一种新的TLR2/TLR4识别分子- - 肺炎链球菌热休克蛋白ClpP诱导宿主呼吸道上皮细胞致炎机制研究

During respiratory bacterial infections, the interactions between bacterial virulence factors and airway epithelial cells initiate and promote immune response and airway inflammation. ClpP, as a serine-type peptidase of Hsp100, is an important virulence factor in a variety of pathogens. However, the molecular mechanisms for the interactions between ClpP and host cells have not been elucidated. We have recently found that the expression of ClpP was up-regulated and maintained during respiratory infection and inflammation caused by Streptococcus pneumoniae, and ClpP could directly induce immune response and inflammatory reactions. Furthermore, ClpP could bind to TLR2 and TLR4, and it was able to increase the expression of TLR2 on the surface of airway epithelial cells, which was blocked by anti-TLR2 or anti-TLR4 monoclonal antibodies. We therefore hypothesize that ClpP is a novel ligand for TLR2 and TLR4, and it may activate MAPKs, PI3K/Akt and NF-κB signaling pathways through binding to TLR2/TLR4, which subsequently regulate the immune response and inflammation of airway epithelial cells. In this study, we would take human primary airway epithelial cells and knockdown mouse models to investigate the activation effects of ClpP on airway epithelial cells and their potential molecular signaling pathways in vitro and in vivo through cDNA microarray analysis, luminex multiplex bead assays, siRNA, EMSA and luciferase reporter system, etc. This work may provide new insight into the molecular mechanisms involved in the interactions between respiratory pathogens and host cells.

呼吸系统病原菌感染中，细菌毒力因子与呼吸道上皮细胞相互作用是宿主免疫应答和炎症反应的起始事件。Hsp100肽酶ClpP是一种普遍存在的细菌毒力因子，但它与宿主细胞相互作用机理尚不明确。我们实验新发现：ClpP在肺炎链球菌呼吸道感染和炎症建立过程中持续表达，并具有直接免疫活化致炎功能；进一步研究显示ClpP可结合TLR2和TLR4两种受体，并依赖于TLR2和TLR4，诱导呼吸道上皮细胞TLR2表达上调。推测：ClpP是一种新的TLR2和TLR4配体，可能激活MAPKs、PI3K/Akt和NF-κB等信号通路，从而调控呼吸道上皮细胞免疫炎症效应。本课题拟利用人原代呼吸道上皮细胞和基因敲除小鼠等模型，采用功能分类表达谱芯片、多重蛋白检测技术、siRNA、EMSA和荧光素酶报告系统等方法，体内外分析ClpP活化呼吸道上皮细胞的免疫炎症效应及相关信号通路，以揭示呼吸道病原菌与宿主细胞相互作用新机制。

细菌毒力因子与呼吸道上皮细胞相互作用是宿主免疫应答和炎症反应的起始事件，本项目原计划研究毒力蛋白ClpP与宿主细胞的相互作用机制。然而实验发现其虽可诱导小鼠的气道炎症反应，但不影响肺炎链球菌对小鼠的感染能力。而我们发现另一个新毒力因子PepO蛋白能诱导宿主细胞的炎症因子高表达，引起严重的肺部损伤，提示其可能是一种通过过强炎症反应引发组织损伤的毒力因子。而缺陷动物试验显示，其可能是通过TLR4通路诱导炎症反应，可能是一种新的TLR4的配体蛋白。因此本课题以PepO为靶蛋白，探讨其诱导小鼠肺部炎症反应，引起组织损伤的机制。本项目的主要发现为：①PepO具有极强的宿主固有免疫激活效应，这种效应部分依赖于宿主TLR2、TLR4受体。对于不同的细胞其效应和诱导的信号通路不尽相同，rPepO可刺激PEMs分泌白介素6（IL-6）、肿瘤坏死因子α（TNF-α）、CXCL1和CXCL10，该效应依赖于TLR2、TLR4以及p38、Akt和NF-κB(p65)的激活。而rPepO主要促进MLE-12细胞分泌 IL-6和 CXCL1，依赖的是MAPKs、PI3K /Akt 和NF-κB信号通路，其促进Beas-2B细胞分泌主要IL-8和CXCL-10，依赖的信号通与MLE-12细胞基本一致。②PepO可通过TLR2-NF-κB信号通路诱导miRNA-155表达增高，增强巨噬细胞的吞噬能力，该作用可能与SHIP1表达的下调以及CR3表达上调相关。同时，我们在肺部炎症反应相关研究中发现，在脓毒血症导致的免疫抑制状态下，缺失IL-4可促进肺部继发感染的铜绿假单胞菌的细菌清除，并上调感染小鼠的生存率。本项目还发现另一个TLR4配体蛋白DanJ的可通过PI3K 和JNK通路激活巨噬细胞细胞，另外还发现转化调节子ComE蛋白可负调控肺炎链球菌关键的毒力因子荚膜多糖的合成。这些研究结果为TLR2/4配体类毒力蛋白作用于宿主细胞的免疫反应提供了新的实验证据，对肺炎链球菌蛋白疫苗、抗菌及抗炎药物研发都有一定的参考价值。