肺炎支原体TLR4配体的鉴定及其经自噬相关通路调控促炎细胞因子分泌的分子机制

Mycoplasma pneumoniae is an extracellular human pathogen that is frequently the causative agent for community acquired pneumonia. We previously demonstrated that M.pneumoniae was recognized by Toll-like receptor (TLR) 2/6 and TLR1/2 by the innate immune system and thus resulted in inflammatory response. However, recently studies indicate that the TLR4 is also involved in the cytokines secretion in macrophages via a TLR4-mediated autophy-associated pathway. In this work, we aim at the key scientific issue of M. pneumoniae and inflammatory resonance, to investigate the mechanism of TLR4-activated autophagy-associated pathway in M.pneumoniae-infected macrophages. Firstly, we focus on the screening and characterization of the TLR4 ligand by thin layer chromatography, reporter gene assays and mass spectrometry, and then investigate the expression of the key moleculars involved in autophagy-associated pathways which located at the downstream of TLR4. After then, we will investigate the effect of the above key moleculars on the activation of the inflammatory-associated kinase and nuclear transcriptional factors by inhibition of their expression, and thus to understand the role of autophagy-associated pathways in regulation of proinflammatory cytokines secretion. The objective of this study is to reveal the molecular mechanism of innate system in eradication of M.pneumoniae, and to improve the knowledge of the interactions between M.pneumoniae and the host cells.

肺炎支原体（Mp）是引起社区获得性肺炎的重要病原体。本课题组前期研究表明机体免疫系统通过Toll样受体（TLR）2/6和TLR1/2识别Mp并参与炎症反应发生。但最新研究表明TLR4也参与识别Mp并能诱导细胞因子分泌，其机制与激活TLR4介导的自噬相关通路有关。本项目瞄准Mp感染与炎症反应的关键科学问题，研究Mp经TLR4激活巨噬细胞自噬相关通路的过程。重点通过薄层色谱、报告基因及质谱分析等方法对Mp中TLR4的配体进行鉴定，并对TLR4下游自噬相关通路中的关键分子开展研究。通过RNA干扰和免疫荧光等方法，深入了解Mp感染后激活自噬相关通路的机制。同时通过抑制自噬相关通路中的关键分子，观察其在介导炎症相关激酶和核转录因子活化中的作用，从而阐明自噬相关通路与促炎细胞因子分泌的关系，最终揭示TLR4介导固有免疫系统清除Mp的分子机制，为进一步完善Mp与宿主细胞相互作用提供理论依据。

肺炎支原体（Mp）是引起社区获得性肺炎的重要病原体。课题组前期研究表明机体免疫系统通过Toll样受体（TLR）2/6和TLR1/2识别Mp并参与炎症反应发生。但最新研究表明TLR4也参与识别Mp并能够诱导巨噬细胞分泌炎症因子，但其机制并不明确。本研究本项目瞄准Mp感染与炎症反应的关键科学问题，研究Mp经TLR4激活巨噬细胞自噬相关通路的过程。首先采用氯仿：甲醇以及葡聚糖G25层析柱提取纯化Mp脂质，通过酶学实验、抗体抑制实验等方法验证脂质为TLR4的配体。随后将脂质与巨噬细胞孵育，研究其激活炎性信号通路的机制，重点研究NF-κB，NLRP3以及自噬通路在诱导促炎细胞因子分泌中的作用，并阐明信号通路之间的串话机制。研究结果发现，肺炎支原体脂质可与TLR4结合，随后诱导小鼠巨噬细胞自噬并激活NF-κB和NLRP3，自噬与NF-κB可形成正反馈环，进一步促进TNF-α和IL-1β分泌。进一步研究发现，脂质也能诱导基质金属蛋白酶的表达，其机制与激活丝裂原活化蛋白激酶、诱导组蛋白乙酰化以及下调抑癌基因RECK表达有关。本项目的开展首次证实了TLR4也是识别Mp脂质的模式识别受体，脂质激活TLR4后可通过多种途径发挥促炎作用，是对传统观点“固有免疫系统经TLR2/6识别支原体”的重要补充。