结核分枝杆菌北京基因型ManLAM诱导Th17细胞极化的分子机制研究

Mycobacterial lipoglycan is a peculiar molecule, which has been known to have both inhibitory and stimulatory effects on host immunity. However, their pleiotropic activity has not been clearly elucidated. Based on our previous work, the preliminary results suggested that the mannose-capped lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis (Mtb) Beijing genotype strain, the most successful Mtb lineage, with geographical wide spread and hypervirulence, had unique glycosylation epitopes. And the experimental results of mouse cells showed that Mtb Beijing genotyping ManLAM may promote the maturation and activation of dendritic cells. Also, after Naïve CD4+T was cultured with DCs elicited by Mtb Beijing genotyping ManLAM, ROR-γt, a key transcription factor in the differentiation of Th17 cells was expressed at higher lever. Therefore, we speculated that Mtb Beijing genotyping ManLAM could elicit the polarization of Th17 cells and play an important role in the regulation of immune balance. In this study, the transgenic mice and antibody blocking experiments were carried out to further elucidate the signal pathway of DCs maturation and Th cell differentiation induced by Beijing genotype ManLAM. Also, the molecular mechanism of Th17 cell polarization induced by the Beijing genotype ManLAM will be discussed. Furthermore, PBMC from latent tuberculosis infection and patients infected with different genotype Mtb strains are collected，and are used to confirm that the Beijing genotype ManLAM could induce the polarization of Th cells. In addition, the correlation between ManLAM glycosylation epitopes and the stimulatory effects on host immunity, and tuberculosis infection status, bacterial load will be discussed. The results of this study will provide a new perspective for elucidating the pathogenic mechanism and immune regulation of mycobacterial lipoglycan, which will be helpful to overcome the current difficult positions in tuberculosis prevention and control, and the development of new vaccines.

分枝杆菌细胞壁特征性的脂聚糖分子对宿主的免疫多效性调节机制仍不清楚。前期我们已初步揭示了全球广泛流行的结核分枝杆菌北京基因型菌株ManLAM具有独特的糖基化位点和连接方式；小鼠实验结果显示，北京基因型ManLAM可促进树突状细胞成熟，并介导Th17细胞分化关键转录因子ROR-γt表达量明显增高。据此我们推测北京基因型ManLAM可诱导Th17细胞极化，对机体的免疫平衡有重要调节作用。在此基础上，本项目拟通过对ManLAM糖基化修饰和识别位点的比较分析，结合转基因小鼠和药物处理关键蛋白变化，进一步揭示北京基因型ManLAM诱导树突状细胞成熟及促使Th17细胞分化的互作机制和信号通路，并通过对不同基因型菌株感染的结核病患者进行体外细胞诱导分析，深入揭示北京基因型ManLAM介导的结核感染者T细胞极化及其与不同感染状态的关联机制。研究结果将为应对当前结核病防治和疫苗研制所面临的困境提供新的视角。

随着糖生物学技术和免疫学的不断发展，深入研究分枝杆菌，特别是结核分枝杆菌（Mtb）和我国主要流行的Mtb北京基因型细胞壁脂阿拉伯甘露聚糖（LAM）与宿主细胞相互作用，揭示其致病性及其潜在的免疫保护与免疫平衡调节作用对于结核病防控具有重要意义。按项目计划，首先应用Triton X-114液相法提取耻垢分枝杆菌M. smegmatis mc2155、牛结核分枝杆菌BCG、减毒株H37Ra、Mtb标准株H37Rv、以及Mtb中国临床分离北京家族株BJ和非北京家族株NBJ株的细胞壁粗糖，经葡聚糖柱Sephacryl S-100 HR分离得到纯化LAM。此后，应用凝集素芯片检测比较几种分枝杆菌间LAM的结构差异，并用Western blot进行验证，结果显示，只能特异性识别末端甘露糖结构的ConA，识别α-甘露糖结构的LCA，识别β-1,3半乳糖结构的ACL，以及识别甘露糖基结构的PSA能与有甘露糖帽子结构的M. tuberculosis H37Ra、H37Rv、NBJ、BJ和M. bovis BCG的脂阿拉伯甘露聚糖相互作用，却不能与有磷酸肌醇帽子结构的M. smegmatis mc2155的LAM相互作用。通过全基因组对部分Mtb BJ和NBJ株测序分析，初步揭示不同分枝杆菌LAM结构差异与糖基转移酶的多态性有关联。进一步应用LAM抗原刺激树突状细胞，结果表明H37Rv、BJ、NBJ和BCG的LAM均可诱导树突状细胞成熟，但耻垢分枝杆菌mc2155和减毒株H37Ra的LAM诱导其成熟的能力较弱。通过刺激后树突状细胞与Naïve CD+4 T细胞共培养，检测Th1和Th17的主要转录因子T-bet和ROR-γ的mRNA表达情况，结果发现耻垢分枝杆菌mc2155、BCG和H37Ra、H37Rv、BJ、NBJ的LAM能不同程度诱导Th1细胞的分化，而只有北京家族株BJ的LAM能明显诱导Th17细胞分化。本项目初步揭示了不同菌株来源分枝杆菌脂聚糖的抗原性与其糖分子大小和结构差异有关，并发现甘露糖帽结构的LAM能诱导树突状细胞成熟以及Th1细胞的分化，其中北京家族菌株的manLAM有很强的诱导Th17细胞分化的能力。本研究成果为进一步深入解析Mtb感染宿主过程中宿主—病原体相互作用的聚糖分子结构和功能机制提供了新的视角，同时也为结核病多糖免疫策略和新型疫苗研制提供了新的理论基础。