Function of human lung mucosal hydrolases during M. tuberculosis infection

描述(申请人提供)：当结核分枝杆菌(M.TB)感染通过空气传播时，杆菌沉积在肺部的肺泡腔中。传统的观点认为，结核分枝杆菌在初始感染过程中是静止的，不会引发免疫反应，它被非激活的肺泡巨噬细胞(AM)吸收，而AM是感染的重要蓄水池。然而，我们假设，一旦在肺泡腔中沉积，结核分枝杆菌就会进入一个动态阶段，在那里它会遇到含有动态酶和抗菌酶(水解酶)的肺表面活性物质，这些酶会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。我们最近发表了在人类肺表面活性物质中存在的水解酶在感染过程中极大地改变了结核分枝杆菌的细胞膜，将细胞壁碎片释放到肺环境中。由于这些对结核分枝杆菌细胞壁的修饰，杆菌与人巨噬细胞的结合显著减少，随后吞噬小体-溶酶体融合增加，这导致结核分枝杆菌在这些细胞内的细胞内存活率显著下降，炎性细胞因子的产生增加，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，我们推测，人肺表面活性物质水解酶释放的结核分枝杆菌细胞壁碎片将影响感染结果。我们的初步数据显示，与肺表面活性物质水解酶接触时释放的结核分枝杆菌细胞壁碎片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的感染。这些发现增加了一个新的概念，即肺环境在不同感染阶段对结核分枝杆菌-宿主相互作用的贡献，例如在感染的初始阶段，从溶解的巨噬细胞释放之后，以及当在肺腔内发现结核分枝杆菌的细胞外时。在所有这些阶段，结核分枝杆菌都与含有水解酶的胞外宿主分泌物密切接触，这些水解酶将改变其细胞壁并释放碎片。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碎片的结构；ii)确定表面活性物质水解酶产生的结核分枝杆菌细胞壁修饰和释放的细胞壁碎片如何影响体外利用人原代肺泡室细胞建立结核分枝杆菌感染；以及iii)确定表面活性物质水解酶产生的结核分枝杆菌细胞壁修饰和释放的细胞壁碎片如何影响体内结核分枝杆菌感染的过程。这一应用在检测肺表面活性物质和结核分枝杆菌感染之间一个重要且鲜为人知的关系方面是创新和独特的，并挑战了我们已在体外阐明的宿主-病原体相互作用的现有知识。结核分枝杆菌在感染过程中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也知之甚少。

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.