Role of RNA-binding protein in immune evasion of Mtb in macrophages

项目摘要/摘要 结核病(TB)仍然是一个主要的全球健康问题，约有900万新病例和近150万人 全球每年有100万人死于与结核病有关的疾病。结核分枝杆菌(Mtb)感染持续存在 当宿主的免疫反应被抑制或逃避时，巨噬细胞。然而，结核病的免疫机制 逃避仍未完成。更好地理解这些机制将促进宿主定向的发展 治疗(HDT)，特别是针对致命的耐药结核杆菌。我们的初步数据表明，RNA结合 蛋白，三氢曲普林(TTP)可促进逃避，并可成为宿主导向的结核病治疗的靶点。TTP是一种RNA -在转录后水平参与调节炎症反应的结合蛋白。TTP绑定 到3‘非翻译区(3’UTR)内的富腺嘌呤尿苷元件(ARs)，导致不稳定 编码几种细胞因子的mRNAs对结核分枝杆菌的清除很重要，我们和其他人已经证明它是一种 维持动态平衡的重要调节因子。然而，TTP是否影响MTB复制或重新激活， 仍然不为人知。我们首次在结核杆菌感染的野生型小鼠的肺中发现了显著的TTP诱导。 而在感染分枝杆菌的巨噬细胞中，删除TTP会导致分枝杆菌的抑制 巨噬细胞的生长。此外，用活结核分枝杆菌攻击TTP缺陷小鼠的细菌显著减少。 肺比受感染的野生型窝产仔多。干酪性肉芽肿组织中TTP mRNA水平显著升高 与正常肺实质相比，肺结核病患者肺实质的密度明显降低。结核病的重新激活和传播涉及干酪型 肉芽肿形成空洞并释放结核杆菌。此外，我们的数据首次显示，mTOR的抑制 雷帕霉素发出的信号几乎完全取消了结核分枝杆菌感染的巨噬细胞中TTP的诱导。基于我们的 结果，我们提出了mTOR/TTP轴在结核病免疫逃避中的新作用：毒力结核分枝杆菌激活mTOR 诱导TTP，导致抗结核免疫抑制、结核分枝杆菌复制和原发和/或潜伏的途径 复活性疾病。TTP可作为逆转结核分枝杆菌免疫逃逸的靶点。我们提出两个目标来 验证这一新的假设：目的1.确定TTP在巨噬细胞中对mTOR介导的Mtb生长的作用 以及靶向TTP对结核分枝杆菌生长的影响，以用于潜在的宿主导向治疗。目的2.探索分子 Mtb诱导巨噬细胞TTP的机制。对TTP诱导机制的鉴定不会 不仅增强了我们对MTB逃避机制的理解，还提供了可用于 开发以宿主为导向的治疗方法，打破结核病逃避。这些探索性和机械性研究的结果将 提供对结核病免疫逃避机制的更好理解，并可能导致TTP靶向治疗， 尤其是耐药的结核分枝杆菌。

Project Summary/Abstract Tuberculosis (TB) remains a leading global health problem with about 9 million new cases and nearly 1.5 million TB-related deaths worldwide each year. Mycobacterium tuberculosis (Mtb) infection persists in macrophages when the host immune response is suppressed or evaded. However, mechanisms of TB immune evasion remain incomplete. Better understanding the mechanisms will facilitate development of host-directed therapy (HDT), especially against deadly drug-resistant Mtb. Our preliminary data indicate that an RNA-binding protein, tristetraprolin (TTP) facilitates evasion and can be a target for host-directed TB therapy. TTP is an RNA -binding protein involved in the regulation of inflammatory responses at the post-transcriptional level. TTP binds to adenine-uridine-rich elements (AREs) within the 3’ untranslated region (3'UTR) causing destabilization of mRNAs encoding several cytokines important for Mtb clearance, and has been shown by us and others to be an important regulator maintaining homeostasis. Whether TTP affects Mtb replication or reactivation, however, remains unknown. We found for the first time a significant TTP induction in lungs of Mtb-infected wild type mice and in macrophages infected with mycobacteria, and deleting TTP resulted in suppression of mycobacterial growth in macrophages. Further, mice deficient in TTP challenged with live Mtb had significantly less bacteria in lungs than infected wild type littermates. TTP mRNA levels were significantly higher in caseous granulomas of TB patients than in normal lung parenchyma. Reactivation and transmission of TB involve developing caseous granulomas that cavitate and release Mtb. In addition, our data show for the first time that inhibition of mTOR signaling by rapamycin almost completely abolishes TTP induction in Mtb-infected macrophages. Based on our findings, we propose a novel role for mTOR/TTP axis in TB immune evasion: Virulent Mtb activates mTOR pathway that induces TTP, resulting in suppression of anti-TB immunity, Mtb replicates and primary and/or latent reactivation diseases. TTP may serve as a target to reverse immune evasion of Mtb. We propose two aims to test this novel hypothesis: Aim 1. Determine the role of TTP in macrophages for mTOR-mediated Mtb growth and the effects of targeting TTP on Mtb growth for potential host-directed therapy. Aim 2. Explore the molecular mechanisms of TTP induction by Mtb in macrophages. Identification of the mechanisms of TTP induction will not only enhance our understanding of Mtb evasion mechanisms, but also provide targets that can be used to develop host-directed therapy breaking TB evasion. Results of these exploratory and mechanistic studies will provide a better understanding of TB immune evasion mechanisms and potentially lead to TTP-targeted therapy, especially with drug-resistant Mtb.