结核分枝杆菌Hsp16.3对巨噬细胞M2型极化的作用研究

Recent studies showed that long-term infection of Mycobacterium tuberculosis (MTB) is closely associated with the polarization of M2 macrophages. However, its underlying mechanism remains unclear. Small heat shock protein Hsp16.3 is one of functional proteins of MTB, and plays an crucial role in the long-term survival of MTB in macrophages. Our previous study revealed that MTB Hsp16.3 played an important role in modulating the function of macrophage. This study aims to construct a technological platform for the induction of M1/M2 polarization of macrophages derived from mouse in vitro, and observe that the effects of MTB Hsp16.3 on M1/M2 polarization. Then, we further construct the Hsp16.3 knockdown strains of MTB H37Rv and establish the murine model of tuberculosis infection of lung. The changes of polarization of alveolar macrophage in vivo and the pathological injuries would be observed. The relationship between Hsp16.3 expression level and macrophage polarization would also be analyzed. Finally, the cDNA chip, RNA interference and Western BLot technology are used to explore the possible influence of the TLRs pathway including TLR4 on the effect of HSP16.3 on M2 polarization of macrophages. The expected results of this subject are to provide important experimental and theoretical basis for subsequent in-depth study on effects and mechanisms of the macrophage polarization by MTB infection.

研究发现结核分枝杆菌(MTB)长期感染与巨噬细胞M2极化关系密切，其具体机制不明。小分子热休克蛋白Hsp16.3是MTB功能蛋白，对其在巨噬细胞中长期生存具有重要作用。我们前期研究发现MTB Hsp16.3可影响巨噬细胞功能。本项目拟建立体外诱导小鼠来源的巨噬细胞M1/M2极化技术平台，观察Hsp16.3对巨噬细胞M1/M2型极化的作用；构建敲减Hsp16.3的结核分枝杆菌株H37Rv感染的小鼠肺结核模型，动态观察体内肺泡巨噬细胞极化的改变和病理损伤，分析Hsp16.3表达水平与巨噬细胞极化的关系；采用cDNA芯片、RNA干扰和WB等技术进一步探讨TLR4等通路在Hsp16.3影响巨噬细胞M2 极化中的可能作用。项目预期结果可为后续深入探讨MTB 感染对巨噬细胞极化的影响及机制提供重要的实验和理论依据。

研究发现结核分枝杆菌(MTB)长期感染与巨噬细胞M2极化关系密切，但结核感染中巨噬细胞的极化相关分子机制至今未阐明。小分子热休克蛋白Hsp16.3是MTB功能蛋白，对其在巨噬细胞中长期生存具有重要作用。在本研究中，我们用纯化的Hsp16.3蛋白体外刺激小鼠巨噬细胞发现MTB Hsp16.3抑制M1型巨噬细胞炎症因子的表达，同时能诱导M0/M1型巨噬细胞向M2样表型转换。并探讨了TLR4信号途径在Hsp16.3调节的巨噬细胞极化中的作用及调控分子机制，发现在MTB Hsp16.3刺激巨噬细胞向M2极化的过程中，TLR2/4可能参与MTB Hsp16.3诱导巨噬细胞向M2型极化这一过程，且可能与MAPK/NF-κB相关信号通路有关。但在既往的研究中，TLR4介导的NF-κB信号通路主要促进巨噬细胞的M1型极化，故推测MTB Hsp16.3除了可能作为TLR4的配体外还可能与其他受体相互作用。因此，我们通过查阅相关文献及结合芯片分析结果利用相关实验技术发现CCRL2/CX3CR1在MTB Hsp16.3刺激M0型巨噬细胞向M2型转化的过程中的发挥重要作用并初步阐明其相关分子机制，即MTB Hsp16.3能够通过激活CCRL2/CX3CR1受体从而促进巨噬细胞向M2型极化，其可能与AKT/ERK/p38-MAPK信号通路有关。并且利用H37Ra野生型菌株(WT)，Hsp16.3基因敲除菌株(MUT)和Hsp16.3基因回补菌株(COMP)进一步探索Hsp16.3对巨噬细胞极化的影响。本项目为深入探讨调控结核感染中巨噬细胞M1/M2极化格局的关键影响因素及相关分子机制奠定了研究基础，对阐明结核感染的免疫发病机制及寻找抗结核感染防治策略新靶点具有重要的研究意义。