宿主IFITM3基因在巨噬细胞抗结核感染中的免疫调控功能研究

With Mycobacterium tuberculosis (Mtb) as the major pathogen, tuberculosis（TB）is one of the greatest public health threat throughout the world. As only 10% of individuals infected with Mtb would develop TB disease，patients with TB disease may have genetic-based defects in their immune response to removing MTB infection. The incidence of TB has been proved to be closely related with phagocytic process, as well as interferon signaling. Interferon inducible transmembrane protein 3 (IFITM3) gene was identified based on its elevated expression after interferon stimulation. Human IFITM3 gene locates in a TB related chromosomal region. By conducting a case-control study using single nucleotide polymorphism analysis to investigate the association of IFITM3 with the on set of TB, we have previously presented data that IFITM3 was associated with pediatric TB and inefficient expression of IFITM3 could be one TB risk factor. As IFITM3 could restrict viral replication and mainly act in the endocytic pathway, we further observed the IFITM3 protein existing on the acidic organelle/lysosomal membrane besides cell membrane in 293T cells. In order to clarify whether IFITM3 could play a pivotal anti-tuberculosis role, we would further study the localization of IFITM3 protein in macrophages,especially its colocalization with phagocytic-lysosome components, and observe the influence of different expression intensity of IFITM3 on macrophage immune defense.We would use gene over-expression and knocking-down techniqus to construct macrophage models with different expression intensity of IFITM3,and use cell biology or molecular biology methods for detecting cell morphologic changes, Mtb adhesion and phagocytose，the amount of NO, mutiple gene expression levels, activation of signal transduction，synthesis of cytokines in the macrophage models during Mtb infection . This topic is of great significance and clinical importance; it is innovative and pioneering in the fields of TB control and TB molecular pathogenesis research. Our work would not only shed light on the mechanism by which IFITM3 could contribute to anti-tuberculosis, but also has a far-reaching significance for new vaccine developing and immuno-therapy.

IFITM3为干扰素（IFN）上调的跨膜蛋白，发挥基础性及IFN诱导的抗病毒作用，将病毒限制在内吞途径的晚期。而结核病与宿主巨噬细胞对结核分枝杆菌（Mtb）的吞噬-溶酶体过程以及IFN通路密切相关。在前期研究中，申请人首次发现宿主IFITM3基因表达量不足是中国儿童结核病发病的一个风险因素，并且在活的293T等细胞内观察到IFITM3蛋白可定位于除细胞膜之外的溶酶体/酸性细胞器膜上。为了明确IFITM3基因在机体抗结核免疫防御中是否起关键性作用，在本课题拟进一步研究IFITM3蛋白在Mtb主要的效应细胞巨噬细胞内的吞噬-溶酶体定位，并采用基因过表达和基因敲低、Mtb攻击、巨噬细胞活化指标检测等技术方法在分子机制上探讨IFITM3基因在巨噬细胞抗结核感染中的免疫调控作用。该课题在结核病控制领域具有创新性，对结核病发病分子机制研究、新疫苗研发以及今后的免疫治疗有深远意义，有重要科学和临床价值。

由结核分枝杆菌感染引起的人类结核病在世界范围内是一个严重的公共卫生问题。由于免疫系统发育尚不成熟，儿童是结核病的特殊易感人群，儿童结核病的现状不容乐观。结核病具有遗传易感性，本课题组前期发现干扰素诱导的跨膜蛋白IFITM3是儿童结核病易感相关基因。本研究深入探讨了宿主IFITM3基因的免疫防御功能，有助于进一步了解儿童结核病的发生和发展的机制。本研究以宿主IFITM3基因为对象，一方面通过IFITM3荧光融合蛋白的表达及荧光牛结核分枝杆菌BCG菌株的攻击，明确了IFITM3蛋白在宿主巨噬细胞和肺泡上皮细胞吞噬-溶酶体通路的亚细胞定位；另一方面通过构建IFITM3不同表达水平细胞模型结合结核分枝杆菌攻击实验明确IFITM3基因表达水平差异所导致的宿主细胞免疫防御功能的变化。本研究部分揭示了宿主IFITM3基因抗结核的分子机制，对新疫苗研发以及今后的免疫治疗有重要价值。