Gain-of-function factor H-related 5 protein impairs glomerular complement regulation resulting in kidney damage.

Genetic variation within the factor H-related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

A novel mutation in EROS (CYBC1) causes chronic granulomatous disease.

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterised by opportunistic infection and sterile granulomatous inflammation. CGD is caused by a failure of reactive oxygen species (ROS) production by the phagocyte NADPH oxidase. Mutations in the genes encoding phagocyte NADPH oxidase subunits cause CGD. We and others have described a novel form of CGD (CGD5) secondary to lack of EROS (CYBC1), a highly selective chaperone for gp91phox. EROS-deficient cells express minimal levels of gp91phox and its binding partner p22phox, but EROS also controls the expression of other proteins such as P2X7. The full nature of CGD5 is currently unknown. We describe a homozygous frameshift mutation in CYBC1 leading to CGD. Individuals who are heterozygous for this mutation are found in South Asian populations (allele frequency = 0.00006545), thus it is not a private mutation. Therefore, it is likely to be the underlying cause of other cases of CGD.

Defining the Glycosaminoglycan Interactions of Complement Factor H-Related Protein 5.

Complement activation is an important mediator of kidney injury in glomerulonephritis. Complement factor H (FH) and FH-related protein 5 (FHR-5) influence complement activation in C3 glomerulopathy and IgA nephropathy by differentially regulating glomerular complement. FH is a negative regulator of complement C3 activation. Conversely, FHR-5 in vitro promotes C3 activation either directly or by competing with FH for binding to complement C3b. The FH-C3b interaction is enhanced by surface glycosaminoglycans (GAGs) and the FH-GAG interaction is well-characterized. In contrast, the contributions of carbohydrates to the interaction of FHR-5 and C3b are unknown. Using plate-based and microarray technologies we demonstrate that FHR-5 interacts with sulfated GAGs and that this interaction is influenced by the pattern and degree of GAG sulfation. The FHR-5-GAG interaction that we identified has functional relevance as we could show that the ability of FHR-5 to prevent binding of FH to surface C3b is enhanced by surface kidney heparan sulfate. Our findings are important in understanding the molecular basis of the binding of FHR-5 to glomerular complement and the role of FHR-5 in complement-mediated glomerular disease.

Factor H-Related Protein 1 Drives Disease Susceptibility and Prognosis in C3 Glomerulopathy.

BACKGROUND: C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear. METHODS: Using in vitro and in vivo assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed CFHR1 copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population. RESULTS: Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome. CONCLUSIONS: Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.

B cell OX40L supports T follicular helper cell development and contributes to SLE pathogenesis.

OBJECTIVES: TNFSF4 (encodes OX40L) is a susceptibility locus for systemic lupus erythematosus (SLE). Risk alleles increase TNFSF4 expression in cell lines, but the mechanism linking this effect to disease is unclear, and the OX40L-expressing cell types mediating the risk are not clearly established. Blockade of OX40L has been demonstrated to reduce disease severity in several models of autoimmunity, but not in SLE. We sought to investigate its potential therapeutic role in lupus. METHODS: We used a conditional knockout mouse system to investigate the function of OX40L on B and T lymphocytes in systemic autoimmunity. RESULTS: Physiologically, OX40L on both B and T cells contributed to the humoral immune response, but B cell OX40L supported the secondary humoral response and antibody affinity maturation. Our data also indicated that loss of B cell OX40L impeded the generation of splenic T follicular helper cells. We further show that in two models of SLE-a spontaneous congenic model and the H2-IAbm12 graft-versus-host-induced model-loss of B cell OX40L ameliorates the autoimmune phenotype. This improvement was, in each case, accompanied by a decline in T follicular helper cell numbers. Importantly, the germline knockout did not exhibit a markedly different phenotype from the B cell knockout in these models. CONCLUSIONS: These findings contribute to a model in which genetically determined increased OX40L expression promotes human SLE by several mechanisms, contingent on its cellular expression. The improvement in pathology in two models of systemic autoimmunity indicates that OX40L is an excellent therapeutic target in SLE.

Partial Complement Factor H Deficiency Associates with C3 Glomerulopathy and Thrombotic Microangiopathy.

The complement-mediated renal diseases C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) strongly associate with inherited and acquired abnormalities in the regulation of the complement alternative pathway (AP). The major negative regulator of the AP is the plasma protein complement factor H (FH). Abnormalities in FH result in uncontrolled activation of C3 through the AP and associate with susceptibility to both C3G and aHUS. Although previously developed FH-deficient animal models have provided important insights into the mechanisms underlying susceptibility to these unique phenotypes, these models do not entirely reproduce the clinical observations. FH is predominantly synthesized in the liver. We generated mice with hepatocyte-specific FH deficiency and showed that these animals have reduced plasma FH levels with secondary reduction in plasma C3. Unlike mice with complete FH deficiency, hepatocyte-specific FH-deficient animals developed neither plasma C5 depletion nor accumulation of C3 along the glomerular basement membrane. In contrast, subtotal FH deficiency associated with mesangial C3 accumulation consistent with C3G. Although there was no evidence of spontaneous thrombotic microangiopathy, the hepatocyte-specific FH-deficient animals developed severe C5-dependent thrombotic microangiopathy after induction of complement activation within the kidney by accelerated serum nephrotoxic nephritis. Taken together, our data indicate that subtotal FH deficiency can give rise to either spontaneous C3G or aHUS after a complement-activating trigger within the kidney and that the latter is C5 dependent.

Dimerization of complement factor H-related proteins modulates complement activation in vivo.

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Trans-ancestral studies fine map the SLE-susceptibility locus TNFSF4.

We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P=1.71 × 10(-34) , OR=1.43[1.26-1.60]) and rs1234317-T (P=1.16 × 10(-28) , OR=1.38[1.24-1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5' region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5' risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait.

Nonfunctional variant 3 factor H binding proteins as meningococcal vaccine candidates.

Neisseria meningitidis is a human-specific pathogen and leading cause of meningitis and septicemia. Factor H binding protein (fHbp), a virulence factor which protects N. meningitidis from innate immunity by binding the human complement regulator factor H (fH) with high affinity, is also a key antigen in vaccines being developed to prevent meningococcal disease. fHbp can be divided into three variant groups (V1, V2, and V3) that elicit limited immunological cross-reactivity. The interaction of fH with fHbp could impair the immunogenicity of this antigen by hindering access to the antigenic epitopes in fHbp, providing the rationale for the development of nonfunctional fHbps as vaccine candidates. Here, we characterized the two nonfunctional V3 fHbps, fHbp(T286A) and fHbp(E313A), which each contains a single amino acid substitution that leads to a marked reduction in affinity for fH without affecting the folding of the proteins. The immunogenicity of the nonfunctional fHbps was assessed in transgenic mice expressing a single chimeric fH containing domains of human fH involved in binding to fHbp. No differences in anti-V3 fHbp antibody titers were elicited by the wild-type V3 fHbp, V3 fHbp(T286A), and V3 fHbp(E313A), demonstrating that the nonfunctional fHbps retain their immunogenicity. Furthermore, the nonfunctional V3 fHbps elicit serum bactericidal activity that is equivalent to or higher than that observed with the wild-type protein. Our findings provide the basis for the rational design of next-generation vaccines containing nonfunctional V3 fHbps.

A novel CFHR5 fusion protein causes C3 glomerulopathy in a family without Cypriot ancestry.

C3 glomerulopathy describes glomerular pathology associated with predominant deposition of complement C3 including dense deposit disease and C3 glomerulonephritis. Familial C3 glomerulonephritis has been associated with rearrangements affecting the complement factor H-related (CFHR) genes. These include a hybrid CFHR3-1 gene and an internal duplication within the CFHR5 gene. CFHR5 nephropathy, to date, occurred exclusively in patients with Cypriot ancestry, and is associated with a heterozygous internal duplication of the CFHR5 gene resulting in duplication of the exons encoding the first two domains of the CFHR5 protein. Affected individuals possess both the wild-type nine-domain CFHR5 protein (CFHR5(12-9)) and an abnormally large mutant CFHR5 protein in which the initial two protein domains are duplicated (CFHR5(1212-9)). We found CFHR5(1212-9) in association with familial C3 glomerulonephritis in a family without Cypriot ancestry. The genomic rearrangement was distinct from that seen in Cypriot CFHR5 nephropathy. Our findings strengthen the association between CFHR5(1212-9) and familial C3 glomerulonephritis and recommend screening for CFHR5(1212-9) in patients with C3 glomerulopathy irrespective of ethnicity. Since CFHR5(1212-9) can result from at least two genomic rearrangements, screening is most readily achieved through analysis of CFHR5 protein.

Complement component C3 plays a critical role in protecting the aging retina in a murine model of age-related macular degeneration.

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid ß (Aß) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aß on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious.

IL-10-producing regulatory B cells induced by IL-33 (Breg(IL-33)) effectively attenuate mucosal inflammatory responses in the gut.

Regulatory B cells (Breg) have attracted increasing attention for their roles in maintaining peripheral tolerance. Interleukin 33 (IL-33) is a recently identified IL-1 family member, which leads a double-life with both pro- and anti-inflammatory properties. We report here that peritoneal injection of IL-33 exacerbated inflammatory bowel disease in IL-10-deficient (IL-10(-/-)) mice, whereas IL-33-treated IL-10-sufficient (wild type) mice were protected from the disease induction. A phenotypically unconventional subset(s) (CD19(+)CD25(+)CD1d(hi)IgM(hi)CD5(-)CD23(-)Tim-1(-)) of IL-10 producing Breg-like cells (Breg(IL-33)) was identified responsible for the protection. We demonstrated further that Breg(IL-33) isolated from these mice could suppress immune effector cell expansion and functions and, upon adoptive transfer, effectively blocked the development of spontaneous colitis in IL-10(-/-) mice. Our findings indicate an essential protective role, hence therapeutic potential, of Breg(IL-33) against mucosal inflammatory disorders in the gut.

Distinct roles for complement in glomerulonephritis and atherosclerosis revealed in mice with a combination of lupus and hyperlipidemia.

OBJECTIVE: Although the accelerating effect of systemic lupus erythematosus (SLE) on atherosclerosis is well established, the underlying mechanisms are unknown. The aim of this study was to explore the hypothesis that lupus autoimmunity modulates the effect of hypercholesterolemia in driving arterial pathologic development. METHODS: Low-density lipoprotein receptor-deficient (Ldlr(-/-) ) mice were crossed with B6.129-Sle16 (Sle16)-congenic autoimmune mice to obtain Sle16. Ldlr(-/-) mice, which were compared with Ldlr(-/-) and Sle16 control mice. All mice were fed either a low-fat or high-fat diet. Groups of mice were compared, by strain and by diet group, for features of accelerated atherosclerosis and autoimmunity. RESULTS: Presence of the Sle16 locus significantly increased the extent of atherosclerosis in Ldlr(-/-) mice. Circulating C3 levels were significantly reduced in Sle16.Ldlr(-/-) mice compared to Ldlr(-/-) control mice and this was paralleled by a marked reduction in arterial lesion C3 deposition despite similar levels of IgG deposition between the groups. Increased numbers of apoptotic cells in plaques were observed in the high-fat-fed Sle16.Ldlr(-/-) mice, consistent with the observed defective clearance of cellular debris. After receiving the high-fat diet, Sle16.Ldlr(-/-) mice developed glomerulonephritis and displayed enhanced glomerular C3 deposition. CONCLUSION: These results indicate that accelerated atherosclerosis and renal inflammation in SLE are closely linked via immune complex formation and systemic complement depletion. However, whereas hyperlipidemia will enhance renal immune complex-mediated complement activation and the development of nephritis, accelerated atherosclerosis is, instead, related to complement depletion and a reduction in the uptake of apoptotic/necrotic debris. These results suggest that aggressive treatment of hyperlipidemia in patients with SLE may reduce the occurrence of lupus nephritis, as well as diminish the risk of accelerated atherosclerosis.

A hybrid CFHR3-1 gene causes familial C3 glomerulopathy.

Controlled activation of the complement system, a key component of innate immunity, enables destruction of pathogens with minimal damage to host tissue. Complement factor H (CFH), which inhibits complement activation, and five CFH-related proteins (CFHR1-5) compose a family of structurally related molecules. Combined deletion of CFHR3 and CFHR1 is common and confers a protective effect in IgA nephropathy. Here, we report an autosomal dominant complement-mediated GN associated with abnormal increases in copy number across the CFHR3 and CFHR1 loci. In addition to normal copies of these genes, affected individuals carry a unique hybrid CFHR3-1 gene. In addition to identifying an association between these genetic observations and complement-mediated kidney disease, these results provide insight into the protective role of the combined deletion of CFHR3 and CFHR1 in IgA nephropathy.

Multi-omics identify falling LRRC15 as a COVID-19 severity marker and persistent pro-thrombotic signals in convalescence.

Patients with end-stage kidney disease (ESKD) are at high risk of severe COVID-19. Here, we perform longitudinal blood sampling of ESKD haemodialysis patients with COVID-19, collecting samples pre-infection, serially during infection, and after clinical recovery. Using plasma proteomics, and RNA-sequencing and flow cytometry of immune cells, we identify transcriptomic and proteomic signatures of COVID-19 severity, and find distinct temporal molecular profiles in patients with severe disease. Supervised learning reveals that the plasma proteome is a superior indicator of clinical severity than the PBMC transcriptome. We show that a decreasing trajectory of plasma LRRC15, a proposed co-receptor for SARS-CoV-2, is associated with a more severe clinical course. We observe that two months after the acute infection, patients still display dysregulated gene expression related to vascular, platelet and coagulation pathways, including PF4 (platelet factor 4), which may explain the prolonged thrombotic risk following COVID-19.

The alternative pathway is critical for pathogenic complement activation in endotoxin- and diet-induced atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The early components of the classical and lectin complement pathways have been shown to protect low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) from early atherogenesis. However, the role of the alternative pathway remained unknown, and that was investigated in this study. METHODS AND RESULTS: Mice lacking factor B (Bf(-/-)), the initiator of the alternative pathway, were crossed with Ldlr(-/-) mice and studied under different proatherogenic conditions. There was no statistically significant difference in lipid profiles or atherosclerotic lesion development between Bf(-/-)/Ldlr(-/-) and Ldlr(-/-) mice fed a low-fat diet. However, in these groups, administration of bacterial lipopolysaccharide led to a significant increase in atherosclerosis only in Ldlr(-/-) and not in Bf(-/-)/Ldlr(-/-) mice, indicating that the alternative pathway is necessary for endotoxin-mediated atherogenesis. Bf(-/-)/Ldlr(-/-) mice also had significantly decreased cross-sectional aortic root lesion fraction area and reduced lesion complexity compared with Ldlr(-/-) animals after a 12-week period of high-fat diet, although this was also accompanied by reduced levels of serum cholesterol. Under both experimental conditions, the atherosclerotic changes in the Bf(-/-)/Ldlr(-/-) mice were accompanied by a marked reduction in complement activation in the circulation and in atherosclerotic plaques, with no statistically significant differences in immunoglobulin G deposition or in the serum antibody response to oxidized low-density lipoprotein. CONCLUSIONS: These data demonstrate that amplification of complement activation by the alternative pathway in response to lipopolysaccharide or high-fat diet plays a proatherogenic role.

Factor I is required for the development of membranoproliferative glomerulonephritis in factor H-deficient mice.

The inflammatory kidney disease membranoproliferative glomerulonephritis type II (MPGN2) is associated with dysregulation of the alternative pathway of complement activation. MPGN2 is characterized by the presence of complement C3 along the glomerular basement membrane (GBM). Spontaneous activation of C3 through the alternative pathway is regulated by 2 plasma proteins, factor H and factor I. Deficiency of either of these regulators results in uncontrolled C3 activation, although the breakdown of activated C3 is dependent on factor I. Deficiency of factor H, but not factor I, is associated with MPGN2 in humans, pigs, and mice. To explain this discordance, mice with single or combined deficiencies of these factors were studied. MPGN2 did not develop in mice with combined factor H and I deficiency or in mice deficient in factor I alone. However, administration of a source of factor I to mice with combined factor H and factor I deficiency triggered both activated C3 fragments in plasma and GBM C3 deposition. Mouse renal transplant studies demonstrated that C3 deposited along the GBM was derived from plasma. Together, these findings provide what we believe to be the first evidence that factor I-mediated generation of activated C3 fragments in the circulation is a critical determinant for the development of MPGN2 associated with factor H deficiency.

C1q enhances IFN-gamma production by antigen-specific T cells via the CD40 costimulatory pathway on dendritic cells.

Dendritic cells (DCs) are known to produce C1q, the initiator of the classical complement pathway. We demonstrate that murine DCs deficient in C1q (C1qa(-/-)) are poorer than wild-type (WT) DCs at eliciting the proliferation and Th1 differentiation of antigen-specific T cells. These defects result from decreased production of IL-12p70 by C1qa(-/-) DCs and impaired expression of costimulatory molecules CD80 and CD86 in response to CD40 ligation. The defective production of IL-12p70 and the reduced expression of CD80 and CD86 by C1qa(-/-) DCs were specifically mediated via CD40 ligation, as normal levels of IL-12p70 and CD80/86 were observed after ligation of Toll-like receptors (TLRs) on C1qa(-/-) DCs. CD40 ligation on C1qa(-/-) DCs, but not TLR ligation, results in decreased phosphorylation of p38 and ERK1/2 kinases. A strong colocalization of CD40 and C1q was observed by confocal microscopy upon CD40 ligation (but not TLR ligation) on DCs. Furthermore, human DCs from 2 C1q-deficient patients were found to have impaired IL-12p70 production in response to CD40L stimulation. Our novel data suggest that C1q augments the production of IL-12p70 by mouse and human DCs after CD40 triggering and plays important roles in sustaining the maturation of DCs and guiding the activation of T cells.

Severe COVID-19 is associated with endothelial activation and abnormal glycosylation of von Willebrand factor in patients undergoing hemodialysis.

BACKGROUND: A major clinical feature of severe coronavirus diease 2019 (COVID-19) is microvascular thrombosis linked to endothelial cell activation. Consistent with this, a number of studies have shown that patients with severe COVID-19 have highly elevated plasma levels of von Willebrand Factor (VWF) that may contribute to the prothrombotic phenotype. In the current study, we investigated the extent of endothelial activation in patients receiving hemodialysis who had either mild or severe COVID-19. METHODS: Plasma VWF, ADAMTS-13, angiopoietin-2 (Ang2), and syndecan-1 levels were determined by ELISA. The sialic acid content of VWF was investigated using a modified ELISA to measure elderberry bark lectin, specific for sialic acid residues, binding to VWF. RESULTS: Patients receiving hemodialysis with severe COVID-19 had significantly higher plasma levels of VWF and lower ADAMTS-13. VWF levels peaked and were sustained during the first 10 days after positive confirmation of infection. While Ang2 trended toward being higher in severely ill patients, this did not reach significance; however, severely ill patients had significantly higher soluble syndecan-1 levels, with high levels related to risk of death. Finally, higher VWF levels in severely ill patients were correlated with lower VWF sialic acid content. CONCLUSIONS: Severe COVID-19 in patients undergoing hemodialysis is associated with both acute and sustained activation of the endothelium, leading to alteration of the VWF/ADAMTS-13 axis. Lower VWF sialic acid content represents altered VWF processing and further confirms the disturbance caused to the endothelium in COVID-19.

Adeno-Associated Virus Vector Gene Delivery Elevates Factor I Levels and Downregulates the Complement Alternative Pathway In Vivo.

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.