NOD信号对肺泡上皮细胞TLR介导抗结核分枝杆菌感染的免疫调节机制

With an increasing incidence in recent years, the tuberculosis is the second leading infectious disease cause deaths in adults, which is caused by Mycobacterium tuberculosis (M. tuberculosis). Ningxia is one of the provinces with higher number of tuberculosis cases in China. We have previous uncovered a NOD (nucleotide-binding oligomerization domain receptor) -mediated signaling transduction between the alveolar epithelial cells and macrophages using an air-liquid interface (ALI) co-culture cell model. However, it is not clear about immunoregulatory mechanism underpinning the interaction between Toll-like receptor (TLR) signaling and NOD signaling in alveolar epithelial cells in response to an infection of M. tuberculosis. Therefore, in order to accomplish this goal, an ALI model of primary human alveolar epithelium cells will be first generated, and stimulated with ligands or inhibitors of TLR and NOD signaling, before alterations of TLR and NOD signaling are ascertained; then, the impact of TLR and NOD signaling on inflammatory responses in the alveolar epithelial cells will be accessed by an approach of gain/loss of TLR and NOD signaling using overexpression/small RNA interface; finally, above finding will be further validated in murine models deficient MyD88 and NOD2, respectively. The results of these studies will provide novel insight into the molecular mechanism of pathogenesis of M. tuberculosis.

结核病是导致成年人死亡的第二大传染病，宁夏作为全国结核病高发重点省区之一，疫情长期处于传染病报告前列。肺泡上皮细胞（AECs）在呼吸道免疫中的作用日趋受到重视，为真实模拟体内肺泡细胞群相互作用的微环境，我们前期建立气液相共培养模型，发现上皮细胞与巨噬细胞之间存在NOD2介导的信号传递。但上皮细胞在抗结核杆菌感染中是否存在TLR与NOD2信号通路的协调互作调控机制尚不清楚。本项目在人AECs气液相3D体外感染模型基础上，利用慢病毒载体介导过表达、小RNA干扰、受体激动剂和抑制剂等刺激手段，借助高通量、高灵敏度抗体芯片技术筛选并分析NOD2和TLR信号网络中关键蛋白的变化与相关功能；同时利用MyD88和NOD2缺陷小鼠进行体内验证。在整体、细胞与分子水平层层解析肺泡上皮细胞中TLR与NOD2通路协调互作的抗菌免疫调控机制，对进一步开展结核免疫机理研究具有重要意义。

结核病是导致成年人死亡的第二大传染病，宁夏作为全国结核病高发重点省区之一，疫情长期处于传染病报告前列。肺泡上皮细胞（AECs）在呼吸道免疫中的作用日趋受到重视，为真实模拟体内肺泡细胞群相互作用的微环境，我们前期建立气液相共培养模型，发现上皮细胞与巨噬细胞之间存在NOD2介导的信号传递。但上皮细胞在抗结核杆菌感染中是否存在TLR与NOD2信号通路的协调互作调控机制尚不清楚。本项目在人AECs气液相3D体外感染模型基础上，利用慢病毒载体介导过表达、小RNA干扰、受体激动剂和抑制剂等刺激手段，借助高通量、高灵敏度抗体芯片技术筛选并分析NOD2和TLR信号网络中关键蛋白的变化与相关功能；同时利用MyD88和NOD2缺陷小鼠进行体内验证。用PCR法对小鼠基因型鉴定，通过气管滴注法建立小鼠分枝杆菌H37Ra感染模型，检测三种小鼠外周血及NOD2-/-小鼠肺组织匀浆中细胞因子TNF-α、IFN-γ、MCP-1的含量；测定MyD88-/-小鼠肺组织中NOD2/RIP2/TRAF6信号轴、NOD2-/-小鼠肺组织中TLR4/MyD88/TRAF6信号轴中关键因子的表达以及这两条信号轴下游NF-κB和MAPK信号通路中蛋白的表达。实验结果发现： 1. NOD2与TLR4/MyD88信号通路均参与小鼠MTB感染后肺组织中NF-κB的调控。2. 小鼠感染MTB后，肺组织内P38的激活主要经NOD2信号通路诱导，而不是TLR4/MyD88信号途径。3. 小鼠感染MTB后，肺组织内JNK的激活主要经经TLR4/MyD88信号通路诱导，而不是NOD2信号途径。根据以上研究成果，发表论著 4 篇，其中 2 篇北大核心期刊，一篇 Medline 收录。发表 sci 综述 1 篇，发表国际学术会议论文 1 篇，国内学术会议论文1篇，培养研究生 4 名。项目划拨直接经费380000元，目前实际支出319109元，剩余60891元，用于后续研究及发表文章。