CARMA3调控金黄色葡萄球菌激活NLRP3炎症复合体的机制研究

Staphylococcus aureus infection leads to NLRP3 inflammasome activation and resultant secretion of pro-inflammatory cytokines, such as IL-1β, promoting occurrence of inflammation, while its mechanisms still need to be further clarified. Our study indicated that S. aureus infection induced upregulation of CARMA3 at the protein level and activated NF-κB. Knockout of CARMA3 decreased the protein level of NLRP3 and release of IL-1β, suggesting that CARMA3 regulates S. aureus-induced NLRP3 inflammasome activation. In this study, target genes will be knocked out using CRISPR Cas9 technology, activities of the NLRP3 inflammasome and NF-κB will be detected using western blotting and ELISA, and colocalization will be observed using immunofluorescence assay. In order to elucidate the regulatory role of CARMA3 in NLRP3 inflammasome activation by S. aureus, the effect of CARMA3 on assembly of the NLRP3 inflammasome, the roles of MAVS and NF-κB in the NLRP3 inflammasome regulated by CARMA3, and the influence of CARMA3 on pyroptosis by means of lactate dehydrogenase activity assay will be explored. This study will contribute to understand the regulation mechanisms of inflammation induced by S. aureus in a perspective of the NLRP3 inflammasome, and provide the potential targets for designing antibacterial agent-independent program of control and prevention.

金葡菌激活NLRP3炎症复合体释放炎症因子IL-1β，促进炎症的发生，但调控机制有待进一步探索。我们的前期研究表明，金葡菌诱导CARMA3蛋白水平升高，NF-κB活化，敲除CARMA3后NLRP3蛋白水平下降，IL-1β释放减少，提示CARMA3调控金葡菌激活NLRP3炎症复合体。本项目使用CRISPR Cas9技术敲除靶基因，western blotting和ELISA法检测NLRP3炎症复合体和NF-κB的活化，免疫荧光技术观察共定位。通过观察CARMA3对NLRP3炎症复合体组装的影响，探究MAVS和NF-κB在CARMA3调控NLRP3炎症复合体中的作用，乳酸脱氢酶活性法检测敲除CARMA3对焦亡的影响，从而明确CARMA3在该过程中的调控作用。本项目有助于从NLRP3炎症复合体角度了解机体感染金葡菌后发生炎症的调控机制，为设计独立于抗菌药的防治方案提供可能靶点。

金黄色葡萄球菌（简称金葡菌）是医学和兽医临床上重要的病原，感染多种动物，引起关节炎、脊椎炎、乳房炎和心内膜炎等多种炎症，给畜牧业带来严重经济损失。金葡菌引起机体发生炎症的机制仍有待进一步阐明。HEK293T细胞过表达NLRP3、ASC、caspase-1、IL-1β和CARMA3后，随着CARMA3量的增加，分泌进入上清的IL-1β呈剂量依赖性增加，LPS+ATP处理过表达细胞后，过表达CARMA3促进IL-1β分泌，表明在体外重组系统中，CARMA3促进NLRP3炎症复合体的激活。过表达ASC、caspase-1、IL-1β、CARMA3和NLRC4或AIM2后，CARMA3促进NLRC4炎症复合体的激活，但对AIM2炎症复合体没有影响。CRISPR/Cas9敲除小鼠CARMA3，分离小鼠骨髓源巨噬细胞（bone marrow-derived macrophage，BMDM），用LPS+ATP或LPS+nigericin处理，IL-1β分泌减少，表明CARMA3促进NLRP3炎症复合体的激活。与野生型BMDM相比，CARMA3敲除BMDM感染金葡菌后IL-1β分泌减少，表明CARMA3参与金葡菌诱导分泌IL-1β。金葡菌感染BMDM后免疫荧光染色可见，NLRP3迁移到线粒体，表明金葡菌感染BMDM后促进NLRP3炎症复合体组装，但CARMA3与线粒体共定位没有明显增加。HEK293T分别过表达CARMA3和NLRP3以及CARMA3和caspase-1，发现CARMA3对NLRP3和caspase-1的蛋白表达没有影响，表明CARMA3不是通过促进NLRP3和caspase-1的蛋白表达促进NLRP3炎症复合体的激活。免疫共沉淀试验发现，CARMA3与ASC互作，并且是通过CARMA3的CARD结构域与ASC互作，CARMA3的coiled-coil和MAGUK与ASC无互作，表明CARMA3通过其CARD结构域与ASC互作促进NLRP3炎症复合体的激活。与野生型BMDM相比，CARMA3敲除BMDM感染金葡菌后释放LDH显著减少，表明CARMA3促进金葡菌诱导焦亡的发生。以上结果表明，CARMA3通过ASC互作促进金葡菌激活NLRP3炎症复合体以及IL-1β的分泌，为临床防治金葡菌提供了理论基础和潜在靶点。