Function of human lung mucosal hydrolases during M. tuberculosis infection

描述（由申请人提供）：当结核分枝杆菌（M. tb）通过空气传播感染时，杆菌沉积在肺的肺泡间隙中。传统观点认为，结核分枝杆菌在初始感染时是“静态的”，不会引起免疫反应，它被非活化的肺泡巨噬细胞（AMs）吸收，而AMs是一个重要的感染库。然而，我们假设结核分枝杆菌沉积在肺泡间隙后进入一个动态阶段，在那里它遇到含有稳态和抗菌酶（水解酶）的肺表面活性剂，这些酶改变结核分枝杆菌细胞壁。这些水解酶将具有生物活性的结核分枝杆菌片段释放到局部环境中，并刺激肺泡内的各种肺细胞，从而改变肺微环境，从而改变感染的结果。我们最近发表的研究表明，人肺表面活性剂中的水解酶在感染过程中显著改变结核分枝杆菌的细胞膜，将细胞壁碎片释放到肺环境中。由于这些结核分枝杆菌细胞壁的修饰，杆菌与人巨噬细胞的相关性显著降低，随后吞噬体-溶酶体融合增加，这导致这些细胞内结核分枝杆菌细胞内存活率显著降低，炎症细胞因子产生增加，从而更好地控制感染。除了宿主肺水解酶对结核分枝杆菌细胞壁的重塑外，我们假设响应人肺表面活性剂水解酶释放的结核分枝杆菌细胞壁片段也会影响感染结果。我们的初步数据表明，释放的结核分枝杆菌细胞壁片段与肺表面活性剂水解酶接触后产生，能够激活原代人肺泡室细胞。此外，暴露于这些片段的人巨噬细胞能够更好地控制结核分枝杆菌感染。这些发现为肺部环境在不同感染阶段（如感染初期）对结核分枝杆菌-宿主相互作用的贡献提供了新的概念；从裂解的巨噬细胞释放后；以及在肺腔内的细胞外发现结核分枝杆菌。在所有这些阶段，结核分枝杆菌与含有水解酶的细胞外宿主分泌物密切接触，这些酶会改变其细胞壁并释放片段。为了解决我们的假设，我们建议：i)确定暴露于人肺泡水解酶后释放的结核分枝杆菌细胞壁片段的结构；ii)确定表面活性剂水解酶产生的结核分枝杆菌细胞壁修饰和释放的细胞壁片段如何影响体外人原代肺泡室细胞结核分枝杆菌感染的建立；iii)确定表面活性剂水解酶产生的结核分枝杆菌细胞壁修饰和释放的细胞壁片段如何影响体内结核分枝杆菌感染的过程。这一应用是创新的和独特的，在检查肺表面活性物质和结核分枝杆菌感染之间的重要的和鲜为人知的关系，挑战我们现有的知识，宿主-病原体相互作用已经阐明了体外。结核分枝杆菌在感染期间所遇到的肺环境的作用尚未得到充分的研究，对其在结核分枝杆菌发病机制中的作用所知甚少。

DESCRIPTION (provided by applicant): When Mycobacterium tuberculosis (M. tb) infection occurs by airborne transmission, bacilli are deposited in the alveolar spaces of the lungs. The traditional view is that M. tb is somewhat "static" during initial infection, does not induce an immune response, and it is taken up by non-activated alveolar macrophages (AMs) that serve as an important reservoir for infection. However, we hypothesize that upon deposition in the alveolar space M. tb enters a dynamic phase where it encounters pulmonary surfactant that contains homeostatic and antimicrobial enzymes (hydrolases) which alter the M. tb cell wall. These hydrolases release biologically active M. tb fragments into the local milieu and stimulate a variety of lung cells within the alveoli that changes the pulmonary microenvironment, and thus the outcome of infection. We recently published that hydrolases present in the human lung surfactant dramatically alter the cell envelope of M. tb during infection releasing cell wall fragments into the lung milieu. As a result of these M. tb cell wall modifications, bacilli had a significant decrease in association with human macrophages followed by an increase in phagosome-lysosome fusion, which translated to a significant decrease in M. tb intracellular survival within these cells and an increase in inflammatory cytokine production leading to better control of infection. In addition to remodeling of the M. tb cell wall by host lung hydrolases, we hypothesize that M. tb cell wall fragments released in response to human lung surfactant hydrolases will influence the infection outcome. Our preliminary data show that released M. tb cell wall fragments generated upon contact with lung surfactant hydrolases are capable of activating primary human alveolar compartment cells. Moreover, human macrophages exposed to these fragments are better able to control M. tb infection. These findings add a new concept to the contribution of the lung environment to M. tb-host interactions at different stages of infection such as at the initial stage of infection; following release from lysed macrophages; and when M. tb is found extracellularly within lung cavities. In all of these stages, M. tb is in intimte contact with extracellular host secretions containing hydrolases that will alter its cell wall and release fragments. To address our hypothesis we propose to: i) Determine the structure of M. tb cell wall fragments released following exposure to the human alveolar hydrolases; ii) Determine how M. tb cell wall modifications and released cell wall fragments generated by surfactant hydrolases influence the establishment of M. tb infection in vitro using human primary alveolar compartment cells; and iii) Determine how M. tb cell wall modifications and released cell wall fragments generated by surfactant hydrolases influence the course of M. tb infection in vivo. This application is innovative and unique in examining an important and little known relationship between lung surfactant and M. tb infection and challenges our existing knowledge of host-pathogen interactions that have been elucidated in vitro. The role of the lung environment that M. tb encounters during infection is understudied and very little is known about its contribution t M. tb pathogenesis.