环状RNAcircTRAPPC6B对结核菌感染巨噬细胞自噬的影响及其在结核病宿主导向治疗中的初步应用

It has long been noted that patients with tuberculosis (TB) are associated with reduced macrophage innate immunity for autophagy to Mycobacterium tuberculosis (Mtb). Despite past extensive studies, the underlying mechanisms, however, largely remain elusive. Given the role of macrophages played in against Mtb infection, they are also considered to be a pivotal target for developing effective host-directed therapy strategies. Circular RNA (circRNA), a new kind of non-coding RNAs, has a variety of regulatory effects on the function of immune cells. We found in patients with active pulmonary tuberculosis (APTB) that the expression of a circRNA, named circTRAPPC6B, in monocyte and macrophage decreased significantly in circRNA-seq assay. Bioinformatics analysis shows that it regulates autophagy by interacting with autophagy associated miRNA or proteins. Importantly, experiments confirmed preliminarily that over-expression of circTRAPPC6B in Mtb infected macrophage could promote autophagy associated protein LC3-II expression. Based on these findings, we herein hypothesize that Mtb impairs macrophage autophagy through down-regulation of the expression of circTRAPPC6B so as to escapes macrophage killing. Up-regulation of circTRAPPC6B expression in macrophage could promote its bactericidal effect of autophagy. To test the hypothesis, we will employ Mtb infected cellular model and Mtb infected animal model to confirm that down-regulation of circTRAPPC6B expression in macrophage is the cause of Mtb escape from intracellular bactericidal effect of autophagy. Then, the molecular mechanisms of circTRAPPC6B interaction with autophagic associated miRNA or proteins by which Mtb infection impairs the autophagic functionality of macrophage will be also investigated. Finally, we will employ a nanometer drug loading system loaded with circTRAPPC6B expression vector to target macrophage intracellular expression of circTRAPPC6B and to explore the feasibility of this approach for the development of host-derected therapy strategies for TB treatment. This project is of great significance in the elucidating the mechanism of Mtb escaping macrophage autophagic bactericidal effect and in exploring host-directed therapy based on macrophage autophagy.

结核菌(Mtb)感染损伤巨噬细胞自噬机制不清。环状RNA对免疫细胞有多种调节作用。我们前期测序分析发现活动性肺结核患者单核/巨噬细胞中circTRAPPC6B表达下调，生物信息学分析表明其可与自噬相关miRNA或蛋白互作，并证实过表达circTRAPPC6B可促进Mtb感染巨噬细胞自噬。据此推测：Mtb通过下调circTRAPPC6B表达来抑制巨噬细胞自噬对其杀灭，上调circTRAPPC6B表达可促进巨噬细胞自噬的杀菌作用。本项目将开展以下研究①在细胞和整体水平论证Mtb抑制circTRAPPC6B表达来逃逸巨噬细胞自噬杀菌作用的机制；②在分子水平阐明circTRAPPC6B通过与miRNA或蛋白互作调控自噬的分子机制；③探讨纳米载药靶向巨噬细胞内circTRAPPC6B表达对结核病实验治疗的效果。本项目对探究Mtb逃逸巨噬细胞自噬杀菌作用和探索基于巨噬细胞自噬宿主导向治疗具有重要意义。

巨噬细胞是结核菌感染的第一道防线，同时也是结核菌潜伏感染的“避风港”。环状RNA是一大类存在于哺乳动物体内的具有多种生物学功能的内源性调节分子，本课题在前期发现结核病患者外周血单核细胞中环状RNA circTAPPC6B下调的基础上，进一步明确其在结核病患者以及结核菌感染巨噬细胞中的表达情况，并探讨其对巨噬细胞杀灭结核菌的调节作用及机制，同时探讨了其作为免疫调节剂靶向巨噬细胞杀灭结核菌的可行性。首先，本课题研究证实了肺结核患者外周血单个核细胞尤其是单核细胞中circTAPPC6B表达下调，治疗后回升；结核菌感染巨噬细胞中circTAPPC6B表达下调。本研究首次发现过表达circTAPPC6B可以抑制结核菌在巨噬细胞中生长，其机制与circTAPPC6B可促进巨噬细胞自噬和诱导巨噬细胞向M1型方向极化并高表达M1型细胞因子有关，从而有利于巨噬细胞的胞内杀菌作用。生物信息学分析发现circTAPPC6B可与多个miRNA分子以及蛋白质互作，RNA pulldown等实验发现其可与miR-874-3P互作，并能解除miR-874-3P对其下游分子ATG16L1表达的抑制作用；同时也证明了circTAPPC6B与miR-892c-3p互作，上调M1型细胞因子IL-6和IL-1β的表达，促进巨噬细胞的胞内杀菌活性。蛋白质质谱分析和RIP等实验也发现circTAPPC6B可与HuR蛋白及ILF2蛋白分子互作，circTAPPC6B通过与ILF2结合上调STAT3的磷酸化，并促进IL-6的表达。为了进一步探讨circTAPPC6B作为结核病宿主导向治疗的调节剂的可行性，本研究制备了高表达circTAPPC6B稳转细胞株，并制备了负载高水平circTAPPC6B细胞外泌体，进一步通过工程化修饰，使外泌体装载抗结核药物利福平并能靶向巨噬细胞，该工程化外泌体在结核菌感染巨噬细胞模型中可靶向巨噬细胞并抑制胞内结核菌的生长。综上所述：本项目的研究结果显示circTAPPC6B可通过促进巨噬细胞自噬和向M1型极化提高其抗结核免疫功能，从而抑制结核菌在巨噬细胞内的存活, 初步证实circTAPPC6B可作为结核病宿主导向治疗的潜在靶点。