PTPN4的表达调控及其在Toll样受体介导的免疫反应中的作用

Toll like receptors (TLRs) play crucial roles in both innate immunity and adaptive immunity. Investigation of the regulatory mechanisms of TLR signaling has important scientific significance and application value in elucidating the mechanism of the immune responses and developing novel ways of immunomodulatory therapy. Under the support of Youth Project of the National Natural Science Foundation, we focused on the modulation of TLR signal transduction, a fundamental scientific issue in immunology, and carried out a series of researches. We reported that multiple molecules including heterogeneous nuclear ribonucleoprotein U (hnRNP-U), E3 ligases tripartite motif-containing protein 38 (TRIM38) and TRAF-interacting protein (TRIP) were involved in the regulation of TLR signaling and clarified their molecular mechanism. In the follow-up experiments, we found the expression of tyrosine-protein phosphatase non-receptor type 4 (PTPN4) was substantially induced with stimulation of LPS (a TLR4 ligand) in thioglycolate-elicited mouse primary peritoneal macrophages. siRNA knockdown of endogenous PTPN4 expression enhanced TLR4-mediated IFN-β expression, while PTPN4 knockdown had no effects on TLR2/3/7/8- and RIG-I-mediated signal transduction. These data suggest that PTPN4 may be specifically involved in the regulation of TLR4 signaling. In this following proposal, we plan to use PTPN4 knockout mice, various cells including macrophages and HEK293 cells combining with varieties of molecular techniques, such as co-immunoprecipitation, cytokine antibody array, dual-luciferase reporter assay etc., to explore the functions of PTPN4 in the regulation of TLR signaling, reveal the possible molecular mechanisms and supply ideal target for novel vaccine development and drug design.

Toll样受体(TLR)对固有免疫及获得性免疫均具有重要的调节作用。TLR信号通路调节机制的研究对于阐明机体免疫反应产生的机理，寻找免疫调节治疗的新途径具有重要科学意义和应用价值。我们前期在青年基金项目资助下，围绕TLR信号通路调控这一重要科学问题展开研究，发现了hnRNP-U、TRIM38、TRIP等多个调节因子并阐明了其作用机制。后续实验发现TLR4可诱导磷酸酶PTPN4的表达；siRNA抑制PTPN4表达，LPS介导的IFN-β表达明显增强，而TLR2/3/7/8和RIG-I介导信号通路均未受影响，提示PTPN4特异性调控TLR4信号通路。本研究利用动物模型(PTPN4基因敲除小鼠)、细胞模型(巨噬细胞及HEK293细胞等)和多种分子技术（免疫共沉淀、细胞因子抗体芯片、报告基因分析等）探讨PTPN4是否参与TLR信号通路调控并揭示其分子机制，为疫苗研制和药物设计提供更为理想的靶点。

固有免疫系统是机体抗感染和抗肿瘤等的“第一道防线”，主要通过多种模式识别受体，启动固有免疫的效应机制，活化、调节后续的获得性免疫应答。固有免疫过度激活和耐受都会对机体造成损害，精细调节其活化强度对机体迅速有效地清除入侵病原微生物及维持免疫稳态均具有重要作用。因此，模式识别受体信号通路调节机制的研究对于阐明免疫反应产生及精确调控的机理，寻找免疫调节治疗的新途径具有重要科学意义和应用价值。在本项目资助下，我们以巨噬细胞为研究模型，对固有免疫调控机制进行研究，并取得了阶段性成果。在J.Exp.Med、Nat.Commun、PNAS、Cell.Rep、J.Immunol等杂志发表论文8篇。在本项目基础上，2014年获得山东省杰出青年基金资助；2016年获得国家自然科学基金优秀青年基金资助。2015年担任中国科协第299次青年科学家论坛执行主席；2016年入选首批山东大学齐鲁(仲英)青年学者。.我们研究发现TRAM在TLR4活化中发生Y167位酪氨酸磷酸化，且在TLR4介导的IFN-β表达中发挥关键作用，为TLR信号通路转导及调控提出了新的认识；明确了PTPN4可作为反馈信号通过靶向TRAM，去除其Y167酪氨酸磷酸化，进而特异性抑制TLR4介导的信号通路；分析并构建了PTPN4磷酸酶活性缺失点突变体，并发现该突变体失去了抑制TLR4信号通路活化功能，为设计合成以PTPN4为干预靶点的药物提供了理论依据。.同时在本项目资助下，我们发现TLR活化或病毒感染可调控E3泛素连接酶TRIM31、Trim40，去泛素化酶USP1，Mint3等多个分子的表达；这些分子可作为反馈信号在不同水平参与TLR、RLR及NLRP3炎症小体等信号通路调控。这一系列研究成果进一步揭示和丰富了固有免疫信号通路调节网络；通过发现这些关键调节分子并揭示其作用的靶点和机制，可为寻找免疫调节治疗的新途径提供理论依据。