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The ability of the complement system to rapidly and broadly react to microbial intruders, apoptotic cells and other threats by inducing forceful elimination responses is indispensable for its role as host defense and surveillance system. However, the danger sensing versatility of complement may come at a steep price for patients suffering from various immune, inflammatory, age-related, or biomaterial-induced conditions. Misguided recognition of cell debris or transplants, excessive activation by microbial or damaged host cells, autoimmune events, and dysregulation of the complement response may all induce effector functions that damage rather than protect host tissue. Although complement has long been associated with disease, the prevalence, impact and complexity of complement’s involvement in pathological processes is only now becoming fully recognized. While complement rarely constitutes the sole driver of disease, it acts as initiator, contributor, and/or exacerbator in numerous disorders. Identifying the factors that tip complement’s balance from protective to damaging effects in a particular disease continues to prove challenging. Fortunately, however, molecular insight into complement functions, improved disease models, and growing clinical experience has led to a greatly improved understanding of complement’s pathological side. The identification of novel complement-mediated indications and the clinical availability of the first therapeutic complement inhibitors has also sparked a renewed interest in developing complement-targeted drugs, which meanwhile led to new approvals and promising candidates in late-stage evaluation. More than a century after its description, complement now has truly reached the clinic and the recent developments hold great promise for diagnosis and therapy alike.

Background The development of age-related macular degeneration (AMD) is influenced by risk factors that contribute to inflammatory processes, cellular stress responses, and a dysregulation of the complement system. Given the incomplete understanding of the pathogenesis of AMD and the necessity for novel therapeutics, biomarker studies investigating aqueous humour from the anterior chamber of the eye serve as a valuable tool. This pilot study aimed to assess inflammatory mediators and complement components in aqueous humour of non-exudative AMD patients in comparison with a control group. Methods The aqueous humour of 12 non-exudative AMD patients and 21 control subjects was collected during cataract surgery. Levels of 78 inflammatory proteins and complement components were measured using multiplex immunoassays. The influence of sex or smoking on the AMD status was assessed using Pearson’s chi-square test. Biomarker levels between AMD patients vs. controls, smokers vs. non-smokers, and females vs. males were compared. Parametric datasets were analysed using independent-means t -test, while non-parametric data analysis was conducted utilising Wilcoxon’s rank-sum test. Spearman’s correlation investigated associations between drusen volume and biomarker levels, as well as biomarker levels and subject age. Results All examined 78 immunological factors were detectable in aqueous humour. The proteins were categorised into high, medium, and low level groups. Aqueous humour contained high levels of complement proteins, including iC3b, FH/FHL-1, C4B, and FI. Non-exudative AMD patients exhibited decreased levels of C4 ( P  = 0.020), IL-10 ( P  = 0.033), and FI ( P  = 0.082). A positive correlation was observed between drusen volume and CCL4 levels ( r S  = 0.78, P  = 0.013). Furthermore, smokers demonstrated significantly increased levels of pro-inflammatory proteins (CCL7, IL-7; P  = 0.027, P  = 0.030). MMP-1 was positively correlated with age ( r S  = 0.44, P  = 0.010), while sex differences were observed in FB ( P  = 0.027) and C4B ( P  = 0.036) levels. Conclusions This pilot study presents an initial overview of inflammation-associated biomarkers in the aqueous humour, highlighting potential roles for C4 and IL-10 in the development of non-exudative AMD. A larger, more-focused follow-up study is in progress to further investigate biomarkers localised to the eye and refine our understanding of AMD.

The alternative pathway is a continuously active surveillance arm of the complement system, and it can also enhance complement activation initiated by the classical and the lectin pathways. Various membrane-bound and plasma regulatory proteins control the activation of the potentially deleterious complement system. Among the regulators, the plasma glycoprotein factor H (FH) is the main inhibitor of the alternative pathway and its powerful amplification loop. FH belongs to a protein family that also includes FH-like protein 1 and five factor H-related (FHR-1 to FHR-5) proteins. Genetic variants and abnormal rearrangements involving the FH protein family have been linked to numerous systemic and organ-specific diseases, including age-related macular degeneration, and the renal pathologies atypical hemolytic uremic syndrome, C3 glomerulopathies, and IgA nephropathy. This review covers the known and recently emerged ligands and interactions of the human FH family proteins associated with disease and discuss the very recent experimental data that suggest FH-antagonistic and complement-activating functions for the FHR proteins.

Factor H-related (FHR) protein 1 and 2 form dimers resulting in FHR-1 and -2 homodimers, and FHR-1/2 heterodimers. Dimerization is hypothesized to further increase their antagonistic function with complement regulator factor H (FH). So far, only FHR-1 homodimers and FHR-1/2 heterodimers could be quantified in a direct way. With the reported genetic associations between CFHR2 and complement-related diseases such as age related macular degeneration and C3-glomerulopathy, direct assessment of FHR-2/2 levels determining the dimer distribution of FHR-1 and -2 is needed to further elucidate their role within complement regulation. Therefore, novel in-house generated FHR-2 antibodies were used to develop a specific ELISA to enable direct quantification of FHR-2 homodimers. Allowing for the first time the accurate measurement of all FHR-1 and -2 containing dimers in a large cohort of healthy donors. By using native FHR-1 and -2 or deficient plasma, we determined the stability, kinetics and distribution of FHR-1 and -2 dimers. Additionally, we show how genetic variants influence dimer levels. Our results confirm a rapid, dynamic, dimer formation in plasma and show FHR-1/2 dimerization rearches a distribution equilibrium that is limited by the relative low levels of FHR-2 in relation to its dimerization partner FHR-1.

Schistosomes are highly efficient evaders of human immunity, as evident by their ability to survive in human blood for years. How they protect themselves against the constant attack by a key element of innate immunity, the complement system, has remained unclear. In this study, new light is shed on the interaction between distinct life-cycle stages of and the human complement system. We demonstrate that schistosomula, the young stage assumed immediately after cercaria penetration of the skin, are extremely vulnerable towards complement-mediated killing as only 10-20% survive. The survival rate increases to 70% already within 30 minutes and reaches close to 100% within two hours. Pathway-specific complement inhibitors revealed the alternative pathway of complement activation as the main contributor to killing and damage of the schistosomula. Moreover, the complement regulator factor H is recruited by the schistosomula in this early stage to evade killing. Surviving parasites appear fully viable despite the ongoing complement attack, as demonstrated by the deposition of C3 fragments. However, when exposed to the widely used schistocidal drug praziquantel, the vulnerability of 24 h-old schistosomula towards complement-mediated killing is notably increased; no such effect was observed for mefloquine or oxamniquine. Similar to the younger life-cycle stages, adult worms remain under complement attack. C3 fragments were found all over the outer surface (tegument), deposited mostly on the ridges and not on the tubercles. The recruitment of factor H merits more detailed studies that pinpoint the molecules involved and elucidate the novel possibilities to intercept the uncovered immune evasion therapeutically. That praziquantel and complement work in synergy is surprising and may in the future result in enhanced understanding of the drug's mechanism of action.

Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous deletions or compound heterozygous missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed , using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Background The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance. Methods In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out ( Cfh −/− ) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model. Results We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh − / − mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes. Conclusion Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.

Dysregulation of the complement alternative pathway (AP) is a major pathogenic mechanism in atypical hemolytic-uremic syndrome (aHUS). Genetic or acquired defects in factor H (FH), the main AP regulator, are major aHUS drivers that associate with a poor prognosis. FH activity has been suggested to be downregulated by homologous FH-related (FHR) proteins, including FHR-3 and FHR-1. Hence, their relative levels in plasma could be disease-relevant. The genes coding for FH, FHR-3, and FHR-1 ( , and , respectively) are polymorphic and located adjacent to each other on human chromosome 1q31.3. We have previously shown that haplotype associates with aHUS and reduced FH levels. In this study, we used a specific enzyme-linked immunosorbent assay to quantify FHR-3 in plasma samples from controls and patients with aHUS genotyped for the three known alleles ( , and ). In the 218 patients carrying at least one copy of , significant differences between genotype groups were found, with patients having the lowest FHR-3 concentration (0.684-1.032 µg/mL), and patients presenting intermediate levels (1.437-2.201 µg/mL), and and patients showing the highest concentration (2.330-4.056 µg/mL) (  < 0.001). These data indicate that is a low-expression allele, whereas , associated with increased risk of aHUS, is a high-expression allele. Our study reveals that the aHUS-risk haplotype generates twofold more FHR-3 than the non-risk haplotype. In addition, FHR-3 levels were higher in patients with aHUS than in control individuals with the same genotype. These data suggest that increased plasma levels of FHR-3, altering the balance between FH and FHR-3, likely impact the FH regulatory functions and contribute to the development of aHUS.