Release And Function Study Of Extracellular UDP In Bacterial Infection

As the first defense of hosts, the innate immune system can respond to the invaded pathogens rapidly by non-specific immune effect to infection. In the process of infection, the cells involved in the innate immune reactions can recognize Pathogen-associated molecular patterns(PAMPs) through their own Pattern recognition receptors(PRRs), and then result in the release of inflammatory cytokines, type I interferons, chemokines to exert other innate immune defenses and induce adaptive immune responds. The Membrane-bound TLRs have been fully studied among the PRRs. However, recent studies have found Damage-Associated Molecular Pattern (DAMP) can also play an important role in immune regulation by activating the corresponding receptors when the bodies react to the injury or stress. Extracellular nucleotides as danger signal molecules can cause the activation of downstream signaling pathway and induce the immune response through their bind with relevant purinoceptors. Our previous studies have found that exogenous UDP can induce macrophages in vivo to release of MCP-1 recruiting more monocytes, and enhance the antimicrobial ability. However, the mechanism of the endogenous UDP release and the mutual regulation mechanism of TLRs have not been reported in detail.In this thesis, macrophages were used as research object. We found that the activation of TLR signal pathway could increase the phosphorylation level of ERK and up-regulate the expression of Cx43 leading to the release of UDP. The released UDP activated P2Y6 contributing to the MCP-1 release in order to recruit more monocytes/macrophages enhancing the ability against the bacterial infection.This paper includes results as follows:1、Bacterial infection can induce UDP releaseIn this thesis, we have established a mouse model by injecting E. coli 0111 and have measured the UDP release of mouse peritoneal fluid through fluorescence polarization. The results show that the UDP release of mouse peritoneal fluid gradually increased with bacterial infection for a longer period and suggest that there will be UDP release during bacterial infection process. At the same time, we used mouse macrophage cell line RAW264.7 and bone marrow derived macrophages(BMDMs) as cell models, Pam3CSK4 and LPS molecule which are regarded respectively as the Gram-positive bacteria and Gram-negative bacteria mode to stimulate macrophages to simulate different bacterial infection process. The results showed that Pam3CSK4 and LPS can both stimulate macrophages to release UDP in a time and dose-dependent manner, which may be accompanied by the activation of TLRs.2、Connexin43 is mediated by Connexin43According to the current studies, UDP release is related with membrane channel and exocytosis. By using the channel and exocytosis inhibitors to pre-treat cells, we found that CBX and FFA can inhibit LPS-mediated UDP release instead of NEM. Compared with CBX, FFA-induced inhibition is more distinct. Then, it shows that Connexin43 is the main macrophage-expressed Connexin by reverse transcription-polymerase chain reaction (RT-PCR) and by quantitative PCR(qPCR) and Western blot respectively, we can see that LPS can up-regulate the expression of Connexin43 in mRNA and protein level.By Western blot, we found that the stimulation of LPS can enhance the phosphorylation level of ERK, AKT and P65. By using the inhibitors U0126 of ERK, LY294002 of AKT, BAY 11 of P65 to treat cells respectively, the results showed that ERK phosphorylation inhibitor U0126 significantly reduced the expression of Conenxin 43 compared with Ly294002 and BAY11. Moreover, we used Cx43 specific inhibitor Gap26 and ERK phosphorylation inhibitor U0126 to pre-treat cells, and the results showed that both Gap26 and U0126 can down-regulate the UDP release, which suggests that LPS-induced UDP release is mainly mediated by the open of Connexin43 channel upregulated by ERK signal pathway. The results suggested that LPS up-regulated the expression of Cx43 through ERK signal pathway, and release UDP to extracellular environment through the Cx43 channel.3、UDP activating P2Y6 receptor contributing to MCP-1 release increasing the body’s anti-bacterial capabilityFinally, because specific receptor of UDP is P2Y6, we have adopted a specific inhibitor MRS2578 and P2Y6 knockout mice to explore the influence of P2Y6 on the MCP-1 expression. Then, we used the Gap26 and U0126 to inhibit the expression and function of Cx43. Results showed that when the function of P2Y6 is inhibited or lacked, it will impair LPS-induced MCP-1 release. And inhibiting the expression and function of Connexin 43 will also reduce the LPS-induced MCP-1 release. Therefore, the expression of MCP-1 is caused by released UDP, leading to recruit more monocytes/macrophages and further strengthen of the body’s anti-bacteria ability.From the mouse peritonitis model, we found that pre-injected MRS2578 and FFA, U0126, Gap26, respectively, can all weaken the mice’s ability to kill the bacteria, which leads to the death of mice. The results showed that, in the bacteria-infection process, the released UDP has a vital role in enhancing the mice’s ability to resist the bacteria infection.In summary, we found that, in the bacterial infection, the activation of TLRs will increase phosphorylation of ERK, and significantly up-regulate the expression of Conenxin43 contributing to the extracellular release of UDP. Released UDP activated specific receptor P2Y6 in extracellular autocrine and paracrine manner, resulting in the expression of MCP-1 and recruiting more monocytes/macrophages, thus improving the body’s antimicrobial capacity. From the perspective of the extracellular release of danger signals, our studies make clear the antibacterial mechanism of the body and verify the function of the released UDP and its receptor. By combining the PAMPs and DAMPs, our studies lay a theoretical foundation for the innate immune regulation and provide theoretical proofs about extracellular nucleotide for designing new drugs.