结核分枝杆菌Hsp16.3通过Fractalkine/CX3CR1信号轴调控巨噬细胞M2型极化的作用机制

The M2 polarization of macrophage is closely related to the long-term infection of MTB, but the molecular mechanism has not been elucidated so far. Our previous study found that MTB Hsp16.3 could induce macrophage M2 polarization, and identified the receptor CX3CR1 which was highly expressed via gene microarray（CX3CR1 is a specific receptor of Fractalkine），whereas after silencing CX3CR1, MTB Hsp16.3 induced a decrease in the proportion of M2 polarization of macrophages. It has been reported that Fractalkine was overexpressed in serum and tubercular focus of tuberculosis patients. Therefore, we hypothesized that MTB Hsp16.3 regulated macrophage M2 polarization via the Fractalkine/CX3CR1 signaling axis. This project aims to further explored the role and molecular regulation mechanism of fractalkine/CX3CR1 in MTB Hsp16.3 induced macrophage M2 polarization via Hsp16.3 stimulates murine macrophage in vitro and establish the CX3CR1-/- tuberculosis murine model，which was infected the Hsp16.3 knockdown strains of H37Rv. The expected results can lay a good foundation for further molecular mechanism of the macrophage polarization in mycobacterium tuberculosis infection, and can also find a new target for anti-tuberculosis infection prevention and control strategies.

巨噬细胞M2型极化与MTB的长期感染密切相关，但相关分子机制至今未阐明。本课题组前期研究发现：MTB Hsp16.3可诱导巨噬细胞M2型极化；基因芯片等筛选出巨噬细胞高表达受体为CX3CR1(Fractalkine特异性受体)；沉默巨噬细胞CX3CR1，M2型巨噬细胞比例明显降低。结合文献报道Fractalkine在肺结核患者血清及病灶中呈高表达，推测MTB Hsp16.3可能通过Fractalkine/CX3CR1信号轴调控巨噬细胞M2型极化。本项目拟利用MTB Hsp16.3体外刺激小鼠来源巨噬细胞及构建H37Rv-si-Hsp16.3菌株感染CX3CR1基因敲除小鼠肺结核模型，深入探讨Fractalkine/CX3CR1信号轴在MTB Hsp16.3调控巨噬细胞M2极化中的重要作用及分子机制。预期成果有助于进一步阐明结核感染中巨噬细胞极化的分子机制，也可为结核感染防治提供新靶点。

小分子热休克蛋白Hsp16.3是MTB功能蛋白，在结核休眠阶段大量表达，结核分枝杆菌长期感染与巨噬细胞M2极化关系密切。研究发现MTB Hsp16.3可诱导巨噬细胞向M2极化，但结核感染中巨噬细胞的极化相关分子机制至今未阐明。前期研究发现，趋化因子受体CX3CR1及其配体Fractalkine可能在MTB Hsp16.3诱导巨噬细胞M2型极化过程中发挥作用，参与MTB感染的发生发展过程，但具体作用及机制不清。. 故本项目在前期研究基础上，成功构建可溶性MTB Hsp16.3重组蛋白，利用纯化的MTB Hsp16.3蛋白体外刺激小鼠骨髓来源巨噬细胞及肺泡巨噬细胞，发现MTB Hsp16.3能诱导M0型巨噬细胞向M2样表型转换，上调M2型巨噬细胞炎症因子的表达，且Fractalkine和CX3CR1呈高表达。利用慢病毒介导的RNA干扰技术(siRNA)反向验证CX3CR1对巨噬细胞M2型极化的调控作用，发现下调 CX3CR1 的表达抑制了MTB Hsp16.3诱导的巨噬细胞M2型极化。为了明确MTB Hsp16.3通过Fractalkine/CX3CR1信号轴影响巨噬细胞M2型极化的的机制，利用RNA-seq技术进行转录组测序后对差异基因进行分析汇总，通过KEGG通路富集分析出MTB Hsp16.3能够通过激活Fractalkine/CX3CR1信号轴从而促进巨噬细胞向M2型极化，可能与JAK/STAT、MAPK以及PI3K/AKT等信号通路有关。最后，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MTB Hsp16.3调控巨噬细胞炎性细胞因子表达、极化、趋化以及抗原呈递功能。. 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。