Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation.

BACKGROUND: Immature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides. RESULTS: In current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC-MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with ß-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes. CONCLUSIONS: Our results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

Targeting Complement C3a Receptor to Improve Outcome After Ischemic Brain Injury.

Ischemic stroke is a major cause of disability. No efficient therapy is currently available, except for the removal of the occluding blood clot during the first hours after symptom onset. Loss of function after stroke is due to cell death in the infarcted tissue, cell dysfunction in the peri-infarct region, as well as dysfunction and neurodegeneration in remote brain areas. Plasticity responses in spared brain regions are a major contributor to functional recovery, while secondary neurodegeneration in remote regions is associated with depression and impedes the long-term outcome after stroke. Hypoxic-ischemic encephalopathy due to birth asphyxia is the leading cause of neurological disability resulting from birth complications. Despite major progress in neonatal care, approximately 50% of survivors develop complications such as mental retardation, cerebral palsy or epilepsy. The C3a receptor (C3aR) is expressed by many cell types including neurons and glia. While there is a body of evidence for its deleterious effects in the acute phase after ischemic injury to the adult brain, C3aR signaling contributes to better outcome in the post-acute and chronic phase after ischemic stroke in adults and in the ischemic immature brain. Here we discuss recent insights into the novel roles of C3aR signaling in the ischemic brain with focus on the therapeutic opportunities of modulating C3aR activity to improve the outcome after ischemic stroke and birth asphyxia.

C3a Receptor Signaling Inhibits Neurodegeneration Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic neonatal encephalopathy due to perinatal asphyxia is the leading cause of brain injury in newborns. Clinical data suggest that brain inflammation induced by perinatal insults can persist for years. We previously showed that signaling through the receptor for complement peptide C3a (C3aR) protects against cognitive impairment induced by experimental perinatal asphyxia. To investigate the long-term neuropathological effects of hypoxic-ischemic injury to the developing brain and the role of C3aR signaling therein, we subjected wildtype mice, C3aR deficient mice, and mice expressing biologically active C3a in the CNS to mild hypoxic-ischemic brain injury on postnatal day 9. We found that such injury triggers neurodegeneration and pronounced reactive gliosis in the ipsilesional hippocampus both of which persist long into adulthood. Transgenic expression of C3a in reactive astrocytes reduced hippocampal neurodegeneration and reactive gliosis. In contrast, neurodegeneration and microglial cell density increased in mice lacking C3aR. Intranasal administration of C3a for 3 days starting 1 h after induction of hypoxia-ischemia reduced neurodegeneration and reactive gliosis in the hippocampus of wildtype mice. We conclude that neonatal hypoxic-ischemic brain injury leads to long-lasting neurodegeneration. This neurodegeneration is substantially reduced by treatment with C3aR agonists, conceivably through modulation of reactive gliosis.

The Role of Complement C3a Receptor in Stroke.

The complement system is a key regulator of the innate immune response against diseased tissue that functions across multiple organ systems. Dysregulation of complement contributes to the pathogenesis of a number of neurological diseases including stroke. The C3a anaphylatoxin, via its cognate C3a receptor (C3aR), mediates inflammation by promoting breakdown of the blood-brain barrier and the massive infiltration of leukocytes into ischemic brain in experimental stroke models. Studies utilizing complement deficient mice as well as pharmacologic C3aR antagonists have shown a reduction in tissue injury and mortality in murine stroke models. The development of tissue-specific C3aR knockout mice and more specific C3aR antagonists is warranted to facilitate our understanding of the role of the C3aR in brain ischemia with the ultimate goal of clinical translation of therapies targeting C3aR in stroke patients.

Functional Activity of the Complement System in Deceased Donors in Relation to Kidney Allograft Outcome.

Complement activation is considered one of the mediators of renal ischemia-reperfusion injury. Elevated levels of C5b-9, C3a, and C5a are detected in sera of deceased kidney donors. The goal of the study was to characterize the functional activity of complement pathways in donor sera and to assess their influence on transplant outcome. MATERIALS AND METHODS: Sixty-four deceased kidney donors (age 45 ± 16 years; 28 female, 36 male) and 27 healthy controls (age 42 ± 12 years; 14 female, 13 male) were enrolled in the study. The results of transplantation for the respective 122 kidney recipients were included in the analysis. The functional activities of classical (CP), lectin (LP), and alternative (AP) pathways were measured using Wielisa-kit (reference normal level = 100%). In most cases, decreased functional activity reflects the activation status of the pathway. RESULTS: The median (interquartile range) functional activities of the pathways in donor sera were CP 118 (89-150)%, LP 80 (20-127)%, and AP 74 (50-89)%, and did not differ from the control values CP 110 (102-115)%, LP 81 (26-106)%, AP 76 (61-88)%. The frequency of pathway activation observed in controls was CP 0%, LP 11%, and AP 0%. Deceased donors did not differ in activation of classical (11%) and lectin (13%) pathways, but presented a higher rate of alternative pathway activation (19%, P = .03). No significant influence of any pathway functional activity or its activation was proved to influence the transplant outcome. CONCLUSION: Complement activation via alternative pathway was observed in diseased donor sera. No predictive potential of donor complement functional activity on the transplant outcome could be proved.

Complement C3a signaling facilitates skeletal muscle regeneration by regulating monocyte function and trafficking.

Regeneration of skeletal muscle following injury is accompanied by transient inflammation. Here we show that complement is activated in skeletal muscle injury and plays a key role during regeneration. Genetic ablation of complement C3 or its inactivation with Cobra Venom Factor (CVF) result in impaired muscle regeneration following cardiotoxin-induced injury in mice. The effect of complement in muscle regeneration is mediated by the alternative pathway and C3a receptor (C3aR) signaling, as deletion of Cfb, a key alternative pathway component, or C3aR leads to impaired regeneration and reduced monocyte/macrophage infiltration. Monocytes from C3aR-deficient mice express a reduced level of adhesion molecules, cytokines and genes associated with antigen processing and presentation. Exogenous administration of recombinant CCL5 to C3aR-deficient mice rescues the defects in inflammatory cell recruitment and regeneration. These findings reveal an important role of complement C3a in skeletal muscle regeneration, and suggest that manipulating complement system may produce therapeutic benefit in muscle injury and regeneration.

Exploiting a novel conformational switch to control innate immunity mediated by complement protein C3a.

Complement C3a is an important protein in innate and adaptive immunity, but its specific roles in vivo remain uncertain because C3a degrades rapidly to form the C3a-desArg protein, which does not bind to the C3a receptor and is indistinguishable from C3a using antibodies. Here we develop the most potent, stable and highly selective small molecule modulators of C3a receptor, using a heterocyclic hinge to switch between agonist and antagonist ligand conformations. This enables characterization of C3 areceptor-selective pro- vs. anti-inflammatory actions in human mast cells and macrophages, and in rats. A C3a receptor-selective agonist induces acute rat paw inflammation by first degranulating mast cells before activating macrophages and neutrophils. An orally administered C3a receptor-selective antagonist inhibits mast cell degranulation, thereby blocking recruitment and activation of macrophages and neutrophils, expression of inflammatory mediators and inflammation in a rat paw edema model. These novel tools reveal the mechanism of C3a-induced inflammation and provide new insights to complement-based medicines.Complement C3a is an important protein in innate and adaptive immunity, but its roles in vivo are unclear. Here the authors develop novel chemical agonists and antagonists for the C3a receptor, and show that they modulate mast cell degranulation and inflammation in a rat paw edema model.

Complement C3a Mobilizes Dental Pulp Stem Cells and Specifically Guides Pulp Fibroblast Recruitment.

INTRODUCTION: Complement activation is considered a major mechanism in innate immunity. Although it is mainly involved in initiating inflammation, recent data reported its involvement in other processes such as tissue regeneration. In the dental pulp, complement C5a fragment has been shown to be involved in the recruitment of dental pulp stem cells (DPSCs). This study sought to investigate the possible role of C3a, another complement fragment, in the early steps of dentin-pulp regeneration. METHODS: Expression of C3a receptor (C3aR) was investigated by immunofluorescence and reverse transcriptase polymerase chain reaction on cultured pulp fibroblasts, STRO-1-sorted DPSCs, as well as on human tooth sections in vivo. The effect of C3a on proliferation of both DPSCs and pulp fibroblasts was investigated by MTT assay. Cell migration under a C3a gradient was investigated by using microfluidic chemotaxis chambers. RESULTS: C3aR was expressed in vivo as well as in cultured pulp fibroblasts co-expressing fibroblast surface protein and in DPSCs co-expressing STRO-1. Addition of recombinant C3a induced a significant proliferation of both cell types. When subjected to a C3a gradient, DPSCs were mobilized but not specifically recruited, whereas pulp fibroblasts were specifically recruited following a C3a gradient. CONCLUSIONS: These results provide the first demonstration of C3aR expression in the dental pulp and demonstrate that C3a is involved in increasing DPSCs and fibroblast proliferation, in mobilizing DPSCs, and in specifically guiding fibroblast recruitment. This provides an additional link to the tight correlation between inflammation and tissue regeneration.

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

Downsizing a human inflammatory protein to a small molecule with equal potency and functionality.

A significant challenge in chemistry is to rationally reproduce the functional potency of a protein in a small molecule, which is cheaper to manufacture, non-immunogenic, and also both stable and bioavailable. Synthetic peptides corresponding to small bioactive protein surfaces do not form stable structures in water and do not exhibit the functional potencies of proteins. Here we describe a novel approach to growing small molecules with protein-like potencies from a functionally important amino acid of a protein. A 77-residue human inflammatory protein (complement C3a) important in innate immunity is rationally transformed to equipotent small molecules, using peptide surrogates that incorporate a turn-inducing heterocycle with correctly positioned hydrogen-bond-accepting atoms. Small molecule agonists (molecular weight <500 Da) examined for receptor affinity and cellular responses have the same high potencies, functional profile and specificity of action as C3a protein, but greater plasma stability and bioavailability.

Obesity-inducing diet promotes acylation stimulating protein resistance.

Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system that is involved in energy homeostasis and inflammation. ASP acts on and correlates positively with postprandial fat clearance in healthy subjects. However, in obesity, ASP levels are elevated and correlate inversely with fat clearance, indicative of a potential resistance to ASP. Using a mouse model, we hypothesized that, over time, diet-induced obesity (DIO) would result in development of ASP insensitivity, as compared to chow-fed animals as controls. Injection of recombinant ASP in DIO mice failed to accelerate fat clearance to the same extent as in chow-fed mice. DIO mice exhibited higher basal levels of plasma ASP and, after 30weeks of diet, showed lower ASP receptor (C5L2) expression in adipose tissue compared to chow-fed mice. Additionally, ex vivo ASP stimulation failed to induce normal Ser(473)AKT phosphorylation in adipose tissue from DIO mice VS chow-fed controls. These results demonstrate for the first time a state of diet-induced ASP resistance. Changes in the ASP-C5L2 pathway dynamics in obesity could alter the development of obesity and co-morbidities such as atherosclerosis and type 2 diabetes.

Complement-mediated chronic inflammation is associated with diabetic microvascular complication.

BACKGROUND: Chronic inflammation is characteristic of type 2 diabetes mellitus (T2DM). Obesity-activated adipocytes release adipocytokines, which induce the secretion of proinflammatory cytokines, resulting in vascular endothelial dysfunction and organ injury. C3a is a candidate to induce tissue inflammation. METHODS: We investigated the association between diabetic microangiopathy and complement-mediated inflammation in 32 obese T2DM patients and 32 normal donors. Plasma levels of complement components and their activation intermediates were examined and related to the level of complication. An incubation study of post-prandial serum was carried out to measure the in vitro production of acylation stimulating protein (ASP/C3a desArg) by chylomicron. RESULTS: Plasma levels of C3, C4, factor B, iC3b, Bb, and ASP were significantly increased in T2DM patients. Levels of C4d and membrane attack complex (C5b-9) were not significantly elevated. The activation rate of these factors indicated that only the early phase of alternative complement pathway was excessively activated. A statistical study revealed close correlation between ASP, body mass index, and highly sensitive C-reactive protein. Plasma ASP was significantly increased in the macroalbuminuric and proliferative retinopathy patient groups. An incubation study revealed that ASP was produced after the in vitro incubation of post-prandial serum from a T2DM patient with hyperchylomicronaemia. CONCLUSIONS: Activation of the alternative complement pathway occurs in obese T2DM patients and is enhanced in the post-prandial hyperchylomicronic condition, which induces overproduction of ASP and C3a-mediated tissue inflammation. Therefore, complement-mediated inflammation may contribute to the acceleration of diabetic microangiopathy in addition to the development of macroangiopathy.

Complement C3a-induced IL-17 plays a critical role in an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness via neutrophilic inflammation in mice.

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.

Role of complement 3a in the synthetic phenotype and angiotensin II-production in vascular smooth muscle cells from spontaneously hypertensive rats.

BACKGROUND: Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMCs) show exaggerated growth with a synthetic phenotype and angiotensin II (Ang II)-production. To evaluate the contribution of complement 3 (C3) or C3a toward these abnormalities in SHR, we examined effects of a C3a receptor inhibitor on proliferation, phenotype, and Ang II-production in VSMCs from SHR and Wistar-Kyoto (WKY) rats. METHODS: Expression of pre-pro-C3 messenger RNA (mRNA) and C3 protein was evaluated by reverse transcription-PCR and western blot analyses, and C3a receptor mRNA was evaluated by reverse transcription-PCR analysis in quiescent VSMCs from SHR and WKY rats. We examined the effects of the C3a inhibitor, SB290157, on proliferation and the expression of phenotype-marker and Krueppel-like factor 5 (KLF-5) mRNAs in VSMCs from SHR and WKY rats. We examined effects of C3a receptor inhibitor, SB290157, on Ang II-production in conditioned medium of VSMCs from SHR and WKY rats by a radioimmunoassay. RESULTS: Expression of pre-pro-C3 mRNA and C3 protein was significantly higher in SHR VSMCs than WKY VSMCs. SB290157 significantly inhibited proliferation of VSMCs from SHR, but not in cells from WKY rats. Relative to WKY VSMCs, SB290157 significantly increased the low expression of SM22α mRNA and decreased the high expression of osteopontin mRNA in SHR VSMCs. SB290157 significantly decreased the high expression of KLF-5 and Ang II-production in VSMCs from SHR, but not in cells from WKY rats. CONCLUSIONS: C3a induces exaggerated growth, a synthetic phenotype and Ang II-production in SHR-derived VSMCs. C3a may be primarily involved in cardiovascular remodeling in hypertension.

Complement fragment C3a controls mutual cell attraction during collective cell migration.

Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.

Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury.

To prevent injury to host tissues, complement activation is regulated by a number of plasma and membrane-associated proteins, most of which limit C3 and C5 activation. An influx of circulating C3 from a syngeneic host into donor kidneys deficient in Crry (a membrane protein that reduces C3 convertase activity) causes spontaneous complement activation, primarily in the tubulointerstitum, leading to renal failure. To determine the roles of the C3a and C5a anaphylatoxins in tubulointerstitial inflammation and fibrosis, kidneys from Crry-/-C3-/- mice were transplanted into hosts lacking the C3a and/or C5a receptor. While unrestricted complement activation in the tubules was not affected by receptor status in the transplant recipient, C3a receptor deficiency in the recipients led to significantly reduced renal leukocyte infiltration and the extent of tubulointerstitial inflammation and fibrosis, all of which led to preserved renal function. The absence of C5a receptors in recipients was not only inconsequential, but the protective effect of C3a receptor deficiency was also eliminated, suggesting distinct roles of C3a and C5a receptor signaling in this model. There was significant infiltration of the tubulointerstitum with 7/4+F4/80+CD11b+ myelomonocytic cells and Thy1.2+ T cells along injured tubules, and interstitial collagen I and III deposition, all of which were C3a receptor dependent. Thus, blockade of C3a receptor signaling is a possible treatment to reduce renal inflammation and preserve renal function associated with complement activation.

Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL-1ß.

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the pro-inflammatory cytokine IL-1ß significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example, during bone healing or in inflammatory bone disorders.

[The effect of C3a, C5a and their receptor antagonists on the expression of beta-catenin in renal tubular cells].

OBJECTIVE: To investigate the effect of C3a and C5a and their receptor antagonists (C3aRA and C5aRA) on the expression of BETAcatenin in renal tubular epithelial cell line HK-2. METHODS: Cells were divided in into C3a group and C5a group which was further divided into four subgroups: C3a group (control, 1 micromol/L TGF-beta1, 50 nmol/L C3a, 1 micromol/L C3aRA); C5a group (control, 1 micromol/L TGF-beta1, 50 nmol/L C5a, 2.5 micromol/L C5aRA). Real time PCR and Western blot were used to detect mRNA and protein expression of beta-catenin. RESULTS: Real-time PCR and Western blot demonstrated that 1 micromol/L TGF-beta1 could increase the expression of beta-catenin; C3a and C5a presented the similar inducible effect as TGF-beta1, which could be blocked by C3aR antagonist and C5aR antagonist (C3aRA and C5aRA). CONCLUSION: Anaphylatoxin C3a and C5a can induce mRNA and protein expression of beta-catenin in renal tubular epithelial cells, which could be blocked by C3aRA and C5aRA. C3a and C5a may participate in tubular epithelial-mesenchymal transition in vitro.

Immune evasion of Moraxella catarrhalis involves ubiquitous surface protein A-dependent C3d binding.

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.