Age-related macular degeneration (AMD) is a chronic, progressive, retinal disease that primarily affects older individuals and is one of the leading causes of blindness worldwide. Both genetic predisposition and environmental factors contribute to its development. Landmark genome-wide association studies (GWAS) positioned the complement system at the center of AMD research, opening new avenues for understanding disease mechanisms and developing targeted therapies. Among the key complement regulators, Factor H and its splice variant FHL-1, are best known for their roles in inhibiting the alternative pathway. Recent research has expanded our understanding of Factor H, revealing a range of non-canonical functions beyond complement regulation which might also affect AMD pathology. These new functions include roles in cell signaling, tissue protection, metabolism, homeostasis, and modulation of inflammation. In contrast, the related protein FHR1 which is also associated with AMD, exhibits pro-inflammatory properties, promoting monocyte recruitment and activation to facilitate clearance processes. In this review, we summarize the canonical and non-canonical functions of Factor H, FHL-1, and FHR1, and we show how the coordinated action of these three proteins integrates into the broader scope of AMD pathogenesis, including complement activation, inflammation, and photoreceptor degeneration. We also describe the current status of approved complement inhibitors in AMD and emerging therapeutic targets within the complement cascade.

Atherosclerosis is the leading cause of heart attack and stroke worldwide. The key characteristic of atherosclerosis is accumulation of LDL cholesterol in artery walls, the subsequent infiltration by monocytes/macrophages, and the development of inflammation. Recently, we reported that plasma protein complement factor H-related 1 (FHR1) binds to the necrotic surfaces of cardiovascular plaques and induces inflammation. Moreover, the concentration of FHR1 is higher, whereas CFHR1 gene deletion frequency is significantly lower in patients with atherosclerosis in comparison to healthy controls. Here we generated muFHR1-/- (the murine homolog of FHR1) knockout mice and then crossed them with ApoE-/- knockout mice (a model of human hyperlipidemia). Notably, deletion of muFHR1 enhanced lipid conversion in the liver as evidenced by RNAseq analysis. This resulted in normalized cholesterol levels, reduced inflammation and plaque formation in muFHR1-/-ApoE-/- mice. These data suggest that muFHR1 directs uptake of oxLDL by macrophages, and supports foam cell formation, plaque development, and inflammation in dyslipidemic mice. As human FHR1 correlates with non-HDL cholesterol concentrations and inflammation markers in patients with atherosclerosis-associated cardiovascular disease (ACVD) we assume that FHR1 plays a key role in the development of atherosclerosis and subsequent events such as stroke and myocardial infarction.

Neurodegeneration is a major driver of disability in multiple sclerosis (MS), the most common chronic inflammatory disease of the central nervous system (CNS)1. Retinal ganglion cells (RGCs), a heterogeneous neuronal population in the eye, undergo degeneration in MS and provide a model to study neuronal subtype-specific resilience to inflammatory injury2. However, the neuron-intrinsic mechanisms underlying differential vulnerability remain unclear. Here we identify a neuroprotective role for intracellular complement factor H (CFH) in neurons. Using single-nucleus RNA-sequencing analysis of RGCs from donors with MS and control individuals, we found that CFH expression was strongly correlated with intrinsic resilience to RGC degeneration. Mechanistically, CFH was induced in retinal and other CNS neurons in response to inflammatory and oxidative stress, where it limited reactive oxygen species accumulation and lipid peroxidation. CFH localized to the endoplasmic reticulum, a major site of lipid peroxidation during neuronal ferroptosis. Its protective activity was dependent on its C-terminal SCR20 domain, was independent of CFH secretion and was preserved in the absence of complement component C3. These findings reveal a non-canonical intracellular function of CFH in neurons. Together, our results identify CFH as a key mediator of neuronal resilience across the CNS in mice and humans and provide mechanistic insight into inflammatory neurodegeneration with implications for MS therapy and neuroprotection more broadly.

Autoimmune factor XIII (FXIII) deficiency is a rare hemorrhagic disease characterized by severe bleeding and a high mortality rate. However, the pathogenesis of this disease remains unclear. FXIII consumption caused by infections is becoming increasingly common. Our clinical investigation, combined with in vivo experiments, revealed that patients and mice with autoimmune FXIII deficiency displayed complement dysfunction and that pathogenic infection and autoantibody generation were positively correlated. Further analysis revealed the presence of combined FXIII-C3 autoantibodies in patients with autoimmune FXIII deficiency. These combined autoantibodies neutralize FXIII, cause bleeding, and form a complex with C3, inhibiting complement activation and complement-mediated adaptive immune responses. Therefore, compromised immune responses increase host susceptibility to pathogenic Candida albicans infections. Consequently, uncontrolled exogenous fungal infections further activate platelets and cause platelet-related CD40 ligand (CD40L) release. By interacting with the CD40 on the B cell surface, the released CD40L further promotes autoreactive B cell activation to produce more autoantibodies, thereby forming a self-amplification loop for the progressive consumption of FXIII. We believe this study provides a perspective on disease pathogenesis and therapeutic guidance for better treatment of autoimmune FXIII deficiency.

Accurate detection of heparin-induced thrombocytopenia (HIT) antibodies is crucial for diagnosing and managing thrombotic events. Conventional immunoassays, however, often lack specificity and require confirmatory testing with fresh human platelets. To address this limitation, we optimized our previously developed cell-based enzyme-linked immunosorbent assay (ELISA) for improved HIT detection under various experimental conditions. Platelet factor 4 was immobilized on breast cancer cells (MDA-MB-231) to capture monoclonal HIT-like (KKO) and non-HIT (RTO) antibodies, which served as models to evaluate assay performance under different pH levels, ionic strengths (NaCl), and fixation methods (ethanol, paraformaldehyde, glutaraldehyde). To identify the most suitable substrate, additional cancer cell lines (HCT-116, MCF-7, HepG2) were tested under live and fixed conditions, with selected conditions validated using human HIT sera. Optimal detection tested with monoclonal antibodies was achieved using 50 mM NaCl and 4% paraformaldehyde fixation. Notably, live MDA-MB-231 and HCT-116 cells demonstrated superior sensitivity and specificity compared to fixed cells. Furthermore, these cell lines enable the efficient detection of HIT antibodies using flow cytometry, a robust and platelet-free diagnostic method. Our findings establish live MDA-MB-231 and HCT-116 cells as highly promising platforms for clinical applications in HIT antibody detection.

Age-related macular degeneration (AMD), a multifactorial type of retinal degeneration represents the most common cause for blindness in elderly. Polymorphisms in complement factor-H increase, while absence of factor-H-related protein-1 (FHR1) decreases the AMD risk, currently explained by their opposing relationship. Here we identify a FHR1-driven pathway fostering chronic cellular inflammation. FHR1 accumulates below the retinal pigment epithelium (RPE) in AMD donor tissue and similarly the murine homolog, muFHR1 is abundant in three AMD-relevant mouse models. These mouse models express the muFHR1 receptor EGF-like module-containing mucin-like hormone receptor 1 (Emr1) on the RPE and on invading mononuclear phagocytes (MP), where both cells form clusters via muFHR1/Emr1. FHR1 ignited EMR2-dependent Ca2+-signals and gene expression in both human RPE cell line and in vivo where muFHR1 affects Emr1+ cells (RPE and MP) gene expression shown by RNAseq analysis. As muFHR1 deletion in mice revealed significantly reduced MP invasion and neoangiogenesis in laser-induced choroidal neovascularization, we hypothesize that FHR1 accumulates, stabilizes and activates MP in the stage of RPE degeneration.

Factor H controls proximal complement activation, and its dysfunction leads to diseases that often manifest in the kidney. Structural and functional analyses have identified 4 distinct functional segments: an N-terminal regulatory unit, a cell binding unit, a segment with combined low-affinity C3b and heparin sites, and a C-terminal recognition or sensor unit with overlapping C3b/C3d and heparin sites. Three segments are linked to diseases. The regulatory segment is affected in C3 glomerulopathy and antineutrophil cytoplasmic antibody–associated vasculitis. The second segment includes the Y402H polymorphism of age-related macular degeneration, is associated with different types of cancer, and is targeted by pathogens. The C-terminal sensor segment is involved in atypical hemolytic uremic syndrome, in FHR1:FHR3 deficient and autoantibody–positive hemolytic uremic syndrome form and is exploited by pathogens. Factor H function is modulated by Factor H like protein 1 and FHR1, 2 plasma proteins that share segments with Factor H. This interplay is critical for fine-tuning local complement. Understanding Factor H’s physiological role, as well as the impact of its absence, mutations, or autoantibody targeting, provides insights into disease mechanisms and provides opportunities for therapeutic intervention by using full-length Factor H, its fragments, or complement-modulatory compounds.

Das Komplementsystem ist eine zentrale Komponente des angeborenen Immunsystems. Eine Fehlregulation dieses Systems trägt wesentlich zur Entstehung verschiedener Nierenerkrankungen und zahlreicher weiterer Organmanifestationen bei. In den letzten Jahren hat bei vielen Erkrankungen das Verständnis der zugrunde liegenden Pathophysiologie erheblich zugenommen. Dadurch ist es heute möglich, komplementvermittelte Erkrankungen gezielt zu diagnostizieren und therapeutisch zu beeinflussen. Dieser Beitrag fokussiert auf die Rolle des Komplements bei systemischen Nierenerkrankungen und erläutert, wie zugelassene Therapeutika die Komplementaktivität modulieren und die immunologische Balance wiederherstellen. Darüber hinaus wird ein Überblick über die breite Palette an Komplementinhibitoren gegeben, die sich derzeit in fortgeschrittener klinischer Entwicklung befinden. In naher Zukunft ist mit der Zulassung weiterer Inhibitoren zu rechnen, die auch bei weiteren Erkrankungen Anwendung finden dürften. Besondere Bedeutung kommt den Sicherheitsaspekten zu: Da das aktivierte Komplementsystem eine Schlüsselfunktion bei der Erkennung und Eliminierung infektiöser Erreger besitzt, erhöht seine pharmakologische Blockade das Risiko für Infektionen mit bekapselten Bakterien. Daher sind bei einer Komplementblockade Impfungen gegen Neisseria meningitidis, Haemophilus influenzae und Streptococcus pneumoniae in vielen Fällen obligat.

Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany Correspondence: Prof. Dr. Peter F. Zipfel, email: [email protected] See related article, "Safety and Efficacy of Avacopan in Patients with Complement 3 Glomerulopathy: Randomized, Double-Blind Clinical Trial," on pages XXX–XXX.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects “self” cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.

C3 glomerulopathy (C3G) is an ultra-rare complement-mediated kidney disease caused by to the deregulation of the alternative pathway (AP) of proximal complement. Consequently, all effector loops of the complement are active and can lead to pathologies, such as C3a- and C5a-mediated inflammation, C3b opsonization, surface C3b-mediated AP C3 convertase assembly, C3 cleavage product deposition in the glomerulus, and lytic C5b-9/MAC cell damage. The most common pathologic mechanisms are defective chronic alternative pathway deregulation, mostly occurring in the plasma, often causing C3 consumption, and chronic complement-mediated glomerular damage. C3G develops over several years, and loss of renal function occurs in more than 50% of patients. C3G is triggered by both genetic and autoimmune alterations. Genetic causes include mutations in individual complement genes and chromosomal variations in the form of deletions and duplications affecting genes encoding complement modulators. Many genetic aberrations result in increased AP C3 convertase activity, either due to decreased activity of regulators, increased activity of modulators, or gain-of-function mutations in genes encoding components of the convertase. Autoimmune forms of C3G do also exist. Autoantibodies target individual complement components and regulators or bind to neoepitopes exposed in the central alternative pathway C3 convertase, thereby increasing enzyme activity. Overactive AP C3 convertase is common in C3G patients. Given that C3G is a complement disease mediated by defective alternative pathway action, complement blockade is an emerging concept for therapy. Here, we summarize both the causes of C3G and the rationale for complement inhibition and list the inhibitors that are being used in the most advanced clinical trials for C3G. With several inhibitors in phase II and III trials, it is expected that effectice treatment for C3G will become availabe in the near future.

Most drugs that target the complement system are designed to inhibit the complement pathway at either the proximal or terminal levels. The use of a natural complement regulator such as factor H (FH) could provide a superior treatment option by restoring the balance of an overactive complement system while preserving its normal physiological functions. Until now, the systemic treatment of complement-associated disorders with FH has been deemed unfeasible, primarily due to high production costs, risks related to FH purified from donors’ blood, and the challenging expression of recombinant FH in different host systems. We recently demonstrated that a moss-based expression system can produce high yields of properly folded, fully functional, recombinant FH. However, the half-life of the initial variant (CPV-101) was relatively short. Here we show that the same polypeptide with modified glycosylation (CPV-104) achieves a pharmacokinetic profile comparable to that of native FH derived from human serum. The treatment of FH-deficient mice with CPV-104 significantly improved important efficacy parameters such as the normalization of serum C3 levels and the rapid degradation of C3 deposits in the kidney compared to treatment with CPV-101. Furthermore, CPV-104 showed comparable functionality to serum-derived FH in vitro, as well as similar performance in ex vivo assays involving samples from patients with atypical hemolytic uremic syndrome, C3 glomerulopathy and paroxysomal nocturnal hematuria. CPV-104 – the human FH analog expressed in moss – will therefore allow the treatment of complement-associated human diseases by rebalancing instead of inhibiting the complement cascade.

IntroductionFHR1 is a multifunctional human plasma protein with three C-terminal domains, namely short consensus repeats (SCR) 3–5, showing 98% sequence-identity with the complement inhibitor Factor H. We show that FHR1 uses all three C-terminal SCR to make surface contact. The conserved C-terminal regions of FHR1 and Factor H are altered in patients with atypical-hemolytic-uremic-syndrome. Therefore, we compared FHR1 isoforms with sequence-variations in SCR3, and pathogenic mutants with sequence variations in SCR5.MethodsFHR1 binding to apoptotic cells was evaluated EM and fluorescent microscopy and in kidney biopsies. FHR1 and Factor H variants and mutants were generated and expressed. The variants and mutant proteins were tested in binding studies to C3b , C3d and heparin, in hemolytic assays and for the induction of inflammatory cytokines. The action profiles of FHR1 and Factor H were calculated and compared.ResultsFunctional data revealed that residues YVQ vs HLE in SCR3 and LA vs SV in SCR5 altered ligand binding and surface interaction, influenced target recognition and complement control. Amino-acid-sequence variations in SCR3 influenced FHR1 contact with surface constituents, such as glycosaminoglycans. By contrast, SCR5, the most C-terminal domain, was more relevant for C3b/C3d contact. Notably, wild-type FHR1LA selected C3d, while pathogenic aHUS-associated alterations FHR1SV selected C3b. In consequence mutant FHR1SV altered fined-tuned FHR1-directed effector functions while pathogenic Factor HLA modified C3-convertase control.DiscussionThis influences timing of complement control and inflammatory effector actions at modified self-surfaces. Pathogenic FHR1SV, directed to C3b-decorated targets, adds inflammatory activity at a time when C3-convertase control is appropriate and conversely, mutant Factor HLA adds C3-convertase control at C3d-coated surfaces when inflammatory effector functions are favorable. Further, our computational modeling approach confirms such distinct effects of FHR1 monomers and dimers as compared to flexible Factor H. These effects may explain inappropriate timing of complement regulation and inflammation of the aHUS-derived mutant proteins FHR1SV and Factor HLA.

Pathogenic platelet factor 4 (PF4) antibodies contributed to the abnormal coagulation profiles in COVID-19 and vaccinated patients. However, the mechanism of what triggers the body to produce these antibodies has not yet been clarified. Similar patterns and many comparable features between the COVID-19 virus and heparin-induced thrombocytopenia (HIT) have been reported. Previously, we identified a new mechanism of autoimmunity in HIT in which PF4-antibodies self-clustered PF4 and exposed binding epitopes for other pathogenic PF4/eparin antibodies. Here, we first proved that the SARS-CoV-2 spike protein (SP) also binds to PF4. The binding was evidenced by the increase in mass and optical intensity as observed through quartz crystal microbalance and immunosorbent assay, while the switching of the surface zeta potential caused by protein interactions and binding affinity of PF4-SP were evaluated by dynamic light scattering and isothermal spectral shift analysis. Based on our results, we proposed a mechanism for the generation of PF4 antibodies in COVID-19 patients. We further validated the changes in zeta potential and interaction affinity between PF4 and SP and found that their binding mechanism differs from ACE2-SP binding. Importantly, the PF4/SP complexes facilitate the binding of anti-PF4/Heparin antibodies. Our findings offer a fresh perspective on PF4 engagement with the SARS-CoV-2 SP, illuminating the role of PF4/SP complexes in severe thrombotic events.