Function And Mechanism Of Extracellular ATP And P2X₇ Purinoceptor In Antibacterial Infection

ATP has only been recognized as the energy substances involved in intracellular metabolism and circulation for a long time, Recently it is discovered as a "danger signal" in the organism physiological and pathological processes (such as:nerve damage, diseases of the cardiovascular system and immune, etc.), which plays an important role. Body against bacterial infection mainly rely on broad-spectrum antibiotics, and the emergence of drug resistance on the abuse of antibiotics pose a severe challenges; scholars have found that the body in the process of anti-bacterial infection in addition to release of cytokines/inflammatory factors, but also release ATP, it’s bound to play an important role in resistance to bacterial infection adjust the body in this process, and which provides a new way of thinking, Make clear the release of ATP in anti-bacterial infection of the body and understand the mechanism that which channel the ATP is released, and ATP regulation of the immune system through its receptor play a role in this process of anti-bacterial infection and its molecular mechanism is critical, but no relevant literature about the system was reported so far.Innate immunity as the first line of the body defense against outside invasion of pathogenic microorganisms such as bacteria, viruses, which is first excited for invasion,the immune cells participating in the process such as macrophages, dendritic cells(DCs) through the expression of cell membrane orpattern recognition molecules of the membrane receptors of PRRs (pattern-recognition receptors),which play a major role in identifing pathogens quickly, preventing infection and clear apoptotic mutations, as well as dead cells against external pathogens invasion and safeguard their own stability and remove abnormal cells.The function of the macrophages secreting inflammatory cytokines and recruit immune cells to migrate to sites of inflammation, phagocytosis of pathogens and presenting antigens,by which play a role in the innate immune response. In this study, we selected the murine macrophage cell line RAW264.7、mouse peritoneal macrophages and bone marrow-derived macrophages as cell model, using Real-time PCR analyzed the ATP receptor family in macrophages cells and we found that the ATP receptor is widely expressed in the macrophages, wherein the P2X7、P2Y2 and P2Y6 showing high expression relative to other members of the receptor family, which implies that they may play an important role in the macrophage-mediated immune function.To further study the ATP through its receptor in the macrophage-mediated innate immune of resistance to bacterial infection function and molecular mechanisms, we applyed the ATP and P2X7 receptor agonist BzATP and its receptor blocker OxATP, activating or blocking ATP receptor signaling pathway, ATP through P2X7 receptors increases the transcription expression of cytokine/chemokine, and significantly enhanced the role of monocyte/macrophage phagocytosis of bacteria by real-time quantitative PCR, ELISA, FACS, thereby increasing the survival rate of the host. And manifested in the following aspects:Firstly, extracellular ATP of macrophage concentration change caused by bacterial infection and the concentration changes over time decreasing trend in the first two peaks in about 15 minutes and 4 hours; Secondly, ATP via its receptor significantly enhance macrophage inflammatory factor /chemotaxis the transcriptional expression, such as CCL-2 and IL-lbeta, etc; Thirdly, the role of ATP in macrophages significantly enhance the ability of its phagocytosis of bacteria, whereas this kinds of effects can be inhibited the ATP receptor inhibitors OxATP; Fourly, ATP activate the downstream signaling pathways through its receptor in the mouse model in vivo, which mainly by accelerating effect of macrophage scavenging the invasive bacteria, and enhance host resistance to bacterial infection, thereby enhance the survival of the host.On the basis of the above research, we used Western blotting analysis of ATP through P2X7 signaling pathways involved in the regulation of macrophage. Results displayed:ATP activation of P2X7 significantly improve the the phosphorylation level of p38 but no significant effect on ERK1/2 and JNK, then exercise regulatory function activation relevant Preparation signal passage, but not by the transcription factor NF-kappaB, the specific mechanism remains to be further studied. These results suggest that ATP receptors in the body’s resistance to infection immunity plays an important role, it can significantly enhance macrophage phagocytosis in the body by activation path of p38 MAPKs, and release inflammatory factors, in order to enhance the body immunity in anti-bacterial infection, so as to protect the host to maintain homeostasis of the body.In summary, this paper mainly systematic studying and discussion on macrophage release of ATP and the release mechanisms in the process of anti-bacterial infection for the first time, ATP/P2X7 regulate production of cytokines and chemokines, such as IL-1β primarily through p38 MAPKs pathway and promote macrophage phagocytosis of pathogens to improve the body’s resistance to infection. This research provides a theoretical basis for elucidation futherly the danger signal molecule ATP and P2X7 receptor in immune inflammation, but also provide a new target for the development of anti-inflammatory drugs.