Increased expression of complement C3c, iC3b, and cells containing CD11b or CD14 in experimentally induced psoriatic lesion.

Psoriasis is a chronic inflammatory skin disease with a characteristic isomorphic reaction, i.e. the Köbner reaction, induced by slight epidermal trauma. In this study, the tape-stripping technique was used to induce the development of Köbner reaction in 18 subjects with psoriasis. Eight subjects developed a positive reaction. To study the early cellular changes, skin biopsies were taken at the baseline and subsequent time points of 2 h, 1 d, 3 d, and 7 d for the immunostaining of complement C3c, iC3b, and cells expressing complement receptor 3 (CD11b/CD18; a receptor of iC3b) or CD14. The results show that the positive Köbner reaction is associated with rapid (2 h-1 d) and sustained (3-7 d) increase in the expression of epidermal C3c and iC3b and dermal C3c. In addition, there was a positive correlation between CD11b+ and CD14+ cells in baseline and 2 h-1 d biopsies with a subsequent increase in CD11b+ and CD14+ cells in 3-7 d biopsies in the Köbner-positive group. In the Köbner-negative group, only a transient increase in epidermal iC3b at 2 h-1 d, as well as rapid (2 h-1 d) and sustained increase (3-7 d) in dermal iC3b and CD14+ cells, was observed. In experiments with cultured monolayer keratinocytes, a slight cell damage already at 30 mJ/cm2 ultraviolet B irradiation led to increased expression of C3c, but not iC3b. Therefore, there are marked differences between Köbner groups in respect to the expression of C3c, iC3b, and cells expressing CD11b or CD14. Of note is the rapid and sustained increase in epidermal C3c and iC3b in the positive Köbner reaction.

CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease.

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

C3 Glomerulopathy and Related Disorders in Children: Etiology-Phenotype Correlation and Outcomes.

BACKGROUND AND OBJECTIVES: Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan-Meier method. RESULTS: Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2-15 (median, 9; interquartile range, 7-11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13-8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on the initial biopsy specimen. CONCLUSIONS: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Biologia Futura: stories about the functions of ß2-integrins in human phagocytes.

Integrins are essential membrane proteins that provide a tightly regulated link between the extracellular matrix and the intracellular cytoskeletal network. These cell surface proteins are composed of a non-covalently bound α chain and ß chain. The leukocyte-specific complement receptor 3 (CR3, αMß2, CD11b/CD18) and complement receptor 4 (CR4, αXß2, CD11c/CD18) belong to the family of ß2-integrins. These receptors bind multiple ligands like iC3b, ICAMs, fibrinogen or LPS, thus allowing them to partake in phagocytosis, cellular adhesion, extracellular matrix rearrangement and migration. CR3 and CR4 were generally expected to mediate identical functions due to their structural homology, overlapping ligand specificity and parallel expression on human phagocytes. Despite their similarities, the expression level and function of these receptors differ in a cell-type-specific manner, both under physiological and inflammatory conditions.We investigated comprehensively the individual role of CR3 and CR4 in various functions of human phagocytes, and we proved that there is a "division of labour" between these two receptors. In this review, I will summarize our current knowledge about this area.

Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans.

We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested in vivo generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) (n = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals (n = 144) (p < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

CRIg plays an essential role in intravascular clearance of bloodborne parasites by interacting with complement.

Although CRIg was originally identified as a macrophage receptor for binding complement C3b/iC3b in vitro, recent studies reveal that CRIg functions as a pattern recognition receptor in vivo for Kupffer cells (KCs) to directly bind bacterial pathogens in a complement-independent manner. This raises the critical question of whether CRIg captures circulating pathogens through interactions with complement in vivo under flow conditions. Furthermore, the role of CRIg during parasitic infection is unknown. Taking advantage of intravital microscopy and using African trypanosomes as a model, we studied the role of CRIg in intravascular clearance of bloodborne parasites. Complement C3 is required for intravascular clearance of African trypanosomes by KCs, preventing the early mortality of infected mice. Moreover, antibodies are essential for complement-mediated capture of circulating parasites by KCs. Interestingly, reduced antibody production was observed in the absence of complement C3 during infection. We further demonstrate that CRIg but not CR3 is critically involved in KC-mediated capture of circulating parasites, accounting for parasitemia control and host survival. Of note, CRIg cannot directly catch circulating parasites and antibody-induced complement activation is indispensable for CRIg-mediated parasite capture. Thus, we provide evidence that CRIg, by interacting with complement in vivo, plays an essential role in intravascular clearance of bloodborne parasites. Targeting CRIg may be considered as a therapeutic strategy.

The Complement System Is Essential for the Phagocytosis of Mesenchymal Stromal Cells by Monocytes.

Mesenchymal stromal cell (MSC) therapy is a promising tool in the treatment of chronic inflammatory diseases. This has been ascribed to the capacity of MSC to release a large variety of immune-modulatory factors. However, all aspects of the mode of therapeutic MSC action in different diseases remain unresolved, mainly because most of the infused MSC are undetectable in the circulation within hours after infusion. The aim of this study was to elucidate the fate of MSC after contact with plasma. We found that upon contact with blood, complement proteins including C3b/iC3b are deposited on MSC. Importantly, we also found that complement bound to MSC enhanced their phagocytosis by classical and intermediate monocytes via a mechanism that involves C3 but not C5. Thus, we describe for the first time a mechanism which might explain, at least partly, why MSC are not found in the blood circulation after infusion. Our results indicate that MSC immune-modulatory effects could be mediated by monocytes that have phagocytosed them.

Secretion of functionally active complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma Multiforme patients.

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.

A trivalent gC2/gD2/gE2 vaccine for herpes simplex virus generates antibody responses that block immune evasion domains on gC2 better than natural infection.

Vaccines for prevention and treatment of genital herpes are high public health priorities. Our approach towards vaccine development is to focus on blocking virus entry mediated by herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) and to prevent the virus from evading complement and antibody attack by blocking the immune evasion domains on HSV-2 glycoproteins C (gC2) and E (gE2), respectively. HSV-2 gC2 and gE2 are expressed on the virion envelope and infected cell surface where they are potential targets of antibodies that bind and block their immune evasion activities. We demonstrate that antibodies produced during natural infection in humans or intravaginal inoculation in guinea pigs bind to gC2 but generally fail to block the immune evasion domains on this glycoprotein. In contrast, immunization of naïve or previously HSV-2-infected guinea pigs with gC2 subunit antigen administered with CpG and alum as adjuvants produces antibodies that block domains involved in immune evasion. These results indicate that immune evasion domains on gC2 are weak antigens during infection, yet when used as vaccine immunogens with adjuvants the antigens produce antibodies that block immune evasion domains.

Asparaginyl Endopeptidase (Legumain) Supports Human Th1 Induction via Cathepsin L-Mediated Intracellular C3 Activation.

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity-but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn-/- mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an "upstream" activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.

Effect of C1-INH on ischemia/reperfusion injury in a porcine limb ex vivo perfusion model.

Revascularization of an amputated limb within 4-6h is essential to avoid extensive ischemia/reperfusion (I/R) injury leading to vascular leakage, edema and tissue necrosis. I/R injury is a pathological inflammatory condition that occurs during reperfusion of an organ or tissue after prolonged ischemia. It is characterized by a complex crosstalk between endothelial cell activation and the activation of plasma cascades. Vasculoprotective pharmacological intervention to prevent I/R injury might be an option to prolong the time window between limb amputation and successful replantation. We used C1-easterase inhibitor (C1-INH) in this study because of its known inhibitory effects on the activation of the complement, coagulation and kinin cascades. Forelimbs of 8 large white pigs were amputated, subjected to ischemia, and then reperfused with autologous whole blood. All limbs were exposed to 9h of cold ischemia at 4°C. After 2h of cold ischemia the limbs were either perfused with of C1-INH (1U/ml in hydroxyethyl starch, n=8) or hydroxyethyl starch alone (n=7). After completion of the 9-h ischemia period, all limbs were ex vivo perfused with heparinized autologous whole blood for 12h using a pediatric heart lung machine to simulate in vivo revascularization. Our results show that I/R injury in the control group led to a significant elevation of tissue deposition of IgG and IgM, complement C3b/c, C5b-9 and MBL. Also, activation of the kinin system was significantly increased, namely bradykinin in plasma, and expression of bradykinin receptors 1 and 2 in tissue. In addition, markers for endothelial integrity like expression of CD31, VE-cadherin and heparan sulfate proteoglycans were decreased in reperfused tissue. Limb I/R injury also led to activation of the coagulation cascade with a significant elevation of fibrin and thrombin deposition and increased fibrinogen-like protein-2 expression. C1-INH treated limbs showed much less activation of plasma cascades and better protection of endothelial integrity compared to the reperfused control limbs. In conclusion, the use of the cytoprotective drug C1-INH significantly reduced I/R injury by protecting the vascular endothelium as well as the muscle tissue from deposition of immunoglobulins, complement and fibrin.

Heme Oxygenase 1 Up-Regulates Glomerular Decay Accelerating Factor Expression and Minimizes Complement Deposition and Injury.

Complement-activation controllers, including decay accelerating factor (DAF), are gaining emphasis as they minimize injury in various dysregulated complement-activation disorders, including glomerulopathies. Heme oxygenase (HO)-1 overexpression or induction has been shown to attenuate injury in complement-dependent models of glomerulonephritis. This study investigated whether up-regulation of DAF by heme oxygenase 1 (HO-1) is an underlying mechanism by using Hmox-1-deficient rats (Hmox1+/-; Hmox1-/-) or rats with HO-1 overexpression targeted to glomerular epithelial cells (GECHO-1), which are particularly vulnerable to complement-mediated injury owing to their terminally differentiated nature. Constitutively expressed DAF was decreased in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. In GECHO-1 rats with anti-glomerular basement membrane antibody mediated, complement-dependent injury, complement component C3 fragment b (C3b) deposition was reduced, whereas proteinuria was diminished. In glomeruli of wild-type rats, the natural Hmox substrate, hemin, induced glomerular DAF. This effect was attenuated in glomeruli of Hmox1-/- rats and augmented in glomeruli of GECHO-1 rats. Hemin analogues differing in either metal or porphyrin ring functionalities, acting as competitive Hmox-substrate inhibitors, also increased glomerular DAF and reduced C3b deposition after spontaneous complement activation. In the presence of a DAF-blocking antibody, the reduction in C3b deposition was reversed. These observations establish HO-1 as a physiologic regulator of glomerular DAF and identify hemin analogues as inducers of functional glomerular DAF able to minimize C3b deposition.

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity.

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Complement Factor H Antibodies from Lung Cancer Patients Induce Complement-Dependent Lysis of Tumor Cells, Suggesting a Novel Immunotherapeutic Strategy.

Characterization of the humoral immune response in selected patients with cancer who uniformly do well may lead to the development of novel therapeutic strategies. We have previously shown an association between patients with early-stage nonmetastatic lung cancer and autoantibodies to complement factor H (CFH). CFH protects normal and tumor cells from destruction by the alternative complement pathway by inactivating C3b, a protein that is essential for formation of a lytic complex on the cell surface. Here, we show that CFH autoantibodies in lung cancer patients recognize a conformationally distinct form of CFH in vitro, are IgG3 subclass, and epitope map to a crucial functional domain of CFH known to interact with C3b. Purified CFH autoantibodies inhibited binding of CFH to A549 lung tumor cells, increased C3b deposition, and caused complement-dependent tumor cell lysis. This work demonstrates that CFH autoantibodies isolated from patients with lung cancer can kill tumor cells in vitro, suggesting that they may perform this function in vivo as well. Development of specific antibodies to the conformationally distinct epitope of CFH may lead to a useful biologic therapy for lung cancer.

H-ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses.

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses.

Atypical aHUS: State of the art.

Tremendous advances in our understanding of the thrombotic microangiopathies (TMAs) have revealed distinct disease mechanisms within this heterogeneous group of diseases. As a direct result of this knowledge, both children and adults with complement-mediated TMA now enjoy higher expectations for long-term health. In this update on atypical hemolytic uremic syndrome, we review the clinical characteristics; the genetic and acquired drivers of disease; the broad spectrum of environmental triggers; and current diagnosis and treatment options. Many questions remain to be addressed if additional improvements in patient care and outcome are to be achieved in the coming decade.

Interplay between fibrinolysis and complement: plasmin cleavage of iC3b modulates immune responses.

BACKGROUND: The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune surveillance. In particular, the C3 proteolytic fragment, iC3b, and its degradation product C3dg, which is generated by cleavage by factor I (FI) and the cofactor complement receptor CR1, are important in bridging innate and adaptive immunity. Via a thioester (TE) bond, iC3b and C3dg covalently tag pathogens, modulating phagocytosis and adaptive immune responses. OBJECTIVE: To examine plasmin-mediated proteolysis of iC3b, and to evaluate the functional consequences, comparing the effects with products generated by FI/CR1 cleavage of iC3b. METHODS: Dose-dependent and time-dependent plasmin-mediated cleavage of iC3b were characterized by analytical gel electrophoresis. The properties of the resultant TE bond-containing fragments on phagocytosis and induction of pro-inflammatory cytokines were measured in cell culture systems. RESULTS: At low concentrations, plasmin effectively cleaves iC3b, but at numerous previously undescribed sites, giving rise to novel C3c-like and C3dg-like moieties, the latter of which retain the TE bond. When attached to zymosan or erythrocytes and exposed to THP-1 macrophages, the C3dg-like proteins behave almost identically to the bona fide C3dg, yielding less phagocytosis as compared with the opsonin iC3b, and more macrophage secretion of the pro-inflammatory cytokine, IL-12. CONCLUSION: Plasmin cleavage of iC3b provides a complement regulatory pathway that is as efficient as FI/CR1 but does not require a cellular cofactor.

The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase.

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.