清金颗粒介导CTL/GzmB免疫清除调控MRSA感染小鼠胞内逃逸/定植的机制研究

MRSA is a multidrug resistant bacteria with high international and domestic morbidity and difficult to treat. The journal of nature and cell published a new mechanism for the pathogenesis of MRSA - intracellular escape and colonization. Based on the former fund, it was found that Qingjin Granules had a significant intervention effect on MRSA-infected rats and mice with increased survival rate and reduced bacteria, but there was no inhibitory bactericidal effect in vitro and no direct innate immune enhancement effect on macrophage. It was found that it can promote CTL activation and PFN secretion and upregulate macrophage antigen presentation to accelerate Tcell activation. Based on this, it is hoped to conduct preclinical studies to further investigate its effect on the clearance of MRSA infection and intracellular escape by chelation of T cell and CTL by straggling MRSA models of rag2-/-, IL-2Rγ-/-, and Prf1-/- mice. We use cell co-culture in vitro and two-way fluorescence MRSA and GzmB by laser confocal to evaluation the mechanism of MRSA escape and colonization mediated by CTL and GzmB. Then we use intracellular MRSA infection macrophage return in vivo experiments to evaluate the effectiveness of decolonization / escape and mechanism.

MRSA是国际/国内发病率高/治疗难度大的MDRO。Nature/cell等发表MRSA致病新机制—胞内逃逸/定植。基于前基金，发现清金颗粒对MRSA感染大/小鼠有明显干预效应（生存率提高/载菌降低），却体外无抑/杀菌效应、无直接固有免疫（巨噬细胞）强化效应，可实验发现其能促进CTL活化及PFN分泌，上调巨噬抗原提呈/Tcell活化基因。基于此，希望接续前研究，通过分层干预rag2-/-、IL-2Rγ-/-、Prf1-/-小鼠MRSA模型，深入研究其介导Tell/CTL免疫清除MRSA感染及胞内逃逸/定植机制，明确CTL/GzmB活化路径清除MRSA机制，并借体外细胞共培养（清金颗粒+CTL/巨噬/组织细胞+MRSA），双向荧光MRSA/GzmB及激光共聚焦，明确其介导CTL及GzmB清除MRSA逃逸/定植机制，且用胞内MRSA感染巨噬细胞返输体内实验，评价去定植/逃逸效能及机制。

MRSA是国际/国内发病率高/治疗难度大的MDRO。Nature/Cell等发表MRSA致病新机制—胞内逃逸/定植。本项目通过分层干预Rag2-/-、IL-2Rγ-/-、Prf1-/-小鼠MRSA模型，深入研究清金颗粒清除MRSA胞内逃逸/定植菌机制，并借助体外细胞共培养和胞内MRSA感染MФ返输体内实验，评价去定植/逃逸效能及机制。结果发现：（1）建立小鼠42天MRSA慢性感染/定植模型，清金颗粒在WT、IL-2Rγ-/-、Prf1-/-感染小鼠中均显示出对MRSA感染显著的去定植效应，但在Rag2-/-小鼠中这种治疗效应被逆转，说明清金颗粒调控适应性免疫发挥效应，且CD8+T细胞介导的Perforin/GzmB细胞毒性作用不影响清金颗粒的去定植作用。（2）通过WT感染小鼠肺组织流式细胞术、qPCR和Western Blot检测分析发现清金颗粒可调控T细胞向Th1分化及激活下游IFN-γ/JAK2/STAT1通路。（3）构建胞内吞噬MRSA的MФ持留菌模型，在未感染/慢性感染小鼠脾脏中分选CTL细胞，细胞共培养显示42d感染组小鼠分选的MRSA抗原刺激活化的CTL细胞较未感染组小鼠相比，显著降低了MФ胞内MRSA持留菌；清金颗粒干预组小鼠分选的CTL细胞，进一步降低了MRSA胞内菌量，且在CTL:MФ（5:1）的共培养模型中，清金颗粒的胞内清菌效应最强。（4）构建尾静脉回输胞内持留菌模型，给予抗生素（利奈唑胺）压力及抗生素压力下清金颗粒的联合干预，发现单纯利奈唑胺干预无法降低胞内持留菌量，符合胞内菌抗生素难以清杀特征，但给予清金颗粒后可显著降低小鼠肺、肝、肾脏胞内持留菌；流式细胞术检测发现清金颗粒可显著提高小鼠肺组织IFN-γ+CD4+T细胞比例。本项目为抗生素治疗棘手的胞内菌感染或慢性肺部感染治疗提供了中药治疗策略，且为中药复方抗感染提供免疫研究范式。