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.

Recognition and control of neutrophil extracellular trap formation by MICL.

Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage1,2. Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS-PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies to MICL inhibit key functions of this receptor. Of note, we also detect similarly inhibitory anti-MICL autoantibodies in patients with other diseases linked to aberrant NET formation, including lupus and severe COVID-19. By contrast, dysregulation of NET release is protective during systemic infection with the fungal pathogen Aspergillus fumigatus. Together, we show that the recognition of NETs by MICL represents a fundamental autoregulatory pathway that controls neutrophil activity and NET formation.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Canonical and noncanonical functions of complement in systemic lupus erythematosus.

For many years complement activation in systemic lupus erythematosus (SLE) was viewed as a major cause of tissue injury. However, human and murine studies showed that complement plays a protective as well as a proinflammatory role in tissue damage. A hierarchy is apparent with early classical pathway components, particularly C1q, exerting the greatest influence. Understanding the mechanisms underlying the protective function(s) of complement remains an important challenge for the future and has implications for the use of complement therapy in SLE. We review recent advances in the field and give a new perspective on the complement conundrum in SLE.

The role of complement in kidney disease: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Uncontrolled complement activation can cause or contribute to glomerular injury in multiple kidney diseases. Although complement activation plays a causal role in atypical hemolytic uremic syndrome and C3 glomerulopathy, over the past decade, a rapidly accumulating body of evidence has shown a role for complement activation in multiple other kidney diseases, including diabetic nephropathy and several glomerulonephritides. The number of available complement inhibitor therapies has also increased during the same period. In 2022, Kidney Diseases: Improving Global Outcomes (KDIGO) convened a Controversies Conference, "The Role of Complement in Kidney Disease," to address the expanding role of complement dysregulation in the pathophysiology, diagnosis, and management of various glomerular diseases, diabetic nephropathy, and other forms of hemolytic uremic syndrome. Conference participants reviewed the evidence for complement playing a primary causal or secondary role in progression for several disease states and considered how evidence of complement involvement might inform management. Participating patients with various complement-mediated diseases and caregivers described concerns related to life planning, implications surrounding genetic testing, and the need for inclusive implementation of effective novel therapies into clinical practice. The value of biomarkers in monitoring disease course and the role of the glomerular microenvironment in complement response were examined, and key gaps in knowledge and research priorities were identified.

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.

Factor H related proteins modulate complement activation on kidney cells.

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney.

Improving Clinical Trials for Anticomplement Therapies in Complement-Mediated Glomerulopathies: Report of a Scientific Workshop Sponsored by the National Kidney Foundation.

Blocking the complement system as a therapeutic strategy has been proposed for numerous glomerular diseases but presents myriad questions and challenges, not the least of which is demonstrating efficacy and safety. In light of these potential issues and because there are an increasing number of anticomplement therapy trials either planned or under way, the National Kidney Foundation facilitated an all-virtual scientific workshop entitled "Improving Clinical Trials for Anti-Complement Therapies in Complement-Mediated Glomerulopathies." Attended by patient representatives and experts in glomerular diseases, complement physiology, and clinical trial design, the aim of this workshop was to develop standards applicable for designing and conducting clinical trials for anticomplement therapies across a wide spectrum of complement-mediated glomerulopathies. Discussions focused on study design, participant risk assessment and mitigation, laboratory measurements and biomarkers to support these studies, and identification of optimal outcome measures to detect benefit, specifically for trials in complement-mediated diseases. This report summarizes the discussions from this workshop and outlines consensus recommendations.

Baseline Clinical Characteristics and Complement Biomarkers of Patients with C3 Glomerulopathy Enrolled in Two Phase 2 Studies Investigating the Factor D Inhibitor Danicopan.

INTRODUCTION: C3 glomerulopathy (C3G) is a rare, progressive kidney disease resulting from dysregulation of the alternative pathway (AP) of complement. Biomarkers at baseline were investigated in patients with C3G who participated in two phase 2 studies with the factor D (FD) inhibitor, danicopan. METHODS: Patients with biopsy-confirmed C3G, proteinuria ≥500 mg/day, and estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 were enrolled into two studies (NCT03369236 and NCT03459443). Biomarker analysis was performed for patients with C3G confirmed by central pathology laboratory re-evaluation. Complement and clinical biomarkers, biopsy composite score, and activity and chronicity indices were assessed at baseline and analyzed by pairwise Spearman correlation analysis. RESULTS: Twenty-nine patients were included in the analysis (median [interquartile range] age: 24.0 [10.0] years). Systemic complement AP activation was evident by reduced median concentrations of C3 and C5, elevated sC5b-9, and normal C4, relative to reference ranges. C3 showed strong pairwise correlations with C5 and sC5b-9 (r = 0.80 and -0.73, respectively; p < 0.0001). Baseline Ba and FD concentrations were inversely correlated with eGFR (r = -0.83 and -0.87, respectively; p < 0.0001). Urinary concentrations of sC5b-9 were correlated with both plasma sC5b-9 and proteinuria (r = 0.69 and r = 0.83, respectively; p < 0.0001). Biopsy activity indices correlated strongly with biomarkers of systemic AP activation, including C3 (r = -0.76, p < 0.0001), whereas chronicity indices aligned more closely with eGFR (r = -0.57, p = 0.0021). CONCLUSION: Associations among complement biomarkers, kidney function, and kidney histology may add to the current understanding of C3G and assist with the characterization of patients with this heterogenous disease.

Clinical Outcomes of Patients with C3G or IC-MPGN Treated with the Factor D Inhibitor Danicopan: Final Results from Two Phase 2 Studies.

INTRODUCTION: C3 glomerulopathy (C3G) is an ultrarare, chronic and progressive nephropathy mediated by dysregulation of the alternative pathway of complement (AP), with poor prognosis and limited treatment options. Targeted inhibition of proximal AP through factor D (FD) blockade represents a rational treatment approach. We present two phase 2 proof-of-concept clinical studies of the orally active FD inhibitor danicopan in patients with C3G and immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) (NCT03369236 and NCT03459443). METHODS: A double-blind, placebo-controlled study in patients with C3G and a single-arm, open-label study in patients with C3G or IC-MPGN treated with danicopan are reported. The studies evaluated pharmacokinetic/pharmacodynamic (PK/PD), efficacy, and safety outcomes. The co-primary endpoints were change from baseline in composite biopsy score and the proportion of patients with a 30% reduction in proteinuria relative to baseline at 6 or 12 months. RESULTS: Optimal systemic concentrations of danicopan were not achieved for complete and sustained inhibition of AP, although there was evidence that blockade of FD reduced AP activity shortly after drug administration. Consequently, limited clinical response was observed in key efficacy endpoints. While stable disease or improvement from baseline was seen in some patients, response was not consistent. The data confirmed the favorable safety profile of danicopan. CONCLUSION: While demonstrating a favorable safety profile, danicopan resulted in incomplete and inadequately sustained inhibition of AP, probably due to limitations in its PK/PD profile in C3G, leading to lack of efficacy. Complete and sustained AP inhibition is required for a clinical response in patients with C3G.

Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

O- and N-Glycosylation of Serum Immunoglobulin A is Associated with IgA Nephropathy and Glomerular Function.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and is a leading cause of renal failure. The disease mechanisms are not completely understood, but a higher abundance of galactose-deficient IgA is recognized to play a crucial role in IgAN pathogenesis. Although both types of human IgA (IgA1 and IgA2) have several N-glycans as post-translational modification, only IgA1 features extensive hinge-region O-glycosylation. IgA1 galactose deficiency on the O-glycans is commonly detected by a lectin-based method. To date, limited detail is known about IgA O- and N-glycosylation in IgAN. METHODS: To gain insights into the complex O- and N-glycosylation of serum IgA1 and IgA2 in IgAN, we used liquid chromatography-mass spectrometry (LC-MS) for the analysis of tryptic glycopeptides of serum IgA from 83 patients with IgAN and 244 age- and sex-matched healthy controls. RESULTS: Multiple structural features of N-glycosylation of IgA1 and IgA2 were associated with IgAN and glomerular function in our cross-sectional study. These features included differences in galactosylation, sialylation, bisection, fucosylation, and N-glycan complexity. Moreover, IgA1 O-glycan sialylation was associated with both the disease and glomerular function. Finally, glycopeptides were a better predictor of IgAN and glomerular function than galactose-deficient IgA1 levels measured by lectin-based ELISA. CONCLUSIONS: Our high-resolution data suggest that IgA O- and N-glycopeptides are promising targets for future investigations on the pathophysiology of IgAN and as potential noninvasive biomarkers for disease prediction and deteriorating kidney function.

Complement activity is regulated in C3 glomerulopathy by IgG-factor H fusion proteins with and without properdin targeting domains.

C3 glomerulopathy is characterized by accumulation of complement C3 within glomeruli. Causes include, but are not limited to, abnormalities in factor H, the major negative regulator of the complement alternative pathway. Factor H-deficient (Cfh-/-) mice develop C3 glomerulopathy together with a reduction in plasma C3 levels. Using this model, we assessed the efficacy of two fusion proteins containing the factor H alternative pathway regulatory domains (FH1-5) linked to either a non-targeting mouse immunoglobulin (IgG-FH1-5) or to an anti-mouse properdin antibody (Anti-P-FH1-5). Both proteins increased plasma C3 and reduced glomerular C3 deposition to an equivalent extent, suggesting that properdin-targeting was not required for FH1-5 to alter C3 activation in either plasma or glomeruli. Following IgG-FH1-5 administration, plasma C3 levels temporally correlated with changes in factor B levels whereas plasma C5 levels correlated with changes in plasma properdin levels. Notably, the increases in plasma C5 and properdin levels persisted for longer than the increases in C3 and factor B. In Cfh-/- mice IgG-FH1-5 reduced kidney injury during accelerated serum nephrotoxic nephritis. Thus, our data demonstrate that IgG-FH1-5 restored circulating alternative pathway activity and reduced glomerular C3 deposition in Cfh-/- mice and that plasma properdin levels are a sensitive marker of C5 convertase activity in factor H deficiency. The immunoglobulin conjugated FH1-5 protein, through its comparatively long plasma half-life, may be a potential therapy for C3 glomerulopathy.

Complement and kidney disease, new insights.

PURPOSE OF REVIEW: In this review, we discuss recent studies showing the importance of the complement pathway in kidney disease. RECENT FINDINGS: Recent findings in C3 glomerulopathy (C3G) include: acute postinfectious glomerulonephritis is characterised by the presence of antifactor B antibodies; human leukocyte antigen type, but not rare complement gene variation, is associated with primary immunoglobulin-associated membranoproliferative GN and C3G. Immunohistochemistry in C3G shows that factor H related protein 5 (FHR5) is the most prevalent complement protein and correlates with kidney function. A multicentre study supported the use of mycophenolate mofetil (MMF) in C3G even after a propensity matching analysis. In immunoglobulin A nephropathy (IgAN) several studies have emphasised the importance of complement. Imbalances of circulating FH and FHR1 and FHR5, which interfere with the regulatory functions of FH, associate with IgAN. Immunohistochemistry has shown associations between glomerular FHR5 deposition and C3 activation; glomerular FHR5 associated with clinical markers of IgAN severity. Data also suggest the lectin complement pathway contributes to IgAN severity. We also discuss complement activation in thrombotic microangiopathy and other kidney diseases. SUMMARY: Complement activity can be detected in a wide range of kidney diseases and this provides pathogenic insight and potential for therapy with the ongoing development of several drugs directed at complement activation.

High Prevalence of Asymptomatic COVID-19 Infection in Hemodialysis Patients Detected Using Serologic Screening.

BACKGROUND: Strategies to minimize the risk of transmission and acquisition of COVID-19 infection in patients with ESKD receiving in-center hemodialysis have been rapidly implemented across the globe. Despite these interventions, confirmed COVID-19 infection rates have been high in the United Kingdom. Prevalence of asymptomatic disease in an adult hemodialysis population has not been reported. Also, to our knowledge, the development of humoral response to SARS-CoV-2 has not been previously reported in this population. Although serologic testing does not provide information on the infectivity of patients, seroprevalence studies may enable investigation of exposure within dialysis units and hence, assessment of current screening strategies. METHODS: To investigate the seroprevalence of SARS-CoV-2 antibodies in a hemodialysis population, we used the Abbott IgG assay with the Architect system to test serum samples from 356 patients receiving in-center hemodialysis for SARS-CoV-2 antibodies. RESULTS: Of 356 patients, 121 had been symptomatic when screened before a dialysis session and received an RT-PCR test; 79 (22.2% of the total study population) tested positive for COVID-19. Serologic testing of all 356 patients found 129 (36.2%) who tested positive for SARS-CoV-2 antibodies. Only two patients with PCR-confirmed infection did not seroconvert. Of the 129 patients with SARS-CoV-2 antibodies, 52 (40.3%) had asymptomatic disease or undetected disease by PCR testing alone. CONCLUSIONS: We found a high seroprevalence of SARS-CoV-2 antibodies in patients receiving in-center hemodialysis. Serologic evidence of previous infection in asymptomatic or PCR-negative patients suggests that current diagnostic screening strategies may be limited in their ability to detect acute infection.

Successful simultaneous liver-kidney transplantation for renal failure associated with hereditary complement C3 deficiency.

Hereditary complement C3 deficiency is associated with recurrent bacterial infections and proliferative glomerulonephritis. We describe a case of an adult with complete deficiency of complement C3 due to homozygous mutations in C3 gene: c.1811delT (Val604Glyfs*2), recurrent bacterial infections, crescentic glomerulonephritis, and end-stage renal failure. Following isolated kidney transplantation he would remain C3 deficient with a similar, or increased, risk of infections and glomerulonephritis. As C3 is predominantly synthesized in the liver, with a small proportion of C3 monocyte derived and kidney derived, he proceeded to simultaneous liver-kidney transplantation. The procedure has been successful with restoration of his circulating C3 levels, normal liver and kidney function at 26 months of follow-up. Simultaneous liver-kidney transplant is a viable option to be considered in this rare setting.