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.

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.

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.

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.

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.

Murine Factor H Co-Produced in Yeast With Protein Disulfide Isomerase Ameliorated C3 Dysregulation in Factor H-Deficient Mice.

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.

Homodimeric Minimal Factor H: In Vivo Tracking and Extended Dosing Studies in Factor H Deficient Mice.

C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH1-5^18-20^R1-2)], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH KD=505 nM; mFH KD=1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH-/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH-/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH-/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G.

Longitudinal proteomic profiling of dialysis patients with COVID-19 reveals markers of severity and predictors of death.

End-stage kidney disease (ESKD) patients are at high risk of severe COVID-19. We measured 436 circulating proteins in serial blood samples from hospitalised and non-hospitalised ESKD patients with COVID-19 (n = 256 samples from 55 patients). Comparison to 51 non-infected patients revealed 221 differentially expressed proteins, with consistent results in a separate subcohort of 46 COVID-19 patients. Two hundred and three proteins were associated with clinical severity, including IL6, markers of monocyte recruitment (e.g. CCL2, CCL7), neutrophil activation (e.g. proteinase-3), and epithelial injury (e.g. KRT19). Machine-learning identified predictors of severity including IL18BP, CTSD, GDF15, and KRT19. Survival analysis with joint models revealed 69 predictors of death. Longitudinal modelling with linear mixed models uncovered 32 proteins displaying different temporal profiles in severe versus non-severe disease, including integrins and adhesion molecules. These data implicate epithelial damage, innate immune activation, and leucocyte-endothelial interactions in the pathology of severe COVID-19 and provide a resource for identifying drug targets.

COVID-19 varies from a mild illness in some people to fatal disease in others. Patients with severe disease tend to be older and have underlying medical problems. People with kidney failure have a particularly high risk of developing severe or fatal COVID-19. Patients with severe COVID-19 have high levels of inflammation, causing damage to tissues around the body. Many drugs that target inflammation have already been developed for other diseases. Therefore, to repurpose existing drugs or design new treatments, it is important to determine which proteins drive inflammation in COVID-19. Here, Gisby, Clarke, Medjeral-Thomas et al. measured 436 proteins in the blood of patients with kidney failure and compared the levels between patients who had COVID-19 to those who did not. This revealed that patients with COVID-19 had increased levels of hundreds of proteins involved in inflammation and tissue injury. Using a combination of statistical and machine learning analyses, Gisby et al. probed the data for proteins that might predict a more severe disease progression. In total, over 200 proteins were linked to disease severity, and 69 with increased risk of death. Tracking how levels of blood proteins changed over time revealed further differences between mild and severe disease. Comparing this data with a similar study of COVID-19 in people without kidney failure showed many similarities. This suggests that the findings may apply to COVID-19 patients more generally. Identifying the proteins that are a cause of severe COVID-19 ­ rather than just correlated with it ­ is an important next step that could help to select new drugs for severe COVID-19.

Immunoglobulin M is required for protection against atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: Immunoglobulin M (IgM) natural antibodies bind oxidatively-modified low-density lipoprotein (LDL) and apoptotic cells and have been implicated as being important for protection against atherosclerosis. We have directly investigated the requirement for IgM by studying the effects of IgM deficiency in LDL receptor-deficient (Ldlr(-/-)) mice. METHODS AND RESULTS: Mice deficient in serum IgM (sIgM) or complement C1q were crossed with Ldlr(-/-) mice and studied on both low-fat and high-fat semisynthetic diets. On both diets, en face and aortic root atherosclerotic lesions in sIgM.Ldlr(-/-) mice were substantially larger and more complex, with accelerated cholesterol crystal formation and increased smooth muscle cell content in aortic root lesions. Combined C1q and IgM deficiency had the same effect as IgM deficiency alone. Increased apoptosis was observed in aortic root lesions of both sIgM.Ldlr(-/-) and C1qa.Ldlr(-/-) mice. Because lesions were significantly larger in IgM-deficient mice than in the absence of C1q, IgM protective mechanisms appear to be partially independent of classical pathway activation and apoptotic cell clearance. Levels of IgG antibodies against copper-oxidized LDL were lower in sIgM.Ldlr(-/-) mice fed a high-fat diet, suggesting compensatory consumption of IgG in the absence of IgM. CONCLUSIONS: This study provides direct evidence that IgM antibodies play a central role in protection against atherosclerosis. The mechanism appears to be at least partly independent of classical pathway complement activation by C1q.