NLRP3炎症小体介导金黄色葡萄球菌溶血素炎症反应及其调控机制的研究

Recent studies revealed that some bacteria hemolysins have a strong proinflammatory function. It has been found that α hemolysin of Staphylococcus aureus could activate NLRP3 inflammasome to trigger inflammatory response, but ways in cell and regulation mechanism remain unknown. Whether β, γ or δ hemolysins has similar proinflammatory function has not reported.Our previous study found that all Staphylococcus aureus hemolysin could penetrate into human monocytes to raise level of IL-1β and NLRP3 inflammasome-specific IL-18. In this project,we try to use laser confocal, flow cytometry, molecular hybridization, gene silencing, fluorescent probes and other advanced technology to determine the roles of four kinds hemolysin damage membrane and activation of NADH/NADPH oxidase to generate ROS, the release of cathepsin B and ROS after the injury in endocytic lysosomal membrane, penetrating into the active intracellular the NLRP3 inflammasome directly, then to identify TLRs and its downstream pathway raised the role of the NLRP3 inflammasome expression of inflammatory reaction, finally we confirm the above results reversely using NLRP3 and TLR gene knockout mice monocytes, and eventually reveal molecular mechanisms of Staphylococcus aureus hemolysin in activating the NLRP3 inflammasome and to amplify the inflammatory response TLR pathways.

近年发现一些细菌溶血素有很强致炎功能。新近发现金黄色葡萄球菌α溶血素激活胞内NLRP3 炎症小体引发炎症反应，但进入细胞方式及调控机制不明，β、γ、δ溶血素有无类似致炎功能也未有报道。我们前期研究发现，所有金黄色葡萄球菌溶血素均能穿膜进入人单核细胞并上调IL-1β和NLRP3 炎症小体特征性IL-18 表达。本项我们拟采用激光共聚焦、 流式细胞、分子杂交、基因沉默、荧光探针等主流先进技术，了解4 种溶血素损伤细胞膜并激活NADH／NADPH 氧化酶产生ROS、内吞后损伤溶酶体膜释放组织蛋白酶B 和ROS、直接穿膜进入胞内激活NLRP3 炎症小体的作用，确定识别溶血素TLRs及其下游通路上调NLRP3 炎症小体表达及炎症反应的作用，另用NLRP3、TLR 基因敲除小鼠单核细胞反向验证上述结果，最终阐明葡萄球菌溶血素激活NLRP3 炎症小体作用并经TLR 通路放大炎症反应的分子机制。

金黄色葡萄球菌是人类最为重要的致病菌之一，可以引起局部化脓感染，也可引起肺炎、伪膜性肠炎、心包炎等，甚至败血症、脓毒血症等全身感染。溶血素作为金黄色葡萄球菌的外毒素是造成金黄色葡萄球菌感染重要的致病因子，能损伤红细胞、血小板、破坏溶酶体引起局部缺血和坏死。近年发现一些细菌溶血素有很强致炎功能。新近发现金黄色葡萄球菌α溶血素激活胞内NLRP3 炎症小体引发炎症反应，但进入细胞方式及调控机制不明，β、γ、δ溶血素有无类似致炎功能尚未有报道。本研究采用T-A克隆方法成功构建金葡菌溶血素基因（hla/hlb/hlg）的原核表达系统（hld未表达成功），亲和层析法提纯各重组溶血素蛋白, 为后续研究金葡菌溶血素功能奠定了良好的基础。实时荧光定量PCR发现金黄色葡萄球菌在感染THP-1细胞过程中溶血素基因（hla/hlb/hlg/hld）表达上调，进一步表明溶血素是金葡菌感染重要的致病因子。同时研究发现重组溶血素蛋白（rHla/rHlb/rHlg）能够刺激THP-1细胞分泌成熟的IL-1β, IL-18，以rHla作用最强，此过程依赖于NLRP3炎症小体的活化，并可通过识别TLR2受体放大炎症反应。特异性抑制剂的预处理THP-1细胞和IL-1β的分泌水平及Caspase-1活性的测定，初步发现金菌溶血素诱导的NLRP3炎症小体活化依赖于cathepsin B的参与和ROS的产生。上述结果表明NLRP3炎症小体在宿主抵抗金黄色葡萄球菌感染的先天免疫反应中发挥关键作用，有望成为金葡萄感染治疗的靶点。