Function And Mechanism Of P2Y₆ Receptor In Innate Immunity

Purinergic receptor P2Y6, a G protein-coupled receptor for UDP, which was found expressed in a variety of cells, including epithelial cells, vascular endothelial cells and most immune cells, plays an important role in inflammatory response and vascular physiology via the up-regulation of phospholipase C (PLC) and the mobilization of Ca2+. P2Y6 has been reported to have different functions, such as promoting the survival of osteoclasts and arresting cell cycle. Recently, several reports indicated that P2Y6 is involved in TLR (Toll-like receptor) 1/2/4-induced neutrophils migration through regulating IL-8 release in human monocytic cells. Different avenues have been pursued to explore the therapeutic potential of P2Y6 receptor as a drug target. However, the systemic functions and mechanisms of P2Y6 in innate immunity are poorly understood.Macrophage is one of the key phagocytes that regulates the process of innate immune response, and aptly positioned to function as the primary line of defense against invading pathogens in many organs, including the lung and peritoneum. The invaded-pathogens or other danger signals are quickly recognized by macrophges through pattern-recognition receptors (PPRs) such as TLRs, NLRs and RLRs, then activate macrophges and other immune cells. The activated macrophges could secret many kinds of cytokines and chemokines, recruit effector immune cells and phagocytose pathogens and dead cells, and then initiate immune response. In our study, it has been observed that the expression level of purinoreceptor P2Y6 was much higher in murine macrophages when compared with other P2Y receptors, implying that P2Y6 receptor may play a crucial role in macrophges-mediated immune response.To address the function and mechanism of P2Y6 in innate immunity, murine macrophage RAW264.7 cells and peritoneal macrophages were employed. By application of P2Y6 selectivity agonist and antagonist, UDP and MRS2578, P2Y6 receptor demonstrated significant effect on macrophages-mediated cytokines and chemokines release and the recruitment of monocytes/macrophages. Firstly, activation of P2Y6 by UDP significantly up-regulated the expression of cytokines and chemokines such as MCP-1, MIP-la/p, IFN-a/p, TNF-a and G-CSF. Moreover, costimulatory molecules CD80/86 are significant induced by UDP/P2Y6 system. Secondly, UDP-treated cell supernatant obviously enhanced the chemotaxis of monocytes/macrophages, and this UDP-mediated activation could be abrogated by P2Y6 selective antagonist MRS2578. Thirdly, the activity of macrophages migration was promoted by activation of P2Y6 with UDP.Next, we further examined the signaling pathways and molecular mechanisms regualted by UDP/P2Y6 in macrophages. Firstly, activation of P2Y6 with UDP significantly increased the phosphorylation of MEKl/2, PI3K, STAT1 and IRF3, but not Src and IKK determined by Western blot. In addition, phosphorylation of MEKl/2 significantly leaded to the activation of ERK1/2 signaling pathway. Secondly, treatment with MAPKs inhibitors respectively, further elucidated that the P2Y6-induced MCP-1 release is primarily through an MEK1/2-ERK1/2-AP1 signaling pathway dependent mechanism. Thirdly, UDP/P2Y6 system could activate IRF3 and STAT1 signaling pathway, and then up-regulated the expression of IFN-a/p, suggesting that P2Y6 is possibly involved in the activity of antivirus.Furthermore, by application of MCP-1 blocking Ab, air-pouch model and peritonitis model, further comfirmed that UDP/P2Y6-induced MCP-1 production significantly enhances the recruitment of monocytes/mcrophages both in vitro and in vivo, thereby contributing to the clearance of invaded bacteria and improve the host survival in peritonitis.In present study, we provide evidence that P2Y6 receptor has a non-redundant role in promoting host survival from invaded-pathogens. UDP/P2Y6 system could regulate production of cytokines and chemokines MCP-1 primarily through a MEK1/2-ERK1/2 pathway and induced the monocytes/macrophages recuitment, then contributed to the clearance of Escherichia coli and promote host survival in vivo. Thus, therapeutic strategies targeting the UDP/P2Y6 signaling system may have the potential in regulation of anti-infection immunity, but further studies are still needed to elucidate the system function and mechanism of UDP/P2Y6 system in various physiological processes.