Key Points• C3aR activation increases ATP efflux, NLRP3 inflammasome activation, and IL-1b secretion in human monocytes.• C3aR-activated monocytes drive Th17 responses in vitro and likely in vivo.Interleukin-1b (IL-1b) is a proinflammatory cytokine and a therapeutic target in several chronic autoimmune states. Monocytes and macrophages are the major sources of IL-1b. IL1b production by these cells requires Toll-like receptor (TLR) and adenosine triphosphate (ATP)-mediated P2X purinoceptor 7 (P2X7) signals, which together activate the inflammasome. However, how TLR signals and ATP availability are regulated during monocyte activation is unclear and the involvement of another danger signal system has been proposed. Here, we demonstrate that both lipopolysaccharide (LPS) and the anaphylatoxin C3a are needed for IL-1b production in human macrophages and dendritic cells, while in monocytes, C3a enhanced the secretion of LPS-induced IL-1b. C3a and LPS-stimulated monocytes increased T helper 17 (Th17) cell induction in vitro, and human rejecting, but not nonrejecting, kidney transplant biopsies were characterized by local generation of C3a and monocyte and Th17 cell infiltration. Mechanistically, C3a drives IL-1b production in monocytes by controlling the release of intracellular ATP into the extracellular space via regulation of as-yet unidentified ATPreleasing channels in an extracellular signal-regulated kinase 1/2-dependent fashion. These data define a novel function for complement in inflammasome activation in monocytes and suggest that C3aR-mediated signaling is a vital component of the IL1b-Th17 axis. (Blood. 2013;122(20):3473-3481) IntroductionInterleukin-1b (IL-1b) is a key proinflammatory cytokine involved in host responses to pathogens and tissue injury. IL-1b has gathered substantial interest in recent years because several autoimmune disorders respond specifically to IL-1 receptor blockade using either soluble IL-1 receptor antagonists or blocking monoclonal antibodies (mAbs).1 In addition to inducing neutrophil activation and histamine release by mast cells and maintaining epithelial cell integrity, 2 IL-1b also plays a central role in modulating adaptive effector T-cell responses by preferentially inducing T helper 1 (Th1) and Th17 differentiation.3 In humans, CD4 1 T cells require IL-1b and IL-6 to differentiate into Th17 cells, 4 which contribute to the pathophysiology of inflammatory and autoimmune diseases 5 as well as ischemia reperfusion injury (IRI) and transplant rejection. Monocytes, macrophages, and dendritic cells (DCs) are major IL-1b sources and release this cytokine in response to stimuli such as pathogen-associated or danger-associated molecular patterns (PAMPs or DAMPs) mediated by signaling via several Toll-like receptor (TLR) pathways. 7 Although consensus exists that IL-1b generation requires processing of the 31 K D inactive pro-IL-1b to the 17 K D active form by caspase-1 activation via an inflammasome complex (eg, NLRP3), 8,9 signals leading to NLRP3 and caspase-1 activation are...

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.

Mannan-binding lectin-associated serine protease 2 (MASP-2) has been described as the essential enzyme for the lectin pathway (LP) of complement activation. Since there is strong published evidence indicating that complement activation via the LP critically contributes to ischemia reperfusion (IR) injury, we assessed the effect of MASP-2 deficiency in an isogenic mouse model of renal transplantation. The experimental transplantation model used included nephrectomy of the remaining native kidney at d 5 post-transplantation. While wild-type (WT) kidneys grafted into WT recipients (n‫)7؍‬ developed acute renal failure (control group), WT grafts transplanted into MASP-2-deficient recipients (n‫)7؍‬ showed significantly better kidney function, less C3 deposition, and less IR injury. In the absence of donor or recipient complement C4 (n‫,)7؍‬ the WT to WT phenotype was preserved, indicating that the MASP-2-mediated damage was independent of C4 activation. This C4-bypass MASP-2 activity was confirmed in mice deficient for both MASP-2 and C4 (n‫,)7؍‬ where the protection from postoperative acute renal failure was no greater than in mice with MASP-2 deficiency alone. Our study highlights the role of LP activation in renal IR injury and indicates that injury occurs through MASP-2-dependent activation events independent of C4.