副猪嗜血杆菌激活TLRs/NF-κB信号通路的分子机制及其生物学效应的研究

Haemophilus parasuis （HPS）is characterized by fibrinous polyserositis, meningitis and polyarthritis. In the past decade, it has received more attention due to the increasing economic losses in the pig industry. Toll-like receptors (TLRs) can elicit conserved inflammatory pathways, culminating in the activation of nuclear factor-kappa B (NF-κB), which is the switch gate of inflammatory reaction by regulating the transcription of many genes. The studies of the applicant indicated that the infection of HPS triggered TLRs/NF-κB signaling pathways that result in the induction of inflammatory and immune reaction. The molecular mechanisms underlying the mechanisms of H. parasuis which induced a severe inflammation of the serous membranes remain undefined. In this study, we will analyze the details and the roles of TLRs/NF-κB signaling pathways by a NF-κB dual-luciferase Assay System, Western blot and confocal laser microscope assay, which will provide a basis for understanding molecular pathways of immune evasion and inflammatory response associated with diseases caused by H. parasuis and the designing of a new effective vaccine and medicine.

副猪嗜血杆菌（Haemophilus parasuis, HPS）是当前危害世界养猪业最重要的病原菌之一，感染猪后引起严重的多发性浆膜炎、脑膜炎和关节炎。Toll样受体（Toll like receptors, TLRs）能通过激活NF-κB信号通路而成为炎症反应的启动闸门，调控许多基因的转录，在免疫应答和炎症反应中发挥重要作用。申请者的前期研究表明，HPS感染能够显著激活NF-κB，而TRAF6的干扰分子能抑制HPS诱导的NF-κB激活，提示HPS可能通过TLR信号通路激活NF-κB。本课题拟采用NF-κB双荧光素酶报告系统、Western blot和激光共聚焦扫描显微镜观察等技术，解析HPS激活TLRs/NF-κB的信号通路的分子细节，阐明TLRs信号通路在HPS激活NF-κB中的作用，为揭示HPS的炎症发生机理和免疫机理奠定基础，为设计药物靶标、研制安全高效的HPS疫苗提供科学依据。

本项目揭示了副猪嗜血杆菌（Haemophilus parasuis, HPS）感染PK-15细胞后激活NF-κB和p38/JNK MAPK信号通路，但不激活ERK MAPK信号通路。TLR1、TLR2、TLR4和TLR6介导激活NF-κB和p38/JNK MAPK信号通路；接头分子MyD88、TRIF、 IRAK4、siIRAK1、siTRAF6、siTAB1和siTAK1参与HPS感染激活NF-κB信号通路。HPS感染PK-15细胞通过TLR1, TLR2, TLR4和TLR6介导的NF-κB和p38/JNK MAPK信号通路来上调IL-8，CCL4和RANTES（CCL5） 的表达。HPS感染PK-15细胞后RANTES启动子活性上调。RANTES启动子上 NF-κB1、NF-κB2位点起到最关键作用，副猪嗜血杆菌感染PK-15细胞产生RANTES与TLR/NF-κB和JNK MAPK信号通路相关。