Complement Regulators Research Articles

78 Characterisation of CSMD1 – A complement regulator associated with schizophrenia risk

78 Characterisation of CSMD1 – A complement regulator associated with schizophrenia risk

216 Association of a new variant of complement regulator FHR2 with C3 glomerulopathy

216 Association of a new variant of complement regulator FHR2 with C3 glomerulopathy

216 Association of a new variant of complement regulator FHR2 with C3 glomerulopathy

216 Association of a new variant of complement regulator FHR2 with C3 glomerulopathy

Properdin inhibition ameliorates hepatic ischemia/reperfusion injury without interfering with liver regeneration in mice.

Hepatic ischemia/reperfusion injury (IRI) often causes serious complications in liver surgeries, including transplantation. Complement activation seems to be involved in hepatic IRI; however, no complement-targeted intervention has been clinically applied. We investigated the therapeutic potential of Properdin-targeted complement regulation in hepatic IRI. Male wild-type mice (B10D2/nSn) were exposed to 90-minute partial hepatic IRI to the left and median lobes with either monoclonal anti-Properdin-antibody (Ab) or control-immunoglobulin (IgG) administration. Since the complement system is closely involved in liver regeneration, the influence of anti-Properdin-Ab on liver regeneration was also evaluated in a mouse model of 70% partial hepatectomy. Anti-Properdin-Ab significantly reduced serum transaminases and histopathological damages at 2 and 6 hours after reperfusion (P <0.001, respectively). These improvements at 2 hours was accompanied by significant reductions in CD41+ platelet aggregation (P =0.010) and ssDNA+ cells (P <0.001), indicating significant amelioration in hepatic microcirculation and apoptosis, respectively. Characteristically, F4/80+ cells representing macrophages, mainly Kupffer cells, were maintained by anti-Properdin-Ab (P <0.001). Western blot showed decreased phosphorylation of only Erk1/2 among MAPKs (P =0.004). After 6 hours of reperfusion, anti-Properdin-Ab significantly attenuated the release of HMGB-1, which provokes the release of proinflammatory cytokines/chemokines (P =0.002). Infiltration of CD11b+ and Ly6-G+ cells, representing infiltrating macrophages and neutrophils, respectively, were significantly alleviated by anti-Properdin-Ab (both P <0.001). Notably, anti-Properdin-Ab did not affect remnant liver weight and BrdU+ cells at 48 hours after 70% partial hepatectomy (P =0.13 and 0.31, respectively). In conclusion, Properdin inhibition significantly ameliorates hepatic IRI without interfering with liver regeneration.

Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses.

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies (mAb), we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 mAb tested in parallel. CaEno1 binding to naïve human CD4+ T cells skewed cytokine secretion towards a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4+ T cell recall responses. Therapeutically, CD4+ T cells transduced with a p41/Crf-1-specific TCR developed for adoptive T cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4+ T cells with CaEno1 modulated host CD4+ T cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through direct modulation of CD4+ T cell responses. This article is protected by copyright. All rights reserved.

Atypical Hemolytic-Uremic Syndrome: Genetic Basis, Clinical Manifestations, and a Multidisciplinary Approach to Management.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) defined by the triad of hemolytic anemia, thrombocytopenia, and acute kidney injury. Microthrombi develop in the glomerular capillaries secondary to endothelial damage and exert shear stress on red blood cells, consume platelets, and contribute to renal dysfunction and failure. Per current understanding of pathophysiology, HUS is classified into infectious, secondary, and atypical disease. The most common etiology is infectious sequelae of Shiga toxin-producing Escherichia coli (STEC); other causative organisms include shigella and salmonella. Secondary HUS arises from cancer, chemotherapy, solid organ and hematopoietic stem cell transplant, pregnancy, or autoimmune disorders. Primary atypical hemolytic-uremic syndrome (aHUS) is associated with genetic mutations in complement and complement regulatory proteins. Under physiologic conditions, complement regulators keep the alternative complement system continuously active at low levels. In times of inflammation, mutations in complement-related proteins lead to uncontrolled complement activity. The hyperactive inflammatory state leads to glomerular endothelial damage, activation of the coagulation cascade, and TMA findings. Atypical hemolytic-uremic syndrome is a rare disorder with a prevalence of 2.21 to 9.4 per million people aged 20 years or younger; children between the ages of 0 and 4 are most affected. Multidisciplinary health care is necessary for timely management of its extra-renal manifestations. These include vascular disease of the heart, brain, and skin, pulmonary hypertension and hemorrhage, and pregnancy complications. Adequate screening is required to monitor for sequelae. First-line treatment is the monoclonal antibody eculizumab, but several organ systems may require specialized interventions and coordination of care with sub-specialists.

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Systemic complement activation influences outcomes after allogeneic hematopoietic cell transplantation: A prospective French multicenter trial.

Complement activation has shown a role in murine models of graft-versus-host disease (GVHD) and in endothelial complications after allogeneic hematopoietic cell transplantation (allo-HSCT). However, its impact on post-transplant outcomes has not been so far fully elucidated. Here, we conducted a prospective multicentric trial (NCT01520623) performing serial measurements of complement proteins, regulators, and CH50 activity for 12 weeks after allo-HSCT in 85 patients receiving a myeloablative conditioning (MAC) regimen for various hematological malignancies. Twenty-six out of 85 patients showed an "activated" complement profile through the classical/lectin pathway, defined as a post-transplant decline of C3/C4 and CH50 activity. Time-dependent Cox regression models demonstrated that complement activation within the first weeks after allo-HSCT was associated with increased non-relapse mortality (hazard ratio [HR]: 3.69, 95% confident interval [CI]: 1.55-8.78, p = .003) and poorer overall survival (HR: 2.72, 95% CI: 1.37-5.39, p = .004) due to increased incidence of grade II-IV acute GVHD and in particular gastrointestinal (GI) GVHD (HR: 36.8, 95% CI: 12.4-109.1, p < .001), higher incidences of thrombotic microangiopathy (HR: 8.58, 95% CI: 2.16-34.08, p = .0022), capillary leak syndrome (HR: 7.36, 95% CI: 2.51-21.66, p = .00028), post-engraftment bacterial infections (HR: 2.37, 95% CI: 1.22-4.63, p = .0108), and EBV reactivation (HR: 3.33, 95% CI: 1.31-8.45, p = .0112). Through specific immune staining, we showed the correlation of deposition of C1q, C3d, C4d, and of C5b9 components on endothelial cells in GI GVHD lesions with the histological grade of GVHD. Altogether these findings define the epidemiology and the clinical impact of complement classical/lectin pathway activation after MAC regimens and provide a rational for the use of complement inhibitory therapeutics in a post-allo-HSCT setting.

Alzheimer’s disease-associated complement gene variants influence plasma complement protein levels

BackgroundAlzheimer’s disease (AD) has been associated with immune dysregulation in biomarker and genome-wide association studies (GWAS). GWAS hits include the genes encoding complement regulators clusterin (CLU) and complement receptor 1 (CR1), recognised as key players in AD pathology, and complement proteins have been proposed as biomarkers.Main bodyTo address whether changes in plasma complement protein levels in AD relate to AD-associated complement gene variants we first measured relevant plasma complement proteins (clusterin, C1q, C1s, CR1, factor H) in a large cohort comprising early onset AD (EOAD; n = 912), late onset AD (LOAD; n = 492) and control (n = 504) donors. Clusterin and C1q were significantly increased (p < 0.001) and sCR1 and factor H reduced (p < 0.01) in AD plasma versus controls. ROC analyses were performed to assess utility of the measured complement biomarkers, alone or in combination with amyloid beta, in predicting AD. C1q was the most predictive single complement biomarker (AUC 0.655 LOAD, 0.601 EOAD); combining C1q with other complement or neurodegeneration makers through stepAIC-informed models improved predictive values slightly. Effects of GWS SNPs (rs6656401, rs6691117 in CR1; rs11136000, rs9331888 in CLU; rs3919533 in C1S) on protein concentrations were assessed by comparing protein levels in carriers of the minor vs major allele. To identify new associations between SNPs and changes in plasma protein levels, we performed a GWAS combining genotyping data in the cohort with complement protein levels as endophenotype. SNPs in CR1 (rs6656401), C1S (rs3919533) and CFH (rs6664877) reached significance and influenced plasma levels of the corresponding protein, whereas SNPs in CLU did not influence clusterin levels.ConclusionComplement dysregulation is evident in AD and may contribute to pathology. AD-associated SNPs in CR1, C1S and CFH impact plasma levels of the encoded proteins, suggesting a mechanism for impact on disease risk.

Complement and coagulation crosstalk – Factor H in the spotlight

The immune complement and the coagulation systems are blood-based proteolytic cascades that are activated by pathway-specific triggers, based on protein–protein interactions and enzymatic cleavage reactions. Activation of these systems is finely balanced and controlled through specific regulatory mechanisms. The complement and coagulation systems are generally viewed as distinct, but have common evolutionary origins, and several interactions between these homologous systems have been reported. This complement and coagulation crosstalk can affect activation, amplification and regulatory functions in both systems. In this review, we summarize the literature on coagulation factors contributing to complement alternative pathway activation and regulation and highlight molecular interactions of the complement alternative pathway regulator factor H with several coagulation factors. We propose a mechanism where factor H interactions with coagulation factors may contribute to both complement and coagulation activation and regulation within the haemostatic system and fibrin clot microenvironment and introduce the emerging role of factor H as a modulator of coagulation. Finally, we discuss the potential impact of these protein interactions in diseases associated with factor H dysregulation or deficiency as well as evidence of coagulation dysfunction.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

#5885 HYPOXIA INDUCES COMPLEMENT ACTIVATION ON HUMAN KIDNEY EPITHELIAL CELLS

Abstract Background and Aims Ischemia-reperfusion injury is an inevitable event associated with kidney transplantation (Tx) and has a major impact on short- and long-term graft survival. Proximal tubular epithelial cells are both a source of complement during Tx and a target of complement activation. The normal kidney has a capacity to protect itself from complement activation through cellular expression of complement regulatory proteins. In this study we investigated whether hypoxia and reoxygenation increase C3 deposition and alter complement regulatory protein expression (CD46, CD59 and CD55). Method In vitro, HKC-8 renal proximal tubular epithelial cells were subjected to 24h of hypoxia (1% O2) and then reoxygenated for 4h (O2 = 21%) in the presence of 40% normal human serum. qRT-PCR was used to estimate the level of VEGF-A (as a positive control), C3, CD46, CD59 and CD55 expression. Similar analysis was performed in untreated, normoxic cells. Immunofluorescence staining was used to determine C3 deposition on the cell surface and intensity quantified by ImageJ. Experiments were repeated three times. Results qRT-PCR analysis of HKC-8 cells in hypoxia revealed significant increases in the expression of VEGF-A after 24h (p = 0.0113). C3 expression following hypoxia and reoxygenation increased insignificantly. Increased expression of CD59 with the presence of serum was observed in epithelial cells (p = 0.026). However, no significant change was seen in the expression of CD55 and CD46. Reoxygenation in human serum led to significantly greater deposition of C3 on hypoxic cells compared with normoxic HKC-8 cells (Figure 1, 2). Conclusion Our preliminary data suggests that hypoxia and reoxygenation significantly increases C3 deposition on the proximal tubular cell surface. CD59, as one of the regulators of complement activation is upregulated at mRNA level after hypoxia followed by 4h reperfusion in the presence of serum. These data demonstrate that hypoxia and reoxygenation activate complement system with upregulation of CD59 as a cell defence. However, expression of CD46 and CD55 do not change. Further study is needed to identify the impact of hypoxia on complement regulation in primary epithelial cells and kidney tissues.

#3979 DIGITAL SPATIAL PROFILING CAN BE USED TO STUDY GLOMERULAR ENDOTHELIAL CELLS IN IGA NEPHROPATHY

Abstract Background and Aims IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide; approximately 30% of cases progress to kidney failure 10-20 years from diagnosis. Five histopathological kidney lesions independently predict a poor prognosis in IgAN (MEST-C score) [1]. Published case series highlight the ‘endocapillary hypercellularity’ (E1) lesion as potentially reversible with systemic immunosuppression, improving clinical outcomes [2]. Delineating differences in the transcriptomes of glomerular endothelial cells (GEnCs) associated with and without E1 (E0) may highlight avenues for safer therapeutic strategies, given the overt risks associated with systemic immunosuppression in IgAN. GEnC transcriptomes have never been profiled in diseased kidneys before. Here we used digital spatial profiling (DSP) to achieve this. Method DSP was performed on a Nanostring GeoMx platform. Single 5μm sections were collected from four formalin fixed paraffin embedded (FFPE) kidney biopsies with E1 and five with E0. Following deparaffinization and antigen retrieval, the tissue was incubated with a whole transcriptome atlas probe set. GEnCs were stained for CD31 (red) and macrophages (which associate with E1[3]) with CD68 (yellow) with conjugated antibodies. Glomeruli were selected as regions of interests (ROIs), and a custom JavaScript function was used to mask over GEnCs and macrophages (segmentation), which were selected as areas of illumination (AOIs) (Fig 1). Photocleaved nucleotide barcodes were sequenced using an Illumina sequencer. Single cell enrichment was assessed using the SpatialDecon algorithm, differential gene expression was explored using a linear mixed effects model, and pathway analysis was performed using Reactome. Results The custom written JavaScript function allowed good segmentation on GEnCs and macrophages (Fig 1). Single cell deconvolution performed using the human kidney cell atlas as a reference showed significant enrichment of GEnCs relative to neighbouring cell types (Fig 2). Exploration of differential gene expressions using a linear mixed effects model found an up-regulation of TRIM23, IL27RA, TMEM139, P14K2B and PSMD9, after P value adjustment, among GEnCs associated with macrophages in glomerular capillary loops compared to those in the absence of macrophages (Fig 3). Pathway analysis based on differential gene expression performed using Reactome revealed that the complement cascade and regulators of the complement cascade were enhanced in GEnCs associated with macrophages (Fig 4). Conclusion This pilot study found DSP on the GeoMx to be effective at enriching GEnC transcript signals from neighbouring cell types in FFPE tissue. This preliminary data also shows that GEnCs associated with macrophages may display a more inflammatory phenotype, which may be related to up-regulation in complement activity and may account for the progressive phenotype associated with E1 in IgAN. With several trials investigating complement system targeting therapeutics in IgAN, validation of these findings might highlight a cohort of patients with IgAN most likely to benefit from treatment.

#6849 CLINICOPATHOLOGIC CHARACTERISTICS IN RENAL THROMBOTIC MICROANGIOPATHY

Abstract Background and Aims Thrombotic microangiopathy (TMA) is a life-threatening condition that can be caused on the background of various mechanisms, converging to a severe endotheliopathy that often affects the kidneys. Morphologic features of TMA on kidney biopsy have not been systematically studied. We studied the diagnostic and prognostic role of a kidney biopsy in patients with TMA, either linked to a coexisting condition (i.e., secondary TMA) or not (i.e., complement-mediated [C-]TMA). Method Patients with TMA on kidney biopsy and an enzymatic activity of ADAMTS13 &amp;gt;10% were recruited from the Limburg Renal Registry. C-TMA was defined as massive ex vivo C5b9 formation on the endothelium and/or rare variants in complement genes [1]; secondary TMA was defined as the presence of a coexisting condition and normal complement regulation. Kidney tissue sections were studied for activity and chronicity; also, C3d staining was performed. Results C-TMA and secondary TMA was diagnosed in 35 and 39 patients, respectively. The diagnosis TMA was based on a kidney biopsy in 52 (70%) out of 74 patients as systemic hemolysis, that is, microangiopathic hemolytic anemia (MAHA) and thrombocytopenia, was lacking. Morphologic activity, such as, glomerular thrombosis and mesangiolysis, was not associated with MAHA (p = 0.116) and/or thrombocytopenia (p = 0.380). Morphologic activity, however, was associated with C-TMA, as was lower age and higher serum creatinine. C3d staining did not differentiate C-TMA from secondary TMA. A Mayo Clinic Chronicity Score (MCCS) of ≥4 was associated with an increased partial- (p = 0.023) and complete renal remission rate (p = 0.030), and decreased end-stage kidney disease (p = 0.050). Conclusions Only a small proportion of patients with TMA on kidney biopsy initially present with systemic hemolysis, suggesting an essential role of histologic examination to prevent underdiagnosing. Glomerular thrombosis and mesangiolysis were associated with C-TMA, however there is no role for staining of C3d in differentiating C-TMA from secondary TMA. An increased MCCS indicates worse renal outcome in patients with TMA.

New Insights into Immunopathology Associated to Bothrops lanceolatus Snake Envenomation: Focus on PLA2 Toxin.

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient's prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals.

Complement factor I: Regulatory nexus, driver of immunopathology, and therapeutic

Complement factor I (FI) is the nexus for classical, lectin and alternative pathway complement regulation. FI is an 88 kDa plasma protein that circulates in an inactive configuration until it forms a trimolecular complex with its cofactor and substrate whereupon a structural reorganization allows the catalytic triad to cleave its substrates, C3b and C4b. In keeping with its role as the master complement regulatory enzyme, deficiency has been linked to immunopathology. In the setting of complete FI deficiency, a consumptive C3 deficiency results in recurrent infections with encapsulated microorganisms. Aseptic cerebral inflammation and vasculitic presentations are also less commonly observed. Heterozygous mutations in the factor I gene (CFI) have been demonstrated to be enriched in atypical haemolytic uraemic syndrome, albeit with a very low penetrance. Haploinsufficiency of CFI has also been associated with decreased retinal thickness and is a strong risk factor for the development of age-related macular degeneration. Supplementation of FI using plasma purified or recombinant protein has long been postulated, however, technical difficulties prevented progression into clinical trials. It is only using gene therapy that CFI supplementation has reached the clinic with GT005 in phase I/II clinical trials for geographic atrophy.

Role of Complement in Kidney Diseases - New Aspects

Complement is a central part of the immune system. In the human body, complement is responsible for recognition of infectious microbes, for coordinating the adaptive immune response, controlling homeotic reactions and for the non-inflammatory removal of modified self-cells and infectious microbes. Complement is also closely linked to another proteolytic cascade, the coagulation system. Defective activation and altered complement regulation drives pathology of several severe human kidney diseases.This manuscript summarizes the latest developments on the role of complement in kidney diseases, on new complement inhibitors and on recent complement targeting therapies. In particular focusing on diseases (1) atypical Hemolytic Uremic Syndrome, (2) C3 Glomerulopathy, (3) Anti Neutrophil Cytoplasmic Antibody Mediated Vasculitis, (4) IgA Nephropathy, (5) Membranous Glomerulopathy, (6) Systemic Lupus Erythematosus, (7) Transplant rejection and (8) COVID 19 Infection-Triggered Kidney Diseases. More excitement is generated in this field, as more and more complement mediated diseases can be treated. Several complement targeting compounds are approved by the EMA and FDA and an increasing number of new candidates are in late phase clinical trials. In addition, clinical guidelines are developed for Diagnosis and Therapy of complement mediated diseases, new biomarkers are evaluated in clinical studies, and diagnostic guidelines are in development. The recent Covid infections showed a clear link of complement in thrombo inflammation, which ultimately results in kidney damage. These aspects have increased further the focus of complement inhibitors in COVID infections.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

In peritoneal dialysis (PD) patients, fungi and Pseudomonas aeruginosa are considered important causative microorganisms for peritonitis with poor prognosis. Our objective was to explore expressions of membrane complement (C) regulators (CRegs) and tissue injuries in the peritoneum of patients with PD-related peritonitis, including fungal and Pseudomonas aeruginosa peritonitis. In peritoneal biopsy tissues obtained at PD catheter removal, we investigated the severity of peritonitis-associated peritoneal injuries and the expression of CRegs, CD46, CD55, and CD59 against peritoneal tissues without any episode of peritonitis. In addition, we evaluated peritoneal injuries among fungal and Pseudomonas aeruginosa-peritonitis (P1) and Gram-positive bacterial peritonitis (P2). We also observed deposition of C activation products such as activated C and C5b-9 and measured sC5b-9 in the PD fluid of patients. As a result, the severity of peritoneal injuries correlated inversely with the expression of peritoneal CRegs. Peritoneal CReg expression in peritonitis was significantly reduced compared to no peritonitis. Peritoneal injuries were more severe in P1 than in P2. CReg expression was further decreased and C5b-9 further increased in P1 than in P2. In conclusion, severe peritoneal injuries due to fungal and Pseudomonas aeruginosa-peritonitis decreased CReg expression and increased deposition of activated C3 and C5b-9 in the peritoneum, suggesting that peritonitis, particularly fungal and Pseudomonas aeruginosa-peritonitis, might induce susceptibility to further peritoneal injuries due to excessive C activation.

Reduced binding of apoE4 to complement factor H promotes amyloid‐β oligomerization and neuroinflammation

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late‐onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform‐specific binding of apoE to FH alters Aβ1‐42‐mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aβ1‐42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement‐resistant oligomers with apoE/Aβ1‐42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aβ1‐42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aβ plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.