糖皮质激素负调控巨噬细胞自噬在结核感染免疫中的作用及机制研究

Mycobacterium tuberculosis (MTB) can limit the acidification and maturation of mycobacterial phagosomes to excape degradation by lysosomal hydrolases, so as to survive and persist within host macropahges, leading to latent infection. Previous studies revealed that long-term use of glucocorticoids (GC) would trigger active tuberculosis (TB) in latently infected individuals. However, it remains largely unknown whether GC affect the mycobatericidal activity of macrophages (Mφ), which have a crucial role in directly killing of invading MTB. Our preliminary data showed that the GC treatment disturbed the co-localization of mycobacterial phagosomes with lysosomes in Mφ, which in turn increased the survival of intracellular MTB. Moreover, GC decreased the phosphorylation level of TBK1, and negatively regulated the intracellular DNA sensor (cGAS) signaling pathway, as well as autophagy induced by MTB infection or MTB DNA stimulation. Thus, we proposed a new hypothesis that “GC inhibits the elimination of intracellular MTB in Mφ by negatively modulating cGAS-mediated autophagy”. To test this hypothesis, research work will be carried out as followings: i) determine whether GC inhibits clearance of MTB in Mφ through suppressing autophagic pathway, ii) elucidate the signaling pathway (cGAS-STING-TBK1) by which GC inhibits autophagy in Mφ after MTB infection, iii) identify candidate phosphatases which are responsible for GC-induced inhibition of DNA-sensing pathway and subsequent autophagy during MTB infection, iv) verify the role of GC-mediated suppression of autophagy during MTB infection. The present project aims to reveal the role and underlying mechanism of GC in Mφ-mediated anti-mycobacterial response, which may provide solid experimental evidence for effective prevention of long-term use of GC to increase the risk of tuberculosis.

MTB能抑制吞噬体酸化成熟而逃逸Mφ的杀伤，从而在Mφ内长期存活，造成潜伏感染。长期使用糖皮质激素（GC）是诱发MTB潜伏感染转为活动性结核的重要因素。但GC是否影响Mφ抗结核免疫，尚不清楚。课题组前期研究发现：①GC抑制Mφ中MTB吞噬体与溶酶体融合，促进胞内MTB存活；②GC抑制激酶TBK1磷酸化，负调cGAS受体通路及其诱导的细胞自噬。据此，我们提出“GC负调cGAS受体介导的细胞自噬，从而抑制Mφ清除胞内MTB”的假说。在此基础上，本项目拟：①诱导细胞自噬进行回复实验，明确GC负调自噬而影响胞内MTB的清除；②激活cGAS受体通路/过表达TBK1，确定GC调控自噬的信号通路；③检测GC诱导磷酸酶的表达，确定GC调控TBK1活化及自噬的机制；④实验验证GC调控自噬在抗结核免疫中的作用。项目将揭示GC在Mφ清除MTB中的调控作用及机制，为降低长期使用激素导致的结核发病风险提供依据。

长期服用糖皮质激素已显示可使个体高度易感分枝杆菌感染并导致潜伏细菌重新激活。然而，糖皮质激素在天然免疫抵抗结核杆菌感染中的作用，特别是在巨噬细胞中，尚不清楚。本研究发现糖皮质激素抑制先天免疫反应，抑制巨噬细胞一氧化氮的产生和自噬发生。同时，糖皮质激素处理后RAW264.7细胞中结核杆菌吞噬体的酸化和成熟减弱。更重要的是，糖皮质激素处理的细胞中结核杆菌存活率增加。进一步研究发现，糖皮质激素抑制能够促进自噬小体成熟的TBK1激酶的活化。并且，抑制TBK1也导致一氧化氮的产生的减少。此外，糖皮质激素处理还导致一些自噬相关基因表达下调；相反地，糖皮质激素处理促进Akt / mTOR信号通路的激活增加。这可能是糖皮质激素抑制被结核杆菌感染巨噬细胞自噬的原因。用激动剂雷帕霉素恢复自噬能够消除糖皮质激素促进的结核杆菌存活，表明糖皮质激素通过阻断了重要的抗结核杆菌的存活的细胞自噬途径而促进结核杆菌的存活。总的来说，本研究表明糖皮质激素抑制细胞自噬和一氧化氮的产生，进而促进巨噬细胞中的结核杆菌的存活。本研究揭示了糖皮质激素介导的免疫抑制新机制，为预防结核病提供重要的科学依据。