结核分枝杆菌ESAT-6蛋白对巨噬细胞NADPH氧化酶活性的调控作用及其分子机制研究

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis infection. Macrophage is an important immune cell which plays critical role during this process. Our previous studies demonstrated that mycobacterium tuberculosis ESAT-6 induced ROS generation via TLR2 on the surface of macrophage. Activation of NADPH oxidase is a key factor for ESAT-6 stimulates macrophage producing ROS, activating JNK/p38 and NF-kB, secreting cytokine such as MCP-1 etc. However, it is unclear which signaling pathways are involved during ESAT-6 inducing NADPH oxidase activation. Thus, we used high throughput method screening high expression molecules after ESAT-6 induced macrophage activation, and have found there are 12 molecules related with signaling transduction and 6 molecules related with mROS generation. These results indicated that ESAT-6 may play multiple roles in regulating ROS generation. This project will investigate 1) how ESAT-6 inducing NADPH oxidase activation and its possible signaling pathway and/or molecular mechanisms by using cell line and animal models; 2) clarify ESAT-6 regulating role in macrophage activation during Mycobacterium tuberculosis infection. These findings will provide new insight for the development of drug target of Mycobacterium tuberculosis and intervention strategies of treating tuberculosis.

结核病是一种由结核杆菌感染引起的传染性疾病。巨噬细胞作为一种重要免疫细胞，在抗结核感染中起着重要作用。我们前期研究发现结核分泌蛋白ESAT-6诱导ROS产生是通过与巨噬细胞表面TLR2结合而发挥作用，NADPH氧化酶活化是ESAT-6刺激巨噬细胞导致ROS产生、JNK/p38和NF-kB活化及细胞因子MCP-l等分泌的关键。但ESAT-6通过哪些信号途径活化NADPH氧化酶尚不清楚。为此我们利用高通量检测平台，对ESAT-6诱导巨噬细胞活化样本进行检测，并筛选出12个具有显著变化的相关信号转导分子和6个直接参与mROS产生的分子，提示ESAT-6在不同程度上多方面调控ROS产生。本课题将从分子、细胞和动物模型等不同层次探讨EAST-6诱导巨噬细胞NADPH氧化酶活化途径及相关分子之间相互作用机制，明确ESAT-6在结核感染过程中对巨噬细胞的调节作用。为药物研发提供新的分子靶标和干预策略。

结核病是一种由结核杆菌感染引起的传染性疾病，巨噬细胞在抗结核感染中起着重要作用。本课题围绕结核杆菌早期分泌蛋白ESAT-6诱导巨噬细胞ROS产生机制及其巨噬细胞相关功能变化展开研究。可分为四部分：首先，证实ESAT-6依赖于TLR2传递信号，快速活化PI3K-Akt信号途径，上调NADPH氧化酶亚单位gp91phox和p22phox表达，从而导致ROS产生。揭示ESAT-6诱导巨噬细胞产生ROS是通过TLR2-PI3K/AKT-NADPH氧化酶活化途径完成的，阐明ESAT-6对早期固有免疫反应的调控具有重要意义。第二，探讨ESAT-6在诱导ROS产生在巨噬细胞凋亡中的作用，结果表明ESAT-6通过TLR2诱导ROS产生，促进MAPKs磷酸化，使Caspase-9和 -3活化，启动内源性细胞凋亡途径，阐明ESAT-6诱导巨噬细胞凋亡的分子机制，为正确理解结核病的病理变化机制提供新的见解。第三，PD-L1是一个重要免疫抑制分子，调控机体免疫应答。首次发现ESAT-6具有上调巨噬细胞表面PD-L1表达的能力，它不仅通过TLR2诱导JNK和PI3K-AKT磷酸化途径上调PD-L1表达，同时还发现也可通过非TLR2依赖途径引起Jak2和STAT3磷酸化，进而上调PD-L1的表达。表明ESAT-6诱导PD-L1上调通过多条信号转导途径完成。为增强结核患者免疫药物研发提供新的靶点。第四，结核性肉芽肿的形成是结核杆菌与宿主免疫应答相互作用的结果。我们发现ESAT-6可通过TLR2诱导CREB磷酸化，继而引起iNOS产生，上调多种上皮细胞标志性分子表达，诱导巨噬细胞上皮样变化，抑制巨噬细胞迁移至结核杆菌感染中心区域，影响巨噬细胞功能的发挥，不利于结核杆菌的清除。总之，ESAT-6在结核致病过程中发挥重要作用，从多方面影响巨噬细胞活性。这为研发抗结核菌药物提供新的靶点。