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The complement activation fragment C5a is a potent proinflammatory mediator that is increasingly recognized as an immune modulator. C5a acts through two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), to powerfully modify multiple aspects of immune cell function. Although C5aR1 is generally acknowledged to be proinflammatory and immune-activating, the potential roles played by C5aR2 remain poorly defined. Despite studies demonstrating C5aR2 can modulate C5aR1 in human cells, it is not yet known whether C5aR2 functionality is limited to, or requires, C5aR1 activation or influences immune cells more broadly. The present study, therefore, aimed to characterize the roles of C5aR2 on the signaling and function of primary human monocyte–derived macrophages, using a C5aR2 agonist (Ac-RHYPYWR-OH; P32) to selectively activate the receptor. We found that although C5aR2 activation with P32 by itself was devoid of any detectable MAPK signaling activities, C5aR2 agonism significantly dampened C5aR1-, C3aR-, and chemokine-like receptor 1 (CMKLR1)–mediated ERK signaling and altered intracellular calcium mobilization mediated by these receptors. Functionally, selective C5aR2 activation also downregulated cytokine production triggered by various TLRs (TLR2, TLR3, TLR4, and TLR7), C-type lectin receptors (Dectin-1, Dectin-2, and Mincle), and the cytosolic DNA sensor stimulator of IFN genes (STING). Surprisingly, activity at the C-type lectin receptors was particularly powerful, with C5aR2 activation reducing Mincle-mediated IL-6 and TNF-α generation by 80–90%. In sum, this study demonstrates that C5aR2 possesses pleiotropic functions in primary human macrophages, highlighting the role of C5aR2 as a powerful regulator of innate immune function.

Complement activation generates the core effector protein C5a, a potent immune molecule that is linked to multiple inflammatory diseases. Two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), mediate the biological activities of C5a. Although C5aR1 has broadly acknowledged proinflammatory roles, C5aR2 remains at the center of controversy, with existing findings supporting both immune-activating and immune-dampening functions. Recent progress has been made toward resolving these issues. Instead of being a pure recycler and sequester of C5a, C5aR2 is capable of mediating its own set of signaling events and through these events exerting significant immunomodulatory effects not only toward C5aR1 but also other pattern recognition receptors and innate immune systems, such as NLRP3 inflammasomes. This review highlights the existing knowns and unknowns concerning C5aR2 and provides a timely update on recent breakthroughs which are expected to have a substantial impact on future fundamental and translational C5aR2 research.

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow–derived macrophages from BVRfl/fl and LysM-Cre:BVRfl/fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVRfl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVRfl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a–C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization–associated chemokines, the levels of which depend in part on C5aR1 signaling.

Intracellular/autocrine complement proteins have emerged as critical regulators of human Th1 induction and contraction. T cells contain both intracellular C3 and C5 activation systems, with intracellular C3aR1 and C5aR1 stimulation driving T cell homeostatic survival and normal Th1 IFN-gamma production, respectively. Here we demonstrate how the intracellular/autocrine C5 system is regulated by using T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses and a profound loss of naïve CD4 T cells (95% of CD4 T cells in the blood have a memory T cell phenotype). Thus, in contrast to intracellular C5aR1 stimulation, cell surface expressed C5aR2 is an important negative regulator of inflammatory Th induction. While both C5aR1 and C5aR2 can bind C5a, we found that the carboxypeptidase-processed form of C5a, C5a-desArg, was twice as potent as C5a in reducing Th1 induction. In addition, carboxypeptidase M (CPM) expression was highly induced upon T cell activation indicating that CPM may be mediating T cell-derived C5a-desArg generation and thus, C5aR2 stimulation. In this vein, activation of T cells in the presence of a CPM inhibitor induced Th1 hyper-induction, which was rescued by addition of a C5aR2 agonist and reduced by adding C5a-desArg, but not C5a. The in vivo importance of T cell-expressed CPM and autocrine C5aR2 activation is demonstrated by the fact that CPM−/− mice have enhanced inflammatory Th responses and CPM−/− T cells cause increased pathology in an in vivo T cell transfer colitis model. These data highlight the complex auto-regulatory functions of complement in T cell responses.

The complement activation product C3a is often described as a proinflammatory mediator, alongside its downstream cousin, C5a. However, emerging studies show that C3a has several anti-inflammatory facets in vivo. For example, in the acute inflammatory response, C3a acts in direct opposition to C5a, through preventing the accumulation of neutrophils in inflamed tissues by independently regulating their mobilization. This acute, protective, and opposing activity of C3a to C5a is also illustrated in models of septicemia. In this article, we reinvestigate the discovery and original classification of C3a as a proinflammatory mediator and highlight the emerging studies demonstrating anti-inflammatory effects for C3a in the immune response. It is our hope that this review illuminates these apparently contradictory roles for C3a and challenges the general dogma surrounding C3a, which, historically, has ubiquitously been described as a proinflammatory mediator. In light of this, we urge investigators to use “inflammatory modulator” as the descriptor for C3a.

Regulation of T cell immunity by C5a has been suggested from recent studies. However, the underlying mechanisms, particularly the involved cells and biochemical basis, are not well defined. In this study, the direct modulation of dendritic cell (DC) activation and its function in T cell stimulation by C5a-C5aR interaction and the involved signaling pathways were investigated. We show that DCs from C5aR−/− mice and normal DCs treated with C5aR antagonist have less-activated phenotype characterized with increased IL-10 and decreased IL-12p70 production in response to LPS stimulation, lowered surface expression of MHC class II, B7.2, and consequently have reduced capacity to stimulate allospecific T cells. Conversely, C5a stimulation up-regulates DC activation and its function in allostimulation. Furthermore, stimulation of C5aR mediates the inhibition of cAMP production and protein kinase A activity and is involved in activation of PI3K/AKT and NF-κB signaling in DCs. These results demonstrate that C5a acts directly on C5aR expressed on DCs resulting in the cell activation and subsequently enhances its capacity for allospecific T cell stimulation. It also suggests that NF-κB signaling induced by down-regulation of cAMP/ protein kinase A pathway and up-regulation of PI3K/AKT pathway following C5a stimulation may contribute to up-regulation of DC function.

Alzheimer’s disease (AD) is an age-related dementia, characterized by amyloid plaques, neurofibrillary tangles, neuroinflammation, and neuronal loss in the brain. Components of the complement system, known to produce a local inflammatory reaction, are associated with the plaques and tangles in AD brain, and thus a role for complement-mediated inflammation in the acceleration or progression of disease has been proposed. A complement activation product, C5a, is known to recruit and activate microglia and astrocytes in vitro by activation of a G protein-coupled cell-surface C5aR. Here, oral delivery of a cyclic hexapeptide C5a receptor antagonist (PMX205) for 2–3 mo resulted in substantial reduction of pathological markers such as fibrillar amyloid deposits (49–62%) and activated glia (42–68%) in two mouse models of AD. The reduction in pathology was correlated with improvements in a passive avoidance behavioral task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau (69%). These data provide the first evidence that inhibition of a proinflammatory receptor-mediated function of the complement cascade (i.e., C5aR) can interfere with neuroinflammation and neurodegeneration in AD rodent models, suggesting a novel therapeutic target for reducing pathology and improving cognitive function in human AD patients.

The NLRP3 inflammasome controls IL-1β maturation in antigen presenting cells but a direct role in human adaptive immune cells has not been described. Here we show that the NLRP3 inflammasome assembles in human CD4+ T cells and initiates caspase-1-dependent IL-1b secretion, thereby promoting IFN-γ production and Th1 differentiation in an autocrine fashion. Importantly, NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which drive reactive oxygen species (ROS) production. The alternative cell surface expressed C5a receptor 2 (C5aR2) negatively regulates this process. Dysregulation of NLRP3 activity in T cells affects inflammatory responses in autoimmune disease or infection. Firstly, CD4+ T cells from patients with cryopyrin-associated periodic syndromes (CAPS), who have constitutively-active NLRP3, exhibit overactive Th1 responses that are normalized by NLRP3 inhibitor treatment. Secondly, IFN-γ production is impaired in T cells from Nlpr3−/− or Il1a/Il1b−/− mice upon viral infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to ‘innate immune cells’ but is an integral component of normal adaptive Th1 responses.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone–dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2–deficient serum. CpG-B increased levels of IL-1β, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1–dependent cytokine release.