall survival. C1q was included in further analysis due to its abundance in MPM microenvironment (2). Protein expression of the selected complement components was evaluated both on TMAs and whole tissue sections of MPM patients, being expressed as percentage of tumour cell positivity, immune cell positivity and deposit. C1s, C1INH, CFB and CFI displayed cytoplasmic positivity of tumour cells, but also of immune cells and deposit, whereas C1q presence was only detected as deposit or positivity of monocytes/macrophages. Univariate analyses were performed in order to correlate the expression of each complement component with MPM histotype, TILs (CD4+, CD8+, CD19+), tumor proliferative activity (Ki-67), PD-L1 expression, overall survival. Survival analysis showed that C1qHIGH (Log-rank test, χ2 = 6.01;p= 0.01) and C1INHHIGH(Log-rank test, χ2 = 5.13;p= 0.02) patients displayed significantly increased survival.
Conclusions: Complement Score could be hopefully used in the future as a tool to stratify MPM patients, directing each patient to a personalized treatment, and its application could be potentially extended to other solid tumours, unveiling the “double-edged sword” role of complement system in cancer.
1. Revelet al., Antibodies 2020, https://doi.org/10.3390/antib9040057
2. Agostiniset al., Front Immunol 2017, https://doi.org/10.3389/fimmu.2017.01559
Background: Inflammation plays a pivotal role in the development and progression of colorectal cancer (CRC). Emerging evidence suggests that complement anaphylatoxin C3a produced upon complement activation and acting via its receptor (C3aR) may play a role in intestinal homeostasis. However, its role in CRC is currently unknown.
Methods: We used a comprehensive approach encompassing analysis of publicly available human CRC datasets, inflammation-driven and newly generated spontaneous mouse models of CRC, and multi-platform high dimensional analysis of immune responses using microbiota sequencing, RNASeq, and mass cytometry.
Results: By mining publicly available datasets, we found that CpG island methylation of c3ar1 occurs in CRC patients and is associated with significant downregulation of C3aR. By reverse-translating this finding, we were able to shift in APCMin/+ mice the tumorigenesis from the small intestine to the colon, therefore, generating a novel mouse model which more closely mirrors CRC in humans. RNAseq analysis on the polyps from our newly developed mouse model (APCMin/+/C3aR-/-) revealed a significant increase in immune signatures. Interestingly, loss of C3aR significantly impacted the fecal and tumor-associated microbiota, which promoted enhanced immune infiltration in typically “cold” tumors. In line with our findings in the mouse model, human CRC with C3aR downregulation showed increased innate and adaptive immune cells. Since immune infiltration is often a favorable prognostic factor in CRC and predisposes to response to immune checkpoint blockade (ICB) therapy, we assessed whether the enhanced immune infiltrate could be exploited to treat the tumors of APCMin/+/C3aR-/- mice. We found that using anti-PD1 in APCMin/+/C3aR-/- but not APCMin/+ mice resulted in significant tumor reduction. Therefore, the lack of C3a in the colon activates a microbiota-mediated pro-inflammatory program, promoting tumors’ development with an immune signature that renders them responsive to ICB therapy.
Conclusions. The complement system in the gastrointestinal tract is essential to avoid overt inflammation in health. However, this regulatory mechanism may restrain the activation of immune responses during tumor development. Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance anti-tumor immunity in CRC. C3aR can thus be exploited to overcome ICB resistance in a larger group of CRC patients.
Despite the substantial preclinical evidence for the role of complement in cancer, complement-based cancer therapies have not yet advanced to the clinic, likely, because of the failure to identify the optimal targets within the complement system and patients that will benefit from complement-based interventions. To address these needs, we identified the subsets of renal cell carcinoma (RCC) that are associated with upregulation of complement genes and can be potentially regulated by complement-mediated mechanisms. These mechanisms include complement anaphylatoxins-mediated inhibition of antitumor immunity and acceleration of angiogenesis in mouse model of cancer. The upregulation of complement genes in patients was associated with T cell exhaustion and myeloid cells’ transcriptomic signatures linked to immune suppression. The complement-dependent subsets of RCC responded poorly to immune checkpoint inhibitors (ICI) and antiangiogenics. Abundance of plasma complement proteins was associated with the response to ICI, pointing to plasma complement as a potential predictive biomarker. In low grade RCC tumors, complement protein expression and deposition were limited to stroma and infiltrating cells, consistent with complement functions in the regulation of infiltrating immune cells. However, tumor cells in aggressive high grade RCC expressed large amounts of C3. In addition, we found expression of complement proteins and evidence of complement activation in several cellular organelles of tumor cells from common human cancers. Downregulation of these genes led to reduced tumor cell proliferation and downregulation of the mammalian target of rapamycin complex 1 (mTORC1) downstream signaling, consistent with the role of intracellular complement activation in T cells. Interestingly, tumors with hyperactivation of mTORC1 expressed high levels of complement proteins, suggesting the signaling regulatory loop involving complement and mTORC1. In conclusion, complement in human malignancies appears to regulate both host components of the tumor microenvironment that contribute to immunosuppression and tumor cell signaling essential for tumor cell growth. Thus, cancer patients are likely to benefit from targeting extracellular complement to improve antitumor immunity and complosome to stop tumor cell proliferation.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a survival rate at 5 years very low. Chemotherapy agents causes only a modest enhancement in the survival and is usually associated to toxic effects. The proteoglycan GPC1 showed a cell surface localization in PDAC tissues but limited or absent expression in the most of adult tissues and in chronic pancreatitis. Exploiting this promising tumor-associated antigen, an anti-GPC1 monoclonal IgM was produced with the aim to induce a strong complement activation on PDAC cells and in tumor microenvironment.
The antibody was characterized both in vitro and in vivo. Biodistribution studies, using near-infrared optical imaging technology, confirmed the capacity of anti-GPC1 IgM to selectively bind its target in a localized PDAC model developed in Nude mice, with a pick after 4 days; this antibody was mainly eliminated by the liver. Moreover, a single injection of anti-GPC1 IgM induced a strong activation of the classical pathway of the mouse complement system; as expected, C1q, C3 and C9 deposits were documented in tumor microenvironment by immunofluorescence analysis, causing extended necrotic areas as well as the recruitment of CD14+ macrophages and CD56+ NK cells. Repeated injection of anti-GPC1 IgM (twice a week) controlled tumor growth in all tumor-bearing mice. All saline-treated animals died in 19 days after the first treatment while 60% of anti-GPC1 IgM treated mice survived at the end of the study (50 days after the first treatment).
Collectively, these findings showed the capacity of anti-GPC1 IgM to strongly activate the complement system on PDAC cells, causing cancer cell death, modifying tumor microenvironment and finally increasing mice survival.
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
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)
Session 11 – Complement deficiency in Immunopathology
Background: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59, leading to loss of function, have been described in 18 patients. Here we investigated single cell RNA sequencing of PBMCs from a patient deficient in CD59.
Methods: Single Cell RNA Sequencing. PBMCs from a patient with CD59 homozygous mutation and an age- and gender-matched control were diluted with FACS buffer to a concentration of 0.6–0.7×106 cells/ml and viability above 90% was confirmed. RNA from the barcoded cells from each sample was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a chromium single cell 3′ v2 reagent kit (10× Genomics) according to the manufacturer’s instructions. Sequencing was performed with the Illumina NextSeq 500 high-output kit (Illumina, San Diego, CA, USA) for 75 cycles with 26bp read from read1 and 58bp from read2. Single-Cell RNA-Seq Analysis. Single-cell RNA-seq analyses were performed used Seurat 4.3.0. Uniform Manifold Approximation and Projection (UMAP) was then performed. Clusters were determined using the shared nearest neighbor (SNN) modularity optimization-based clustering algorithm. Differentially expressed markers were identified.
Results: PBMCs from the patient with homozygous CD59 deficiency showed marked depletion of NK cells (36% of normal) and marked elevation in CD4:CD8 ratio; 6.14 compared to 3.75 in normal control. Tregs were twice in number in the patient and both CD4 and CD8 cells expressed differentially gene clusters. B cells were a little bit elevated in the patient, but naïve B cells were elevated 2.5 folds. In one cluster SNHG5, RPS28, ARL4C, HLA-DPB1, AES, ABRACL, CSTB, BAX, CAP1, were elevated along with others that were differentially negatively expressed. Monocytes were relatively depleted in the patient. These gene changes fitted to known patterns in antigen presentation, TH17 signaling pathway, type I diabetes and other autoimmunity, GvHD, and some viral and bacterial infections.
Conclusion: SCA reveals expected and unexpected changes in PBMC subpopulations from membrane CD59 deficient patient. These finding may elucidate unknown functions of both canonic and non-canonic CD59 functions.
Background: C3 glomerulopathy (C3G) is a rare kidney disorder associated with complement alternative pathway (AP) dysregulation and C3 deposition within the glomerulus.
Proteinuria and nephrotic syndrome are present at onset in 90% and 30% of C3G patients, respectively. A morphological hallmark of nephrotic syndrome is effacement of foot processes in podocytes, highly specialized cells that play a main role in glomerular permeability.
Through exome sequencing, we identified in a proband from a trio with C3G and nephrotic syndrome a variant in a new candidate gene, IQGAP1, encoding for the IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). IQGAP1 is a scaffold protein largely expressed in the podocytes, where it interacts with proteins of the slit-diaphragm complex and cytoskeleton, regulating podocytes morphology, motility and permeability. The novel de novo heterozygous 1-base pair deletion leads to a frameshift with the introduction of a premature stop codon (p.L159Rfs*4).
This preliminary finding led us to investigate the link between IQGAP1 alterations and complement activation in podocytes.
Methods: Conditionally immortalized human podocytes were transfected with null or IQGAP1 small interfering RNA (siRNA). IQGAP1 mRNA expression levels were evaluated by real-time PCR, and IQGAP1 and cytoskeleton proteins localization by immunofluorescence. C3 deposition was evaluated, using confocal microscopy, by incubating transfected podocytes with 10% normal human serum (NHS).
Results: Transfection of podocytes with IQGAP1 siRNA, which recapitulates the effect of the frameshift identified in C3G patient, induced a decrease of IQGAP1 mRNA expression by 80% and caused a reorganization of podocyte cytoskeleton proteins, such as integrin-linked kinase (ILK) and podocin, and a cortical accumulation of F-actin. Incubation of siRNA transfected podocytes with NHS induced a three-fold increase in C3 deposition as compared to podocytes transfected with null siRNA (n=5; IQGAP siRNA: 1259 pixels2; null siRNA:377 pixels2; p=0.01). C3 deposits were abrogated by sCR1.
Conclusions: Altogether, these findings support the hypothesis that intrinsic defects altering the podocyte structure and/or function may predispose to a secondary complement attack on the glomerular cells, contributing to C3G onset. Further studies are needed to validate this hypothesis and to shed light on the molecular players involved in C3G.
Background: Ficolin-3 is the main initiator of the lectin pathway in humans. Case reports of ficolin-3 deficient patients have suggested that ficolin-3 deficiency may be enriched in patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease where complement plays an important role. Therefore, this study aimed to investigate the activity levels of ficolin-3 and to identify potential ficolin-3 deficient individuals in two Swedish SLE cohorts.
Methods: Serum or plasma samples from SLE patients (n=810) and matched controls (n=566) were collected from the Karolinska Institute (KI) and Umeå University Hospital. The ficolin-3 activity levels were measured by an in-house developed functional ELISA with a pooled normal human serum sample as a reference. Serial samples were analyzed for ficolin-3 deficient patients when available. Sequencing data were analyzed for FCN3 frame-shift mutation +1637delC (rs532781899) and other potential loss-of-function (LoF) variants.
Results: This screening revealed that the level of ficolin-3 activity varies largely in patients with SLE. The activity levels also show that SLE patients seem to generally have elevated ficolin-3 activity compared to the control group (p<0.0001). Out of 810 patients with SLE, four patients were determined to be ficolin-3 deficient. For two of these patients, the ficolin-3 activity was at normal levels at the time of diagnosis and thereafter depleted over time, indicating an acquired deficiency. For deficient patients, no or very low ficolin-3 protein levels and no lectin pathway-dependent complement activation could be detected. Autoantibodies against ficolin-3 were not detectable. No patients were homozygous for the +1637delC frameshift mutation, whereas in total 10 patients were determined to be heterozygous carriers. These heterozygous patients displayed lower levels of ficolin-3 activity but did not include the deficient patients. Additional possible LoF variants were analyzed but none were enriched in either patients or controls.
Conclusions: Contrary to the classical pathway of the complement system we show that genetic ficolin-3 deficiency is not a risk factor for SLE. Instead, acquired ficolin-3 deficiency was observed in a subgroup of SLE patients, possibly due to a potent activation of the lectin pathway that depleted ficolin-3 plasma levels in these individuals.
C3 is the most abundant protein of the Complement System and plays an important role in the immune response. C3-deficient patients are more susceptible to recurrent and severe infection diseases. Several studies have demonstrated the importance of C3 in controlling infection, but its role in leukocyte differentiation and cellular response is still poorly understood. This study aimed to evaluate several cellular parameters in bone marrow differentiated macrophages (BMDM) and thioglycollated-elicited peritoneal macrophages (TEPM) from C57Black/6 C3-/- (B6.C3-/-; C3KO) and C57Black/6 (B6.C3+/+; wild type) mice. The total number of cells in the peritoneal exudate was not affected by the absence of C3. TEPM F4/80high cells, resident macrophages that do not depend on the differentiation of new circulating monocytes, are present at higher levels in B6.C3-/- mice than in B6.C3+/+ mice. The morphology of the macrophage cytoskeleton plays a crucial role in migration, adhesion and engulfment of particles. We observed an increase in cell roundness and more solid morphology (fewer invaginations) in TEPM B6.C3-/- animals, which may suggest that these cells would be less activated. We did not observe the same phenomenon in BMDM. Microtubules are essential for cell displacement into the inflammatory site. In this study, we observed that in the absence of C3, the presence of F-tubulin filaments increases in both BMDM and TEPM. In addition, a significant increase in CR4 (CD11c/CD18) and CD64 (FcgammaRI) expression was detected in both BMDM and TEPM from B6.C3-/- mice compared to the same cells from B6.C3+/+ mice. ROS production and MAPK activation after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) was lower in B6.C3-/- macrophages than their wild type counterparts, suggesting the importance of C3 for cell signaling. As expected, BMDM and TEPM from B6.C3-/- mice phagocytosed less serum-opsonized zymosan particles than the corresponding cells from B6.C3+/+ mice. Together our results suggest that C3 plays a role in macrophage phenotype and activation.
Plenary 3: Muzz Haniffa – Immune Cell Regulation of Skin Morphogenesis
Session 12 – Translational models of disease
Excessive activation of complement, a crucial component of innate immunity, causes many diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and age-related macular degeneration (AMD). Among the complement components, C3 is central for all complement activation pathways, thus an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for treating PNH and dry AMD. However, most, if not all of these C3 inhibitors are primate C3-specific, making evaluating their treatment efficacies in vivo before a clinical trial extremely difficult and costly. We previously developed a C3 knockout (KO) rat. By supplementing purified human C3 protein into the blood of the C3 KO rats, we found that human C3 is compatible to the rat complement system. We thus developed a human C3 knockin (KI) rat by knocking in a human C3 expression construct into the rat C3 loci using CRISPR/Cas 9 technology. The resultant human C3 KI rats expressed human but not rat C3 as examined by respective ELISA, Western blot and/or RT-PCR, and possessed complement activities both in vitro and in vivo. More importantly, the complement hemolytic activity of the human C3 KI rats was inhibited by compstatin, a C3 inhibitor known to be specific only to primate C3. The successful development of human C3 KI rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.
Background: Complement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin.
Methods: Tail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR).
Results: Tail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed. Neither FH complement regulatory function nor complement haemolytic activity was altered by thrombin binding.
Conclusion: Complement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis.
Background: Polytrauma as a life threatening multiple injury leads to rapid activation of the cross-talking coagulation- and complement systems. However, excessive complement activation can contribute to systemic inflammation and organ dysfunction. In the case of sepsis, blockade of central complement components and TLR molecules appeared to be beneficial as we also documented recently in a pig model with 72 hrs observation using C5 and CD14 inhibition. Therefore, we hypothesized that absence of the central complement component C3 and the TLR 4-coreceptor CD14 will improve cellular and organ function post trauma in genetically modified mice.
Methods: Polytrauma plus hemorrhagic shock (HS) was modelled in C57bl/6 mice by blunt traumatic brain injury, thoracic trauma, femur fracture and soft tissue injury. After the trauma impact, animals were hemodynamically monitored in our mouse intensive-care unit. Sham procedures and polytrauma/HS was applied in genetic absence or presence of C3, or CD14 or both, C3/CD14 (n=8/group). As read-outs, we investigated the hemodynamic and inflammatory response aas well as multi-organ function 4 h after injury. Statistical analyses were performed by ANOVA with post hoc testing.
Results: The hemodynamic changes after the polytrauma/HS did not significantly alter between the C3/CD14 sufficient and deficient mice. Systemic inflammation assessed by IL-6 plasma concentrations, multi-organ performance measured by the established biomarkers CC16, IFABP, GFABP, NGAL, among others, and clinical outcome (reflected by mortality rate) did not significantly alter between the C3 deficient, CD14 deficient and C3/CD14 double knock out mice in the early observation period up to 4 h post polytrauma/HS. Of note, the polytrauma-induced increase in the endothelial damage marker syndecan-1 was significantly reduced in C3-/- mice.
Conclusion: Whereas long-term effects after severe tissue trauma seem to benefit from blockade of complement (C5) and TLRs (CD14) , our current data in mice suggest, that very early after polytrauma/HS neither complement nor TLR inhibition alter the inflammatory and organ response, except for the endothelial damage. These differential-temporal findings indicate that the therapeutic effect of this inhibition is not observed early, but might have a major impact at a later stage.
Background: Traumatic brain injury (TBI) gives rise to a secondary neuroinflammation that can last years after injury and is associated with the development of progressive cognitive impairment and chronic disabilities. We and others have shown that activation of an immune response, including activation of complement, plays a central role in this process by mediating the aberrant phagocytosis of perilesional neurons and synapses. However, since the immune system can promote both brain repair and brain damage, identifying the cellular components and their temporal distribution would contribute to our understanding of the neuroinflammatory process and help identify TBI treatment strategies.
Methods: Using a controlled cortical impact TBI model, we leveraged mass cytometry to evaluate how complement and complement inhibition affects the immune landscape after TBI. We designed a 35-antibody panel to identify resident and peripheral immune cells and their functional receptors involved in phagocytosis, chemotaxis, and complement signaling at various time points after TBI. We used the injury-site targeted complement inhibitor, CR2-Crry, consisting of the complement inhibitor (Crry) linked to a fragment of complement receptor 2 (CR2), which increases complement inhibition bioavailability and efficacy without exerting systemic immunosuppressive effects.
Results: We found an increase in the percentage of infiltrating peripheral immune cells at acute time points and identified distinct microglia sub-populations, among which one subpopulation expressing complement receptor 4 (CR4) showed progressive increase through at least 28 days after injury. CR2-Crry treatment after TBI improved cognitive functions, impacted the expression of functional receptors on infiltrating cells, and reduced the abundance of brain resident immune cells. Conversely, inhibition of C5aR effectively reduced the infiltration of immune cells, but did not improve histological or behavioral outcomes, which suggests the neuroprotective effect of CR2-Crry is not mediated via inhibition of C5a generation.
Conclusions: CR2-Crry improved post-TBI outcomes and altered immune cell infiltration, as well as complement receptor and phagocytic receptor expression. Together with data from previous studies and the current C5aR inhibition data, the results indicate that the neuroprotective effect of CR2-Crry is likely via modulating C3 opsonization and complement receptor expression.
Background: Mannan-binding lectin-associated serine protease 3 (MASP3) converts pro-factor D (pro-FD) to mature FD. It was reported earlier that only mature FD was catalytically active, making MASP3 a key component of the alternative pathway (AP) complement and an attractive drug target. However, the role of MASP3 in AP complement has been controversial as various levels of AP activity were detected in the absence of MASP3, both in human and in mice. We have recently demonstrated significant AP complement activity in a newly generated Masp3-/- mouse strain at high (20-50%) but not low (5-10%) plasma concentrations and attributed this activity to intrinsic catalytic activity of pro-FD (1). The aim of this study is to assess the functional significance of this partial activity in AP complement-mediated tissue injury.
Methods: We crossed the Masp3-/- mouse with a factor H mutant mouse (FHR/R) that develops AP complement-dependent atypical hemolytic uremic syndrome and systemic thrombophilia (2) and assessed the effect of MASP3 deficiency on disease development by evaluating FHR/R and FHR/RMasp3-/- littermates.
Results: We found that MASP3 deficiency significantly reduced mortality, and ameliorated thrombocytopenia, anemia, and renal injury in FHR/R mice. It also prevented systemic thrombophilia and macro-vessel thrombosis. However, about 30% of FHR/RMasp3-/- mice (5/17) still died prematurely because of extra-renal organ injury including intestinal and pulmonary hemorrhage and cerebral ischemia. These findings contrasted with the phenotype of other AP complement and FHR/R double knockout mice such as FHR/RC3-/- or FHR/RFD-/- mice which showed complete disease rescue. AP complement activity in FHR/RMASP3-/- mice, as measured in 20% serum using an LPS-based ELISA assay, was significantly lower than that of FHR/Rmice but significantly higher than that of FHR/RFD-/- mice.
Conclusion: Partial AP complement activity afforded by pro-FD in the absence of MASP3-/- was sufficient to cause tissue injury and therapeutic targeting of MASP3 may not be completely efficacious in all settings of AP complement-mediated diseases.
1. Gullipalli D, Miwa T, Golla M, Sato S, Angampalli S, Song WC. J Immunol. 2023 Mar 29:ji2200932. doi: 10.4049/jimmunol.2200932.
2. Ueda Y et al Blood. 2017 Mar 2;129(9):1184-1196.
Complement as an essential part of innate immunity maintains immune homeostasis and plays a key role in several biological processes, such as removal of injured cells and debris, host defense injuries and repair. However, excessive complement activation can lead to diseases. Therefore, this cascade needs to be tightly regulated. Factor H-related protein 1 (FHR1) is a member of Factor H protein family involved in complement control and in the regulation of the innate immune response. In humans, homozygous deletion of FHR1 and FHR3 genes (∆∆FHR3/FHR1) has been reported to be protective against diseases such as immunoglobulin A nephropathy (IgAN) and age-related macular degeneration (AMD), but has also been shown as a risk for atypical hemolytic-uremic syndrome (aHUS) and atherosclerotic cardiovascular diseases (ACVD) (Skerka et al., 2021; Irmscher et al., 2021). Recently, our group also revealed that FHR1 is an inflammatory mediator inducing proinflammatory response of monocytes (Irmscher et al., 2021). Currently, the role of FHR1 has not been fully explored in animal models. Here, we generated a FHR-E-/- (FHR1 murine homolog) knock-out mouse model for investigating further the role of FHR-E in vivo. We first measured plasma levels of complement C3 and the regulator Factor H in FHRE-/- and WT mice. We observed related levels in both type of animals. In addition expression of pro- and anti-inflammatory cytokines in FHR-E-/- and in WT mice was evaluated in several tissues including kidney, liver and heart. Our data revealed that in most of these tissues, cytokines expression such as IL-1β and TNFα are significantly reduced both at RNA and protein levels in FHR-E-/- mice as compared to WT animals. Further analyses also revealed altered levels of reactive oxygen species in FHRE-/- mice. Taken together, the present study suggests that deletion of the FHR-E gene is more protective against inflammation, confirming our previous observations made in humans. Since this study is conducted in vivo, it provides real insights into the mechanisms how FHR-E regulates the complement system and inflammation and lays the groundwork for further studies.
Please note that a Dinner ticket needed to be booked in advance. If you aren’t sure if you are booked to attend the dinner, please email firstname.lastname@example.org
It is a 7pm arrival and drinks with dinner to start at 7.30pm. We have a fantastic Ceilidh band organised for you during the evening. A Ceilidh Band is a traditional folk band with a caller that will walk you through a series of dance moves!
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The dinner will take place at:
Newcastle Civic Centre, Banqueting Hall, Barras Bridge ,Newcastle upon Tyne, NE1 8QH
Dress Code: smart/casual (we recommend wearing comfortable shoes to join in with the Ceilidh)
Session 13 – Preclinical complement therapeutics
Background: ADX-097 is an antibody fusion protein that targets the alternative pathway (AP) complement negative regulator factor H to diseased tissue. ADX-097 was designed to bind tissue-fixed C3d-containing C3 activation fragments and inhibit complement in diseased tissue while minimizing systemic blockade.
Methods: Having demonstrated C3 activation and C3d deposition in human renal and skin disease biopsies, we generated and characterized ADX-097, a humanized anti-C3d monoclonal antibody linked to five N-terminal repeats of the AP inhibitor factor H (fH1-5). We further evaluated circulating and tissue PK/PD of ADX-097 in mouse and non-human primate (NHP) models of tissue complement activation. Finally, we tested ADX-097 PK/PD and efficacy in the rat Passive Heymann Nephritis (PHN) model of membranous nephropathy.
Results: We characterized in vitro binding of ADX-097 to C3d, demonstrating that at high C3d densities, use of a bivalent anti-C3d antibody yields binding affinities that are ~1000x greater than those of comparable monovalent binders, suggesting a substantial avidity-based targeting advantage. Consistent with this highly efficient targeting, in rodent and NHP models of local complement activation, ADX-097 dosed subcutaneously (SC) as low as 1 mg/kg distributes to C3d+ tissue and inhibits complement activation. In factor H-/- mice, a single 1 mg/kg SC dose achieves >75% glomerular complement C3 inhibition for at least 7 days while avoiding systemic complement blockade. In a rat model, 1 mg/kg dosed SC or IV inhibited glomerular complement activation and significantly reduced urine protein-creatinine ratios without inhibition of systemic complement, and with an efficacy that was equivalent to full systemic complement blockade using daily injections of cobra venom factor. We also show that urinary C5b-9 (uC5b-9) correlates strongly with decreased glomerular C3 activation and is independent of systemic blockade or proteinuria-associated renal leakage, suggesting the utility of uC5b-9 as a biomarker for local complement regulation in the kidney.
Conclusions: These data demonstrate the therapeutic potential of ADX-097 and show that C3d-mediated tissue targeting of fH1-5 in preclinical models results in potent, durable, and efficacious local AP complement blockade without systemic complement inhibition, and that this is an effective strategy for re-regulating tissue complement in disease.
Background: Epidermolysis Bullosa Acquisita (EBA) is an autoimmune skin blistering disease characterized by strong production of autoantibodies (AAbs) against type VII collagen (COL7) at the dermal epidermal junction (DEJ). Local complement activation drives C5a generation associated with neutrophil recruitment and activation resulting in skin lesions and inflammation. The clinical effector phase of EBA can be mimicked by repeated injection of rabbit COL7-specific IgG into mice. Here we tested the impact of prophylactic or therapeutic C5a or combined C5/C3 targeting on disease development and skin inflammation in this model.
Methods: C57BL/6 mice were immunized s.c. with purified rabbit anti-mouse-COL7 IgG. An anti-mouse C5a/C5adesArg IgG1 mAb (M031), a fusion protein between an anti-mouse C5 IgG1 mAb and FH-SCR1-5 (M014), or an IgG1 isotype control (n=12/group) were injected (i.p.) prophylactically on days -1, 2, 5 and 8 or therapeutically on days 5 and 8. Then, formation of skin lesions was evaluated every other day. DEJ separation was assessed in cryosections from day 12 and stained with H&E. Tissue-bound IgGs AAbs, C3/C3b deposition as well as MPO+ or Ly6G+ neutrophil infiltration was identified by direct immunofluorescence.
Results: Isotype-treated mice developed first skin lesions at day 4 peaking at day 12. Prophylactic treatment with either M031 or M014 markedly reduced the development of skin lesions. Also, dermal/epidermal separation as well as neutrophil infiltration on day 12 were significantly lower upon M031 or M014 treatment. Surprisingly, C5/C3 inhibition by M014 but not C5a inhibition by M031 markedly reduced the development of skin lesions and the dermal/epidermal separation after therapeutic treatment as well as the infiltration with MPOhi neutrophils. IgG and C3/C3b deposition did not differ in the three treatment groups.
Conclusion: Prophylactic C5/C3 and C5a targeting is equally effective in reducing the development of skin lesions and tissue inflammation in a preclinical model of EBA. In contrast, only combined C5/C3 but not C5a inhibition protects from the development of skin lesions and skin inflammation after therapeutic administration at day 5 when first skin lesions become evident. Our findings point toward an important role of C5a as well as other complement effectors during disease development in EBA.
Background: Ischaemia reperfusion injury is an inevitable consequence of transplant with the complement system, in particular the alternative pathway being one of the key drivers of damage. The main regulator of the alternative pathway is factor H. We hypothesised that homodimeric mini-factor H (HDM-FH; PMID:29588430) would protect kidneys when administered during normothermic machine perfusion (NMP) with whole blood which mirrors the clinical setting of transplantation.
Methods: A model of porcine whole blood NMP with extended warm and cold ischemic times was optimised to induce complement activation and ischaemic injury in kidneys so that the full efficacy of HDM-FH could be assessed. Utilising this model, both kidneys were retrieved from female white landrace pigs. One kidney from each pair was randomised to receive 5mg of HDM-FH (~8μg/mL). Kidneys were perfused at 37°C with autologous blood for 6 hours. HDM-FH binding within kidneys was confirmed using immunofluorescence. HDM-FH levels in perfusate and urine were measured using ELISA. Complement activation was measured by quantifying Bb deposition in tissue and C5a levels in urine. Fibrosis, inflammatory cytokines, and apoptosis were measured as indicators of downstream ischaemic injury. ‘Cold binding’ of HDM-FH was assessed by flushing kidneys with 4°C saline containing HDM-FH.
Results: 25 minutes warm ischaemic time followed by 16hrs cold ischaemic time led to an increase in complement activation and markers of ischaemic injury. ~4mgs of HDM-FH bound from perfusate during perfusion at 37°C, with <10% lost in urine suggesting saturation was achieved. HDM-FH localised to glomeruli with deposition increasing during the perfusion. Complement activation was reduced in kidneys receiving HDM-FH as demonstrated by reduced Bb deposition in tissue and reduced C5a levels in urine. Fibrinogen deposition, inflammatory cytokine and apoptosis levels were reduced. HDM-FH inhibits complement activity in serum in a dose-dependent manner. HDM-FH also binds at 4°C, experiments are ongoing using this approach.
Conclusion: Infusion with HDM-FH during simulated kidney transplant conditions reduced complement activation and downstream ischaemic injury to the organ. Therefore, organ perfusion with HDM-FH is highly likely to help prolong graft survival after transplant and this will be assessed in future studies.
Background: Iptacopan is a selective low-molecular-weight inhibitor of the alternative complement pathway (AP) that is currently in clinical development for multiple complement-mediated diseases, including aHUS. It inhibits the protease activity of C3bBb by binding to its active site within Bb.
Approximately 3-5% of aHUS patients express one of 18 point-mutations in FB. The aim of this study was to determine if Iptacopan inhibits protease activity of these FB variants.
Methods: Eight FB variants (K533R, E566A, D279G, F286L, K323Q, K323E, K350N and M458I) were selected based on mutations in proximity to the active site of Bb and their capacity to stimulate the AP. We tested activity of iptacopan on these mutants as follows:
- Binding affinity to FB mutants via displacement of an active site binding probe.
- Ability to inhibit an activated C3 convertase formed by cobra venom factor (CVF) bound to wtFB or FB mutants.
- Activity to inhibit lysis of sheep red blood cells (SRBCs) coated with C3 convertases containing wtFB or FB mutants.
- Activity to inhibit zymosan-induced AP activation in Factor B depleted human serum supplemented with wtFB or FB mutants.
Results: Iptacopan bound to all FB mutants and wtFB with comparable affinity. CVF-Bb C3 convertase activity was increased with two of the mutants (FB(K350N) and FB(D279G)) and reduced in FB(F286L) compared to CVF-Bb containing wtFB, but iptacopan inhibited C3 cleavage by all convertases with similar IC50. Cell surface C3/C5 convertase activity in the hemolytic assay was also increased for FB(K350N) and FB(D279G) and decreased for FB(F286L). Iptacopan inhibited wtFB and all FB variants in this assay, albeit less potently for FB(D279G). While serum containing FB(350N) and FB(279G) caused only limited MAC-formation upon zymosan stimulation, Iptacopan showed similar potency in the zymosan assay for wtFB and all mutants.
Conclusion: This in vitro study demonstrates that Iptacopan binds to and inhibits common FB variants identified from aHUS patients. Therefore, patients with FB variants may be included in clinical aHUS trials with iptacopan.
2023 Lambris Complement Training Award
International Complement Society Early Career Researcher Award
Session 14 – Clinical complement therapeutics
Background: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE) disease is an ultra-rare autosomal recessive disorder caused by loss-of-function variants of the CD55 gene, leading to overactivation of the terminal complement system.1 Clinical and laboratory features of CHAPLE disease include hypoalbuminemia and edema; hypogammaglobulinemia; and gastrointestinal symptoms such as abdominal pain, loss of appetite, vomiting, and diarrhea. Patients also present with micronutrient deficiency, anemia, and growth retardation.1 Currently, there is no approved treatment for CHAPLE disease. We assessed the efficacy and safety of pozelimab, an investigational anti-C5 antibody, in patients with CHAPLE disease.
Methods: This is an interim report of an open-label, single-arm, historically-controlled study in patients with CHAPLE disease (ClinicalTrials.gov, NCT04209634). Patients received intravenous pozelimab 30 mg/kg followed by subcutaneous, weight-based dosing once weekly. The primary endpoint was the proportion of patients who achieved normalization of serum albumin and demonstrated improvement or no worsening in clinical outcomes (abdominal pain, bowel movement frequency, facial edema, and peripheral edema) at week 24. Secondary/exploratory efficacy endpoints and safety are also reported.
Results: Ten patients were enrolled in the study, and had ≥48 weeks of efficacy measurements as of the cut-off date for this analysis. All 10 patients (100%) experienced serum albumin normalization and improvement/no worsening in clinical outcomes. Over the 48-week treatment period, most patients experienced remarkable catch-up growth. Following treatment with pozelimab, patients had reduced all-cause hospitalization days; the mean number of hospitalization days across all 10 patients decreased from 26.8 days in the 48 weeks prior to treatment to 0 days by week 48. Four patients (40%) started the treatment period on corticosteroids and all were withdrawn as of the cut-off date for this analysis. Complete inhibition of complement activity (CH50) was achieved. Seven patients (70%) experienced adverse events; none were severe, and only one patient experienced adverse events that were considered related to study drug.
Conclusion: Pozelimab inhibits complement overactivation and resolves the clinical and pathophysiological manifestations of CHAPLE disease.
Reference: 1. Ozen A, et al. N Engl J Med. 2017;377:52–61.
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disorder characterized by hemolysis due uncontrolled complement activation. In vitro, factor D (FD) inhibitors block C3 fragment deposition and lysis of erythrocytes collected from patients with PNH, providing evidence for its potential to inhibit both intravascular and extravascular hemolysis (IVH and EVH). Vemircopan, a second-generation oral FD inhibitor with improved potency against FD and a prolonged T1/2, is being investigated as monotherapy for patients with PNH.
Methods: This ongoing, phase 2, open-label proof-of-concept study (NCT04170023) assesses efficacy, safety, and PK/PD of vemircopan monotherapy in patients with PNH. The study comprises a 60-day screening period, 12-week treatment period, and 96-week long-term extension. Key inclusion criteria for treatment-naïve group: lactate dehydrogenase [LDH] ≥1.5 × upper limit of normal [ULN], absolute reticulocyte count ≥100×109/L, and anemia (hemoglobin [Hgb] <10.5 g/dL). Patients receive vemircopan 120 mg BID (can escalate to 180 mg BID per investigator discretion based on protocol). Primary endpoint is change from baseline to week 12 in Hgb. Safety is reported as treatment-emergent adverse events (TEAEs).
Results: At data cutoff, 11 treatment-naïve patients were enrolled (mean [SD] age, 44.4 [17.8] y; 63.6% male; 72.7% Asian, 54.5% received transfusions during screening period). Nine patients completed 12 weeks in the study and were included in this analysis. From baseline to week 12, mean (SD) Hgb increased by 3.9 (1.11) g/dL from 7.9 (1.29) g/dL to 11.8 (1.25) g/dL, LDH decreased by 81% from 7 × ULN to 1.4 × ULN, absolute reticulocyte count decreased from 212.4 (86.47) x 103/uL to 120.0 (51.44) x 103/uL. No patients needed transfusions except 1 case on day 2 owing to low Hgb (5.1 g/dL). Of 31 TEAEs (n=9 patients) most (25/31; 80.6%) were considered unrelated to study drug. No serious TEAEs, grade ≥3 TEAEs, discontinuations, or deaths were reported. There were no thrombotic events, seizures, or meningococcal infections.
Conclusions: This interim analysis of treatment-naïve patients with PNH suggests that vemircopan monotherapy controlled IVH and prevented EVH. No new safety signals were identified during the 12-week evaluation period. This analysis provides proof-of-concept for FD inhibition in PNH.
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease characterized by chronic intravascular hemolysis. Pozelimab is an investigational monoclonal antibody against complement component C5. Final results from a phase 2 study (NCT03946748) and subsequent open-label extension (OLE; NCT04162470), evaluating long-term pozelimab monotherapy in patients with PNH are presented.
Methods: During the phase 2 study, patients received an intravenous loading dose of pozelimab 30 mg/kg, then weekly subcutaneous pozelimab 800 mg. All 24 patients completed phase 2 and entered the OLE, where patients received weekly subcutaneous pozelimab 800 mg up to Week 104.
Results: Overall, 87.5% of patients were Asian; 54.2% were male. In phase 2, 18/24 (75.0%) patients achieved adequate control of intravascular hemolysis (defined as LDH ≤1.5 x upper limit of normal; 95% confidence interval [CI]: 57.7-92.3%) at every scheduled timepoint between Weeks 4-26 inclusive. During the OLE, 22/23 (95.7%) patients achieved control of intravascular hemolysis at all timepoints through Week 26 (95% CI: 87.3-100.0%); with 15/16 (93.8%; 95% CI: 81.9-100.0%) at Week 78.
In phase 2, 21/24 (87.5%; 95% CI: 74.3-100.0%) patients achieved transfusion avoidance (no red blood cell transfusion). During the OLE, 22/23 (95.7%) patients achieved transfusion avoidance through the initial 26 weeks, and 15/16 (93.8%) through to Week 78. No patients experienced a breakthrough hemolysis event in either trial. Importantly, one patient with a C5 variant resistant to eculizumab/ravulizumab had adequate control of intravascular hemolysis. All patients showed a rapid decrease in total complement hemolytic activity to near zero, maintained to Week 104.
In phase 2, 21 (87.5%) patients experienced 72 treatment-emergent adverse events (TEAEs); two (8.3%) patients experienced severe TEAEs; and 10 (41.7%) patients experienced TEAEs considered related to pozelimab. During the OLE, 15 (62.5%) patients experienced 41 TEAEs; two (8.3%) patients experienced serious TEAEs; two (8.3%) patients experienced severe TEAEs; two (8.3%) patients experienced TEAEs considered related to pozelimab; and no deaths or discontinuations due to TEAEs occurred.
Conclusion: Patients with PNH receiving pozelimab (up to 130 weeks) experienced improvement in intravascular hemolysis control, with no breakthrough hemolysis, and achieved transfusion avoidance. Pozelimab was also generally well tolerated.
Background: In complement mediated atypical haemolytic uraemic syndrome (aHUS) dysregulated complement activation occurs on endothelial cell surfaces and results in thrombotic microangiopathy. Historically management comprised predominantly supportive care with or without plasma exchange, and outcomes were poor. Trials of the terminal complement inhibitor eculizumab published in 2013 suggested efficacy and revolutionized management. Given the high cost of the drug a national specialized service was commissioned by NHS England. We report the real life experience of treating individuals with suspected aHUS with eculizumab in a national cohort and compare outcomes with a control cohort.
Methods: 1956 individuals have been referred with suspected aHUS. Of these, 243 were treated with eculizumab. The control cohort comprised 279 individuals referred with suspected aHUS prior to the availability of eculizumab in whom a pathogenic complement gene mutation or autoantibody was identified. Clinical data were collected in collaboration with local clinicians and from RaDaR. Outcomes were compared using Kaplan-Meier analysis (IBM Statistical Package for Social Sciences (SPSS)). Multivariate analysis used the logistic regression model (R).
Results: The 5-year cumulative estimate (Kaplan-Meier) of end stage kidney disease (ESKD) free survival was 39.5% in the control cohort and 85.5% in the eculizumab treated cohort subgroup with a mutation or FHAA; HR 4.95 (95% CI 2.75-8.90), p=0.000, number needed to treat 2.17 (NNT) (95% CI 1.81-2.73). The magnitude of the improvement with eculizumab depends upon mutation type or autoantibody. The relapse rate upon eculizumab withdrawal was 1 per 9.5 person years for those with a pathogenic mutation. No relapses were recorded in 67.3 person years off eculizumab in those with no rare genetic variant.
Conclusion: Eculizumab costs ~£328000 per person (adult) per year and increases the risk of meningococcal sepsis. It was introduced into clinical practice on the basis of small single-arm clinical trials. In this genotype matched study of aHUS we show ESKD-free survival is significantly improved with eculizumab, and we believe that the magnitude of the improvement in outcomes justifies the high cost and potential complications of terminal complement blockade. We also demonstrate that eculizumab withdrawal in aHUS is safe and relapse is predicted by genotype.
Prizes /Awards for ICW2023
Announcement – ICW2025
Announcement – EMCHD 2024
Lunch – Grab Bag
RONALD N. GERMAIN M.D, PH.D
Ronald N. Germain received his M.D. and Ph.D. from Harvard University. Since then he has investigated basic immunobiology, first on the faculty of Harvard Medical School, then in the Laboratory of Immunology, NIAID, NIH, and most recently at NIAID, NIH as Chief of the Laboratory of Immune System Biology. He has made key contributions to understanding MHC class II molecule structure–function relationships, the cell biology of antigen processing, the molecular basis of T cell recognition, and the application of systems biology to understanding immune function. More recently, his laboratory has explored the immune system using dynamic and static in situ microscopic methods that his laboratory helped pioneer. He has published more than 400 scholarly research papers and reviews.
Among numerous honors, he was elected Associate member of EMBO (2008), elected to the National Academy of Medicine (2013), received the Meritorious Career Award from the American Association of Immunologists (2015), chosen as NIAID Outstanding Mentor (2016), elected to the National Academy of Sciences (2016), designated an NIH Distinguished Investigator and named a Distinguished Fellow of the AAI. He has trained more than 70 postdoctoral fellows, many of whom hold senior academic and administrative positions at leading universities and medical schools.
PROFESSOR MUZLIFAH HANIFFA
Muzlifah Haniffa is a Wellcome Senior Clinical Research Fellow, Senior Group Leader at the Wellcome Sanger Institute and Professor of Dermatology and Immunology at Newcastle University.
She graduated from medical school in Cardiff, trained as a junior doctor in Cambridge and received her dermatology specialist training in Newcastle. Muzlifah is a Fellow of the Academy of Medical Sciences and a recipient of the Academy of Medical Sciences Foulkes Foundation Medal and the European Federation of Immunological Societies ACTERIA Prize in Immunology and Allergology. She is co-Lead of the NIHR Newcastle Biomedical Research Centre Skin and Oral Disease and Immunogenomics Theme.
Muzlifah is a leading member of the Human Cell Atlas initiative and pioneered the application of single cell genomics to decode the developing human immune system, and the human skin in health and disease.
PROFESSOR LUKE O’NEILL
Luke O’Neill is Professor of Biochemistry in the School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute at Trinity College Dublin, Ireland. He is a world expert on innate immunity and inflammation. His main research interests include Toll-like receptors, Inflammasomes and Immunometabolism. He is listed by Thompson Reuters/ Clarivates in the top 1% of immunologists in the world, based on citations per paper. Professor O’Neill is co-founder of Sitryx, which aims to develop new medicines for inflammatory diseases. Another company he co-founded, Inflazome was recently acquired by Roche.
He was awarded the Royal Dublin Society / Irish Times Boyle Medal for scientific excellence, the Royal Irish Academy Gold Medal for Life Sciences, The Society for Leukocyte Biology (SLB) Dolph O. Adams award, the European Federation of Immunology Societies Medal, the Milstein Award of the International Cytokine and Interferon Society and the Landsteiner Award from the Austrian Academy of Sciences. He is a member of the Royal Irish Academy, EMBO (European Molecular Biology Organisation) and a Fellow of the Royal Society.
Organisation) in 2005.