Determinants of Tuberculosis Severity

 描述（由申请人提供）：结核病（TB）仍然是全球感染死亡的主要原因。结核分枝杆菌（Mtb）与宿主巨噬细胞（MΦ）之间的相互作用是结核病发病机制的核心。我们的初步数据表明，毒性结核分枝杆菌抑制宿主保护性凋亡反应的感染MΦ，以使用这些细胞作为一个受保护的复制生态位。在生长到最佳细胞内细菌负荷后，Mtb然后诱导MΦ坏死，使细菌逃逸并扩散到幼稚吞噬细胞。我们认为，感染诱导的MΦ坏死是结核病病灶募集中性粒细胞的主要信号来源。当TB疾病受到限制但当Mtb复制控制不佳或当存在降低MΦ坏死阈值的条件时，中性粒细胞可能增强宿主防御，中性粒细胞可能积累超过清除能力并原位死亡。这建立了由嗜中性粒细胞衍生的水解酶介导的进行性炎症和肺组织损伤的前馈机制。在此基础上，我们计划鉴定Mtb触发MΦ坏死所需的细菌基因，并鉴定与MΦ坏死相关的导致肺中TB疾病部位中性粒细胞积聚的特异性信号。我们的初步数据表明，Mtb PhoPR调节子中的一个或多个基因是这种高细菌负荷“爆发大小”MΦ细胞溶解所需的。在目标1中，我们将利用这一发现，通过系统地测试候选PhoPR调控基因的缺失突变体的细胞溶解功能的丧失。这些突变体将与Christopher Sassetti博士（马萨诸塞大学）合作，使用最先进的重组工程方法产生。作为一种补充方法，我们将对Mtb转座子文库进行无偏筛选，以确定细胞溶解功能表型的丧失。目的2研究将MΦ坏死与中性粒细胞募集联系起来的信号传导机制，在此利用关于白三烯B4和12/15-脂氧合酶在将中性粒细胞募集到细胞损伤的局部位点中的作用的新知识。目的3在小鼠气溶胶TB模型中以及通过与陈新春博士（深圳市第三人民医院和深港传染病研究所）合作获得的肺TB患者的支气管肺泡灌洗液和细胞样品中，研究嗜酸性炎症和组织损伤之间的关系。基于Mtb诱导的MΦ坏死促进中性粒细胞运输到肺以及中性粒细胞是组织损伤的主要介质的相关假设，该项目有可能揭示病原体和宿主定向治疗的新靶点，以限制TB中的嗜中性粒细胞炎症和肺损伤。

 DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a leading cause of death from infection worldwide. The interaction between Mycobacterium tuberculosis (Mtb) and host macrophages (MΦ) lies at the heart of TB pathogenesis. Our preliminary data indicate that virulent Mtb suppresses a host-protective apoptotic response of infected MΦ in order to use these cells as a protected replication niche. After growing to an optimal intracellular bacillary load, Mtb then induces MΦ necrosis that allows bacteria to escape and spread to naïve phagocytes. We propose that infection-induced MΦ necrosis is the predominant source of signals that recruit neutrophils to TB lesions. Neutrophils likely enhance host defense when TB disease is constrained but when Mtb replication is poorly controlled or when conditions exist that reduce the threshold for MΦ necrosis, neutrophils may accumulate in excess of clearance capacity and die in situ. This establishes a feed-forward mechanism for progressive inflammation and lung tissue damage mediated by neutrophil-derived hydrolases. On this basis we plan to identify the bacterial gene(s) required by Mtb to trigger MΦ necrosis and to identify the specific signals linked to MΦ necrosis that lead to the accumulation of neutrophils at sites o TB disease in the lung. Our preliminary data suggest that one or more genes in the Mtb PhoPR regulon are required for this high bacterial load "burst size" MΦ cytolysis. In Aim 1 we will leverage that finding by systematically testing deletion mutants of candidate PhoPR-regulated genes for loss of cytolytic function. These mutants will be generated using state of the art recombineering methods in collaboration with Dr. Christopher Sassetti (UMass). As a complementary approach we will perform an unbiased screen of an Mtb transposon library for the loss of cytolytic function phenotype. Aim 2 investigates the signaling mechanisms linking MΦ necrosis to neutrophil recruitment, here leveraging new knowledge about the role of leukotriene B4 and 12/15-lipoxygenase in recruiting neutrophils to local sites of cell damage. Aim 3 investigates the relationship between neutrophilic inflammation and tissue injury in the mouse aerosol TB model and in samples of bronchoalveolar lavage fluid and cells obtained from pulmonary TB patients made available through collaboration with Dr. Xinchun Chen (Shenzhen Third People's Hospital and Shenzhen-Hong Kong Institute for Infectious Diseases). Based on the linked hypotheses that Mtb-induced MΦ necrosis promotes neutrophil trafficking to the lung and that neutrophils are dominant mediators of tissue injury, this project has the potential to reveal new targets for pathogen and host-directed therapies to limit neutrophilic inflammation and pulmonary impairment in TB.