吸烟激活的信号调节蛋白α调控结核菌易感性的分子机制研究

Tuberculosis (TB) is one of the leading infectious disease worldwide due to the pathogenic acid-fast, rod-shaped bacillus mycobacterium tuberculosis, which invades and multiplies within host macrophages. There is overwhelming epidemiologic evidence that exposure to cigarette smoke (CS) is an independent risk factor for tuberculous sensitivity, progression of primary tuberculosis, development of reactivation tuberculosis, more severe cavitary disease, and death from tuberculosis. However, the biological mechanisms by which CS predisposes individuals to tuberculosis have not been described. Studies have recommended critical roles for CS in promoting the growth of mycobacteria and enhancing the sensitivity in vivo and in vitro. To date, the regulatory mechanism of this complex system is not clear. We have identified Signal regulatory protein (SIRP) α as a key protein in regulating smoke induced factors using microarray. SIRPα is a transmembrane regulatory protein which originally identified in rat cells by its association with cytoplasmic tyrosine phosphatase Src homology region domain-containing phosphatase SHP-2 and was later shown to be highly conserved in other mammals, including human, mouse, and cattle. The cytoplasmic region of SIRPα contains two ITIMs with four tyrosine residues that are phosphorylated in response to a variety of growth factors and ligand binding. This phosphorylation enables recruitment and activation of SHP-1 and SHP-2 that in turn dephosphorylates specific protein substrates involved in mediating various physiological effects. Our previous findings have also indicated that knockdown SIRPα could significantly enhance the autophagy progress. Autophagy is an evolutionarily conserved process which is involved in maintaining cytoplasmic homeostasis by degrading damaged organelles or misfolded proteins. The autophagic cascade is initiated by the engulfment of cytoplasmic cargoes by an autophagosome, which then fuses with a late endosome to form the autolysosome, exposing the inner compartment to lysosomal hydrolases for degradation. Recently, studies suggested that autophagy may link the multistep process of intracellular MTB clearance. Based on recently report and the previous work of our team, we raised the deduce that smoke induced SIRPα enhancement might play a key role in killing intracellular MTB through autophagy. To further explore the mechanism about this, we plan to perform this project on the following aspect: 1) to investigate the relation between the expression of SIRPα and the proliferation of MTB from the various aspects. 2) to determine the downstream signal pathway of SIRPα by sequencing and gene knockout. 3) to explore that the funtion pattern among the change of SIRPα, the infection of MTB, and the reaction of host cell by yeast two-hybrid and co-immunoprecipitation. In conclusion, the study of the change of SIRPα by CS and the regulation of SIRPα to infection of MTB through autopagy could further uncover the molecular mechanism of the MTB susceptivity of smoker and apply to the research of potential drug target of antituberculosis therapy.

研究表明吸烟者更易感染结核菌；我国多地长期处在雾霾天气，其主要成分PM2.5与烟雾中的有害物质一致，这种变相吸烟可能是我国结核病高发的因素之一。因此，阐明烟雾改变结核菌易感性的作用机制尤为重要。本课题组前期发现，烟雾刺激小鼠与结核抗性小鼠基因表达存在相似的变化，其中SIRPα在二者中均显著高表达。进一步研究发现，SIRPα可通过抑制自噬相关基因的表达，负性调控抗结核菌的免疫反应，从而提高结核菌易感性。本项目拟于细胞、动物、临床等不同层面研究烟雾引起的SIRPα表达与结核菌增殖的关系；利用测序、基因敲除等手段研究SIRPα下游信号通路；采用酵母双杂交、免疫共沉淀等方法探讨SIRPα变化、结核菌感染与宿主细胞反应的作用模式，从而深入研究烟雾如何引起SIRPα变化以及SIRPα如何通过调控自噬应对结核菌感染；进一步揭示吸烟/霾者结核菌易感的分子致病机制，并为其作为抗结核治疗潜在药物靶点提供可能。

信号调节蛋白α（SIRPα）是一种表达于单核/巨噬细胞的免疫抑制因子，它在结核分枝杆菌（MTB）感染时上调。本研究表明SIRPα水平与肺结核（PTB）患者的治疗效果密切相关。我们发现PTB患者肺组织中SIRPα表达与PTK2B磷酸化呈负相关。巨噬细胞是抵御结核分枝杆菌的第一道防线。综合运用META分析和多中心临床实验验证，发现MTB能显著诱导巨噬细胞SIRPα的表达。我们证明SIRPα的缺失通过增强巨噬细胞自噬和减少坏死性凋亡来增强巨噬细胞对MTB的杀伤能力。SIRPα通过其细胞内C端结构域与PTK2B形成直接相互作用，并破坏PTK2B和SHP-1之间的相互作用，从而抑制巨噬细胞中PTK2B的激活。有趣的是，这表明SIRPα在巨噬细胞自噬和坏死性凋亡中都起作用，二者对细胞存活有相反的影响。SIRPα缺失导致的坏死性凋亡抑制需要PTK2B活性。SIRPα缺陷型骨髓源性巨噬细胞移植到野生型小鼠后，肺部细菌负荷下降，但炎症性肺损伤加重。联合乌司他丁和SIRPα-/-→WT治疗可减轻炎症反应，维持杀菌能力。本研究将SIRPα定义为MTB感染的新指标和靶点，并描述了通过自噬和坏死性凋亡维持巨噬细胞稳态的潜在机制。此外，我们收集了80例肺癌患者（34例不吸烟，46例吸烟），实验发现尼古丁降低了非小细胞肺癌(NSCLC)细胞中miR-218的表达，促进了NCSCLC细胞的增殖。吸烟的非小细胞肺癌患者肿瘤中miR-218的表达低于不吸烟的非小细胞肺癌患者。我们发现miR-218在NSCLC细胞中直接靶向CDK6 mRNA 3’UTR并抑制其表达，在肺癌组织中也观察到miR-218和CDK6 mRNA表达呈负相关。尼古丁介导的miR-218低表达通过上调CDK6的表达促进NSCLC细胞增殖。我们认为尼古丁通过调控miR-218/CDK6轴促进NSCLC细胞增殖，其可能是肺癌的潜在治疗靶点。