Autoantibodies Against the Complement Regulator Factor H in the Serum of Patients With Neuromyelitis Optica Spectrum Disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by pathogenic, complement-activating autoantibodies against the main water channel in the CNS, aquaporin 4 (AQP4). NMOSD is frequently associated with additional autoantibodies and antibody-mediated diseases. Because the alternative pathway amplifies complement activation, our aim was to evaluate the presence of autoantibodies against the alternative pathway C3 convertase, its components C3b and factor B, and the complement regulator factor H (FH) in NMOSD. Four out of 45 AQP4-seropositive NMOSD patients (similar to 9%) had FH autoantibodies in serum and none had antibodies to C3b, factor B and C3bBb. The FH autoantibody titers were low in three and high in one of the patients, and the avidity indexes were low. FH-IgG complexes were detected in the purified IgG fractions by Western blot. The autoantibodies bound to FH domains 19-20, and also recognized the homologous FH-related protein 1 (FHR-1), similar to FH autoantibodies associated with atypical hemolytic uremic syndrome (aHUS). However, in contrast to the majority of autoantibody-positive aHUS patients, these four NMOSD patients did not lack FHR-1. Analysis of autoantibody binding to FH19-20 mutants and linear synthetic peptides of the C-terminal FH and FHR-1 domains, as well as reduced FH, revealed differences in the exact binding sites of the autoantibodies. Importantly, all four autoantibodies inhibited C3b binding to FH. In conclusion, our results demonstrate that FH autoantibodies are not uncommon in NMOSD and suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage in NMOSD.Peer reviewe

Heterogeneity but individual constancy of epitopes, isotypes and avidity of factor H autoantibodies in atypical hemolytic uremic syndrome

Factor H (FH) autoantibodies are present in 6-10% of atypical hemolytic uremic syndrome (aHUS) patients, most of whom have homozygous deficiency of the FH-related protein FHR-1. Although the pathogenic role of the autoantibodies is established, little is known about their molecular characteristics and changes over time. Here, we describe the specificity and other immunological features of anti-FH autoantibodies in the Spanish and Hungarian aHUS cohorts. A total of 19 patients were included and serial samples of 14 of them were available. FH autoantibodies from FHR-1 deficient patients (n=13) mainly recognized FH, its SCR19-20 fragment and FHR-1, but autoantibody specificity in patients who are homo- or heterozygous for the CFHR1 gene (n=6) was heterogeneous. No significant changes apart from total antibody titer were observed during follow-up in each patient. Fine epitope mapping with recombinant FH SCR19-20 containing single amino acid mutations showed significantly reduced binding in 6 out of 14 patients. In most cases, autoantibody binding to residues 1183-1189 and 1210-1215 was impaired, revealing a major common autoantibody epitope. Avidities showed variations between patients, but in most cases the avidity index did not change upon time. Most autoantibodies were IgG3, and all but three presented only with kappa or with lambda light chains. Although the pathogenic role of anti-FH autoantibodies in aHUS is well established, this study shows autoantibody heterogeneity among patients, but no significant variation in their characteristics over time in each patient. The presence of a single light chain in 16 out of 19 patients and the limited number of recognized epitopes suggest a restricted autoantibody response in most patients

Microbes Bind Complement Inhibitor Factor H via a Common Site

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19–20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens viautilization of a “superevasion site.

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the “common microbial binding site” on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.Peer reviewe

Immunogenicity of subcutaneous TNF inhibitors and its clinical significance in real-life setting in patients with spondyloarthritis

Key messages Considerable proportion of patients with SpA have been immunized to the subcutaneous anti-TNF drug they are using. Concomitant use of MTX protects from immunization, whereas SASP does not. Patients with SpA using subcutaneous anti-TNF drugs can benefit from monitoring of the drug trough levels. Immunization to biological drugs can lead to decreased efficacy and increased risk of adverse effects. The objective of this cross-sectional study was to assess the extent and significance of immunization to subcutaneous tumor necrosis factor (TNF) inhibitors in axial spondyloarthritis (axSpA) patients in real-life setting. A serum sample was taken 1-2 days before the next drug injection. Drug trough concentrations, anti-drug antibodies (ADAb) and TNF-blocking capacity were measured in 273 patients with axSpA using subcutaneous anti-TNF drugs. The clinical activity of SpA was assessed using the Bath AS Disease Activity Index (BASDAI) and the Maastricht AS Entheses Score (MASES). ADAb were found in 11% of the 273 patients: in 21/99 (21%) of patients who used adalimumab, in 0/83 (0%) of those who used etanercept, in 2/79 (3%) of those who used golimumab and in 6/12 (50%) of those who used certolizumab pegol. Use of methotrexate reduced the risk of formation of ADAb, whereas sulfasalazine did not. Presence of ADAb resulted in decreased drug concentration and reduced TNF-blocking capacity. However, low levels of ADAb had no effect on TNF-blocking capacity and did not correlate with disease activity. The drug trough levels were below the consensus target level in 36% of the patients. High BMI correlated with low drug trough concentration. Patients with low drug trough levels had higher disease activity. The presence of anti-drug antibodies was associated with reduced drug trough levels, and the patients with low drug trough levels had higher disease activity. The drug trough levels were below target level in significant proportion of patients and, thus, measuring the drug concentration and ADAb could help to optimize the treatment in SpA patients.Peer reviewe

Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.Peer reviewe

Complement in glomerular injury

In recent years, research into the role of complement in the immunopathogenesis of renal disease has broadened our understanding of the fragile balance between the protective and harmful functions of the complement system. Interventions into the complement system in various models of immune-mediated renal disease have resulted in both favourable and unfavourable effects and will allow us to precisely define the level of the complement cascade at which a therapeutic intervention will result in an optimal effect. The discovery of mutations of complement regulatory molecules has established a role of complement in the haemolytic uremic syndrome and membranoproliferative glomerulonephritis, and genotyping for mutations of the complement system are already leaving the research laboratory and have entered clinical practice. These clinical discoveries have resulted in the creation of relevant animal models which may provide crucial information for the development of highly specific therapeutic agents. Research into the role of complement in proteinuria has helped to understand pathways of inflammation which ultimately lead to renal failure irrespective of the underlying renal disease and is of major importance for the majority of renal patients. Complement science is a highly exciting area of translational research and hopefully will result in meaningful therapeutic advances in the near future

Advances in our understanding of the pathogenesis of glomerular thrombotic microangiopathy

Glomerular thrombotic microangiopathy is a hallmark feature of haemolytic uraemic syndrome, the leading cause of acute renal failure in childhood. This paper is a review of the different mechanistic pathways that lead to this histological picture in the kidney. It will focus on atypical HUS and complement dysregulation, but will also highlight some other recent advances in our understanding of this condition, including the potential role of the molecule vascular endothelial growth factor- A (VEGF-A)

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β2-glycoprotein I and interferes with innate immune recognition