Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis.

Coronavirus disease 2019 (COVID-19) is a disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in a pandemic1. The C5a complement factor and its receptor C5aR1 (also known as CD88) have a key role in the initiation and maintenance of several inflammatory responses by recruiting and activating neutrophils and monocytes1. Here we provide a longitudinal analysis of immune responses, including phenotypic analyses of immune cells and assessments of the soluble factors that are present in the blood and bronchoalveolar lavage fluid of patients at various stages of COVID-19 severity, including those who were paucisymptomatic or had pneumonia or acute respiratory distress syndrome. The levels of soluble C5a were increased in proportion to the severity of COVID-19 and high expression levels of C5aR1 receptors were found in blood and pulmonary myeloid cells, which supports a role for the C5a-C5aR1 axis in the pathophysiology of acute respiratory distress syndrome. Anti-C5aR1 therapeutic monoclonal antibodies prevented the C5a-mediated recruitment and activation of human myeloid cells, and inhibited acute lung injury in human C5aR1 knock-in mice. These results suggest that blockade of the C5a-C5aR1 axis could be used to limit the infiltration of myeloid cells in damaged organs and prevent the excessive lung inflammation and endothelialitis that are associated with acute respiratory distress syndrome in patients with COVID-19.

C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization.

Degenerative alterations in articular cartilage are involved in the pathogenesis of osteoarthritis. The present study aimed to evaluate the role of complement component 5a (C5a) in osteoarthritic alterations in the articular cartilage and synovialis via a joint immobilization (IM) rat model. Rats were assigned to three groups: Control, IM and IM+anti­C5a antibody (IM+anti­C5a) groups. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were used to evaluate the morphological alterations in the articular cartilage and synovialis. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, immunohistochemical analysis and western blotting were used to evaluate C5a expression in the articular cartilage and synovialis. An ELISA was used to evaluate C5a­induced alterations in interleukin (IL)­1ß, IL­17A and tumor necrosis factor (TNF)­α levels in the serum and joint fluid. The results demonstrated that knee joint immobilization induced destruction of knee joint synovial fluid and cartilage in the IM and IM+anti­C5a antibody groups. Immobilization significantly increased the expression levels of C5a in serum and joint fluid in the IM group. Immunohistochemistry, western blotting and RT­qPCR analysis illustrated markedly increased expression of C5a in the IM group. Immobilization markedly increased the IL­1ß, IL­17A and TNF­α expression levels in the serum and joint fluid in the IM group. Anti­C5a was able to decrease immobilization­induced alterations in morphology and cytokines compared with the IM group. The expression of C5a was increased in synoviocytes and joint cartilage in the IM model. Pro­inflammatory cytokines, including TNF­α and IL­1ß were released in the activated synoviocytes via the induction of C5a, suggesting that C5a serves an important role in joint inflammatory processes.

Macrophage scavenger receptor 1 contributes to pathogenesis of fulminant hepatitis via neutrophil-mediated complement activation.

BACKGROUND & AIMS: The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis. METHODS: Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion. RESULTS: Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection. CONCLUSIONS: Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH. LAY SUMMARY: Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

TLR-Induced Murine Dendritic Cell (DC) Activation Requires DC-Intrinsic Complement.

Induction of proinflammatory T cell immunity is augmented by innate dendritic cell (DC) maturation commonly initiated by TLR signaling. We demonstrate that ligation of TLR3, TLR4, and TLR9 induces murine DC production of complement components and local production of the anaphylatoxin C5a. In vitro, ex vivo, and in vivo analyses show that TLR-induced DC maturation, as assessed by surface phenotype, expression profiling by gene array, and functional ability to stimulate T cell responses, requires autocrine C3a receptor and C5a receptor (C3ar1/C5ar1) signaling. Studies using bone marrow chimeric animals and Foxp3-GFP/ERT2-Cre/dTomato fate-mapping mice show that TLR-initiated DC autocrine C3ar1/C5ar1 signaling causes expansion of effector T cells and instability of regulatory T cells and contributes to T cell-dependent transplant rejection. Together, our data position immune cell-derived complement production and autocrine/paracrine C3ar1/C5ar1 signaling as crucial intermediary processes that link TLR stimulation to DC maturation and the subsequent development of effector T cell responses.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood.

Enhanced levels of platelet/granulocyte aggregates (PGAs) are found in patients suffering from many different inflammatory vascular diseases, and their formation in animal models of vascular disease is associated with increased thromboinflammation and worsened outcomes. The complement system, a part of the innate immune system, influences PGA formation, but the mechanisms for its effects are unknown. In this study, we have defined complement-mediated mechanisms that enhance PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP) using ex vivo flow cytometry assays. We demonstrate that physiological properdin, a positive regulator of complement alternative pathway activity, increases PGA formation when added to TRAP-stimulated blood. All physiological properdin forms increase PGA formation, but properdin tetramers are the most efficient at increasing complement activity and PGA formation. Inhibition of endogenous properdin, either circulating in the blood or produced locally by leukocytes, impairs TRAP-mediated PGA formation to the same level as specific inhibition of either the alternative or classical pathway. Additionally, blocking the interaction of C5a with its cellular receptor prevents properdin-mediated increases in PGA formation. Adding either properdin tetramers or C5a to whole blood increases CD11b expression on granulocytes, and this increase is prevented by blockade of the C5a-C5a receptor axis. Finally, we demonstrate that the effects of properdin on PGA formation are tightly regulated by Factor H. Cumulatively, our data indicate that properdin enhances PGA formation via increased production of C5a, and that inhibition of properdin function has therapeutic potential to limit thromboinflammation in diseases characterized by increased PGA formation.

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin.

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin-antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

Neutrophil microvesicles resolve gout by inhibiting C5a-mediated priming of the inflammasome.

OBJECTIVES: Gout is a highly inflammatory but self-limiting joint disease induced by the precipitation of monosodium urate (MSU) crystals. While it is well established that inflammasome activation by MSU mediates acute inflammation, little is known about the mechanism controlling its spontaneous resolution. The aim of this study was to analyse the role of neutrophil-derived microvesicles (PMN-Ecto) in the resolution of acute gout. METHODS: PMN-Ecto were studied in a murine model of MSU-induced peritonitis using C57BL/6, MerTK(-/-) and C5aR(-/-) mice. The peritoneal compartment was assessed for the number of infiltrating neutrophils (PMN), neutrophil microvesicles (PMN-Ecto), cytokines (interleukin-1ß, TGFß) and complement factors (C5a). Human PMN-Ecto were isolated from exudates of patients undergoing an acute gouty attack and functionally tested in vitro. RESULTS: C5a generated after the injection of MSU primed the inflammasome for IL-1ß release. Neutrophils infiltrating the peritoneum in response to C5a released phosphatidylserine (PS)-positive PMN-Ecto early on in the course of inflammation. These PMN-Ecto in turn suppressed C5a priming of the inflammasome and consequently inhibited IL-1ß release and neutrophil influx. PMN-Ecto-mediated suppression required surface expression of the PS-receptor MerTK and could be reproduced using PS-expressing liposomes. In addition, ectosomes triggered the release of TGFß independent of MerTK. TGFß, however, was not sufficient to control acute MSU-driven inflammation in vivo. Finally, PMN-Ecto from joint aspirates of patients with gouty arthritis had similar anti-inflammatory properties. CONCLUSIONS: PMN-Ecto-mediated control of inflammasome-driven inflammation is a compelling concept of autoregulation initiated early on during PMN activation in gout.

The Complement Receptor C5aR Controls Acute Inflammation and Astrogliosis following Spinal Cord Injury.

This study investigated the role of the complement activation fragment C5a in secondary pathology following contusive spinal cord injury (SCI). C5ar(-/-) mice, which lack the signaling receptor for C5a, displayed signs of improved locomotor recovery and reduced inflammation during the first week of SCI compared with wild-type mice. Intriguingly, the early signs of improved recovery in C5ar(-/-) mice deteriorated from day 14 onward, with absence of C5aR ultimately leading to poorer functional outcomes, larger lesion volumes, reduced myelin content, and more widespread inflammation at 35 d SCI. Pharmacological blockade of C5aR with a selective antagonist (C5aR-A) during the first 7 d after SCI improved recovery compared with vehicle-treated mice, and this phenotype was sustained up to 35 d after injury. Consistent with observations made in C5ar(-/-) mice, these improvements were, however, lost if C5aR-A administration was continued into the more chronic phase of SCI. Signaling through the C5a-C5aR axis thus appears injurious in the acute period but serves a protective and/or reparative role in the post-acute phase of SCI. Further experiments in bone marrow chimeric mice suggested that the dual and opposing roles of C5aR on SCI outcomes primarily relate to its expression on CNS-resident cells and not infiltrating leukocytes. Additional in vivo and in vitro studies provided direct evidence that C5aR signaling is required during the postacute phase for astrocyte hyperplasia, hypertrophy, and glial scar formation. Collectively, these findings highlight the complexity of the inflammatory response to SCI and emphasize the importance of optimizing the timing of therapeutic interventions.

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.

Complement components as potential therapeutic targets for asthma treatment.

Asthma is the most common respiratory disorder, and is characterized by distal airway inflammation and hyperresponsiveness. This disease challenges human health because of its increasing prevalence, severity, morbidity, and the lack of a proper and complete cure. Asthma is characterized by T(H)2-skewed inflammation with elevated pulmonary levels of IL-4, IL-5, and IL-13 levels. Although there are early forays into targeting T(H)2 immunity, less-specific corticosteroid therapy remains the immunomodulator of choice. Innate immune injury mediated by complement components also act as potent mediators of the allergic inflammatory responses and offer a new and exciting possibility for asthma immunotherapy. The complement cascade consists of a number of plasma- and membrane-bound proteins, and the cleavage products of these proteins (C3 and C5) regulate the magnitude of adaptive immune responses. Complement protein are responsible for many pathophysiological features of asthma, including inflammatory cell infiltration, mucus secretion, increases in vascular permeability, and smooth muscle cell contraction. This review highlights the complement-mediated injury during asthma inflammation, and how blockade of active complement mediators may have therapeutic application.

Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway.

Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.

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 c5a generation by staphylococcal biofilms.

Biofilms production is a central feature of nosocomial infection of catheters and other medical devices used in resuscitation and critical care. However, the very effective biofilm forming pathogen Staphylococcus epidermidis often produces a modest host inflammatory response and few of the signs and symptoms associated with more virulent pathogens. To examine the impact of bacterial biofilm formation on provocation of an innate immune response, we studied the elaboration of the major complement anaphylatoxin C5a by human serum upon contact with S. epidermidis biofilms. Wild-type S. epidermidis and mutants of sarA (a regulatory protein that promotes synthesis of the biofilm-forming polysaccharide intercellular adhesin [PIA]) and icaB (responsible for postexport processing of PIA) were studied. C5a release, as a function of exposed biofilm surface area, was on the order of 1 fmol · cm · s and was dependent on the presence of PIA. Experimental results were used to inform a physiologically based pharmacokinetic model of C5a release by an infected central venous catheter, one of S. epidermidis' primary means of causing human disease. These simulations revealed that the magnitude of C5a release on a superior vena cava catheter completely covered with S. epidermidis would be lower than necessary to alert circulating leukocytes. Combined, the experimental and computational results are highly consistent with clinical observations in which the clinical signs of central line-associated bloodstream infection are often muted in association with this important pathogen.

Complement activation fragment C5a receptors, CD88 and C5L2, are associated with neurofibrillary pathology.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative dementia characterized by the decline of cognition and the presence of neuropathological changes including neuronal loss, neurofibrillary pathology and extracellular senile plaques. A neuroinflammatory process is also triggered and complement activation has been hypothesized to have a relevant role in this local inflammatory response. C5a, a proinflammatory anaphylatoxin generated after complement activation, exerts its chemotactic and inflammatory functions through the CD88 receptor while the more recently discovered C5L2 receptor has been postulated to have an anti-inflammatory role. Previously, we reported that a CD88 specific antagonist (PMX205) decreased the pathology and improved cognition in transgenic models of AD suggesting that C5a/C5aR interaction has an important role in the progression of the disease. METHODS: The present study characterizes the expression of the two receptors for C5a in human brain with confirmed post mortem diagnosis of vascular dementia (VD) or AD as well as age matched controls by immunohistochemistry and Western blot analysis using several antibodies against different epitopes of the human receptors. RESULTS: The CD88 and C5L2 antibodies revealed increased expression of both receptors in AD samples as compared to age-matched controls or VD brain tissue by Western blot and immunohistochemistry, using multiple antibodies and distinct cohorts of brain tissue. Immunostaining showed that both the C5L2 and CD88 antibodies similarly labeled abundant neurofibrillary tangles, neuropil threads and dystrophic neurites associated with plaques in the hippocampus and frontal cortex of AD cases. In contrast, little or no neuronal staining, tangles or dystrophic neurites associated with plaques were observed in control or VD brains. CD88 and C5L2 receptors are associated with both early (AT8) and mature (PHF1) neurofibrillary tangles and can be found either independently or colocalized with each other. CONCLUSIONS: The observed association of CD88 and C5L2 with neurofibrillary pathology suggests a common altered pathway of degradation.

Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic.

Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a, which activates these receptors, is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucose deprivation, or oxygen-glucose deprivation (OGD), neurons demonstrated increased apoptosis, up-regulation of CD88, and increased levels of C5a in the media. Exogenous murine C5a (100 nM) added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53 (100 nM) significantly blocked ischemia-induced apoptosis (∼50%), and neurons from CD88(-/-) mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion (MCAO), we found that C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88(-/-) mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences.