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Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to

The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli–generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified “native” form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor–independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.

Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2−/− mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2−/− mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF–mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2−/− IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.

C5aR2 (C5L2/gp77) is a 7-transmembrane spanning receptor that binds to C5a but lacks motifs essential for G-protein coupling and associated signal transduction. C5aR2 is expressed on immune cells, modulates various inflammatory diseases in mice, and has been shown to facilitate murine and human regulatory T cell (iT(REG)) generation in vitro. Whether and if so how, C5aR2 impacts in vivo T(REG) generation and pathogenic T cell-dependent disease models have not been established. Herein we show that murine T cells express and upregulate C5aR2 during iT(REG) generation and that the absence of T cell-expressed C5aR2 limits in vivo iT(REG) generation following adoptive transfer of naïve CD4(+) T cells into rag1(−/−) recipients. Using newly generated C5aR2 transgenic mice (C5aR2-tg) we show that overexpression of C5aR2 in naïve CD4(+) T cells augments in vivo iT(REG) generation. In a model of T(REG)-dependent cardiac allograft survival, recipient C5aR2 deficiency accelerates graft rejection associated with lower T(REG)/T(EFF) ratios while overexpression of C5aR2 in immune cells prolongs graft survival associated with an elevation in T(REG)/T(EFF) ratios. T cell-expressed C5aR2 modulates T(REG) induction without altering effector T cell proliferation or cytokine production. Distinct from reported findings in neutrophils and macrophages, T(REG)-expressed C5aR2 does not interact with β-arrestin or inhibit ERK1/2 signaling. Rather, cumulative evidence supports the conclusion that C5aR2 limits C5aR1-initiated signals known to inhibit T(REG) induction. Together, the data expand the role of C5aR2 in adaptive immunity by providing in vivo evidence that T cell-expressed C5aR2 physiologically modulates iT(REG) generation and iT(REG)-dependent allograft survival.

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato–C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato–C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18–induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.

The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a–C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4+ T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4+ T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAFV600E mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium–induced colitis. Following infection, properdin knockout (PKO) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell–derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to PKO mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of PKO mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid–deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.

The complement system is implicated in the pathogenesis of human inflammatory bowel disease, but the specific role of C5a has never been examined. We have compared the efficacy of an orally active human C5a receptor antagonist (AcPhe[Orn-Pro-d-cyclohexylalanine-Trp-Arg]), prednisolone, and infliximab against trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The drugs were administered either 2 days before or 24 h after TNBS instillation, and rats were then examined after 8 days. Drug-free colitis control rats showed severe disease pathology with significant mortality (39%). Rats pre or posttreated with the C5a antagonist (10 mg/kg/day peroral, 0.3 mg/kg/day s.c.) had reduced mortality and significantly improved macroscopic scores, colon edema, colon myeloperoxidase levels, reduced concentrations of TNF-α levels in the colon and serum, and had greater food intake resulting in greater weight gains than colitis-only rats. Rats pretreated with prednisolone (1 mg/kg/day s.c.) displayed significant improvement in parameters measured, but posttreatment was ineffective. Single dose pretreatment with the TNF-α inhibitor infliximab (3 mg/kg i.v.) also had significant improvements in the parameters measured. Rats pretreated with a combination of the C5a antagonist and prednisolone showed no greater improvements than either drug alone. These findings suggest a central role for complement, particularly C5a, in the pathology of TNBS-induced colitis in rats, indicating a possible therapeutic role for C5a antagonists in inflammatory bowel disease.

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease. Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-α, as well as polymorphonuclear leukocyte migration, increased TNF-α levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.