P2X4受体介导HSP70提高罗非鱼巨噬细胞呈递无乳链球菌抗原的机制

Heat shock protein 70 (HSP70) play key roles on antigen presentation process. Though reports indicate that mammal HSP70 is involved in the phagocytosis process of macrophages by interaction with receptors on them, that whether P2X4 receptor (P2X4R) participates the antigen presentation process has not been documented yet, and it has not been reported in any fish macrophages. Our previous works showed that the tilapia P2X4R tightly combined with HSP70 protein by CHIP-seq technique, and the P2X4Rand MHC IIβ mRNAs were significantly increased post the stimulation with HSP70 and antigen derives from Streptococcus agalactiae. Therefore, we proposed that the tilapia P2X4R may mediate the increased S. agalactiae antigen presentation to macrophages induced by HSP70. To prove the hypothesis, the functional roles of tilapia P2X4R on macrophage mature, phagocytosis and antigen presentation are going to be investigated, and the interactions between HSP70 and P2X4R are going to be confirmed by GST pull down, EMSA and DNase I foot printing methods. Also the cellular signaling pathways which may be involved in regulation of tilapia macrophage mature, phagocytosis and antigen presentation will be determined by using specific cellular signaling pathway blocking agents and specific antibody against P2X4 receptor combined with the techniques of QPCR,WB, flow cytometry (FCM), and so on. This item will build solid foundation for the development of efficient tilapia HSP70-peptide vaccine.

HSP70在抗原呈递过程中发挥重要功能。虽报道哺乳动物HSP70可与巨噬细胞受体结合而介导胞吞作用，但未见其与P2X4受体作用而参与抗原呈递过程的报道，在鱼类更未见相关研究。我们前期采用CHIP-seq技术发现罗非鱼P2X4R与HSP70紧密结合，且HSP70和链球菌抗原刺激巨噬细胞后P2X4R和MHCIIβ mRNA显著增加。据此提出“罗非鱼P2X4R可介导HSP70提高巨噬细胞呈递链球菌抗原过程”的科学假说，拟在研究P2X4R在HSP70介导巨噬细胞成熟、吞噬和呈递抗原过程中的功能后，采用pull down、EMSA和DNase I 足迹法探究HSP70与P2X4R间互作及作用位点，重点通过特异性信号通路阻断和受体阻断策略结合QPCR、WB和FCM等方法，研究HSP70与P2X4R结合后通过何种细胞信号机制调节巨噬细胞吞噬和呈递链球菌抗原，为开发高效罗非鱼HSP70-肽疫苗奠定基础。

巨噬细胞作为抗原呈递细胞，在固有免疫和适应性免疫反应中发挥重要功能。我们的前期结果表明，罗非鱼巨噬细胞上的受体分子P2X4R可能在呈递链球菌抗原过程中发挥重要调节作用，且暗示P2X4R与HSP70的互作对链球菌抗原呈递过程具有重要影响。由此提出“罗非鱼P2X4R可介导HSP70提高巨噬细胞呈递链球菌抗原过程”的科学假说。我们从优化所建立的罗非鱼腹腔巨噬细胞系入手，对该巨噬细胞的的稳定性和增殖活性等进行了优化，获得了改良后永生化巨噬细胞系。在此基础上，高效表达了罗非鱼Hsc70\Lck\MyD88\TGFβ等7个蛋白，并制备了相应的高滴度抗体，为研究P2X4R的信号通路机制提供了抗体材料。试验发现P2X4R在HSP70提高巨噬细胞呈递链球菌抗原过程中的功能可能与HSC71、ZAP70、PPARδ和ENSONIT00000015480等通路相关，并发现XLOC_061304 lncRNA与ZAP70存在共表达调控关系。在成功构建P2X4R的shRNA病毒颗粒载体后，试验发现P2X4R可能通过IL1β-NFƘB1信号通路对HSP70刺激的巨噬细胞起调控作用。TMT标记的定量蛋白质组学和qPCR检测验证发现HSP70通过激活Toll样受体信号通路促进罗非鱼巨噬细胞抗原呈递的机制。同时，用Medip-PCR 验证发现甲基化修饰基因可能在罗非鱼腹腔巨噬细胞链球菌抗原呈递过程中可能发挥重要作用。在完成以上工作的同时，开展了罗非鱼高质量转录注释工作，并对罗非鱼感染无乳链球菌过程中的mRNA和非编码RNA进行了较为系统的分析。这项研究生成了一个改进了的罗非鱼转录注释，并揭示了尼罗鱼罗非鱼感染无乳链球菌的过程中存在由非编码 RNA 组成的调控网络。本课题为P2X4R介导罗非鱼腹腔巨噬细胞呈递链球菌抗原的作用机制提供了参考，为后续更加深入阐述鱼类相关机制提供了理论依据和新的思路。