结核分枝杆菌HBHA调控巨噬细胞自噬诱导M2型极化的机制研究

Exploring the innate immune response mechanism of macrophages is of great value for discovering new strategies for the control of tuberculosis. Mycobacterium tuberculosis (M. tuberculosis, Mtb) can regulate macrophage autophagy and polarization to disrupt host immune response and facilitate its infection. Heparin-binding hemagglutinin (HBHA) plays important role in regulating the host immune response to Mtb, but the mechanism remains unclear. Our previous study found that HBHA could induce macrophage M2 polarization in addition to inhibiting autophagy in macrophages. Besides, the number of intracellular viable bacteria increased significantly in rMS-HBHA group compared with wild type MS group. Based on these evidences, we hypothesized that HBHA may regulate autophagy and mediate macrophage M2 polarization, thereby inhibiting the clearance of Mtb by macrophages, promoting its survival and replication, and facilitating its immune evasion. In this study, Western Blotting, HBHA knockout and recombinant strains and bacteria macrophages co-culturing system will be used to investigate the immune response of macrophages, to reveal the molecular mechanism of HBHA regulating macrophage autophagy and inducing M2 polarization. This study will not only help to reveal the regulatory mechanism of autophagy and polarization, but also provide new ideas for the mechanism of immune evasion of Mtb, and explore new ways for the clinical treatment of tuberculosis.

探索巨噬细胞对结核分枝杆菌（Mtb）的免疫应答效应机制对于发现结核病防治新策略具有重要价值。Mtb可通过调控巨噬细胞自噬与极化干扰宿主免疫反应，进而促进结核病的发生与发展。HBHA在Mtb宿主免疫反应中发挥重要调控作用，但对其确切机制的认识还存在局限性。我们前期研究发现，HBHA能够抑制巨噬细胞自噬，诱导M2型极化，增加重组耻垢分枝杆菌胞内活菌数。基于此，我们推测，HBHA可能通过调控自噬，介导巨噬细胞向M2亚型极化，从而抑制巨噬细胞清除Mtb，促进其在巨噬细胞内存活并增殖，辅助其免疫逃逸。本课题将利用免疫印迹、HBHA敲除及重组表达菌株、巨噬细胞共培养等技术，开展HBHA与巨噬细胞免疫应答效应研究，以期揭示HBHA调控巨噬细胞自噬诱导M2型极化的分子机制及相互作用。阐明上述问题，不仅有助于揭示自噬与极化间的调控机制，也将为Mtb免疫逃逸机制研究提供新思路，为结核病的临床治疗探索新途径。

探索巨噬细胞对结核分枝杆菌（Mtb）的免疫应答效应机制对于发现结核病防治新策略具有重要价值。Mtb可通过调控巨噬细胞自噬与极化干扰宿主免疫反应，进而促进结核病的发生与发展。HBHA在Mtb宿主免疫反应中发挥重要调控作用，但对其确切机制的认识还存在局限性。本项目研究结果表明，HBHA能够通过抑制巨噬细胞自噬体的形成，阻滞自噬流，进而抑制自噬的发生。野生型耻垢分枝杆菌感染巨噬细胞后促进自噬体形成并诱导自噬的发生，而重组表达HBHA的耻垢分枝杆菌（rMS-HBHA）感染巨噬细胞抑制巨噬细胞自噬体形成及自噬的发生。野生型卡介苗（BCG）感染巨噬细胞后抑制自噬体形成及自噬的发生，而HBHA敲除株（BCG-ΔHBHA）感染巨噬细胞后促进自噬体形成并诱导自噬的发生。巨噬细胞感染试验结果显示，重组表达HBHA促进耻垢分枝杆菌在巨噬细胞中的存活，而敲除HBHA能够促进巨噬细胞对胞内BCG的清除。siRNA敲减TLR4后能够有效阻断HBHA对巨噬细胞自噬的抑制作用，免疫荧光结果显示HBHA与TLR4存在共定位，提示HBHA抑制巨噬细胞自噬由调控TLR4介导。Western blot的进一步结果表明，HBHA通过TLR4-PI3K-AKT-mTOR通路抑制巨噬细胞自噬。检测巨噬细胞极化相关特异性分子，结果显示低浓度HBHA长时间作用巨噬细胞后诱导巨噬细胞M2极化，并能够将M1型巨噬细胞转化成M2型巨噬细胞。HBHA可能通过上调CUEDC2的表达诱导巨噬细胞M2极化。综上所述，本研究发现HBHA可以通过调控巨噬细胞自噬，诱导M2型极化，抑制巨噬细胞清除胞内分枝杆菌，促进其在巨噬细胞内存活并增殖，进而辅助分枝杆菌免疫逃逸。研究结果可能为结核分枝杆菌免疫逃逸机制研究提供新思路和新切入点，为结核病的临床治疗探索新途径。