Dysregulation of the inflammatory response by Francisella tularensis

描述（由申请人提供）： 土拉菌病是由兼性胞内细菌土拉弗朗西斯菌引起的一种潜在致命的人畜共患病，吸入10种微生物就足以引起严重的肺炎、组织坏死和死亡。中性粒细胞（多形核白细胞，PMNs）在肺中的积聚对于感染该病原体的动物的严重疾病的发展是必不可少的，并且随着更多的肺泡和细支气管被感染的PMNs和碎片堵塞，组织破坏稳步进行。中性粒细胞有助于兔热病进展和发病机制而不是有效的宿主防御的直接证据被以下事实证明：阻断PMN流入肺是保护性的，并且动物在否则将是致命感染的情况下存活。然而，大多数对F.土拉热的研究集中在这种生物在巨噬细胞中的命运上。我们的长期目标是详细确定人类中性粒细胞在兔热病发病机制中的作用。为此，我们有了几个重要的发现。我们已经表明，天然IgM是F的调理作用所必需的。土拉菌感染，并鉴定了介导嗜中性粒细胞和巨噬细胞感染的受体。我们发现，逃避氧化宿主防御是通过能力的F。tularensis在多个水平上破坏NADPH氧化酶的组装和活性，并确定了相关的毒力因子。我们也是第一个证明F。土拉热菌可以逃脱吞噬体并在嗜中性粒细胞胞质溶胶中复制，证实了其成功感染多种吞噬细胞类型的能力。特别相关的是我们发现了PMN特异性的毒力。嗜中性粒细胞是通过组成性细胞凋亡预先编程而死亡的短寿命细胞，组成性细胞凋亡是一种通常通过吞噬作用加速的过程，并且对于炎症反应的消退至关重要。与此相反，我们发现F.土拉热菌抑制PMN凋亡并显著延长细胞寿命，这部分是通过 对中性粒细胞基因表达的影响这些数据是值得注意的，因为PMN周转的缺陷指示无效和失调的炎症反应。死亡的中性粒细胞及时清除巨噬细胞是必不可少的，以防止坏死和组织损伤，我们的初步数据表明，这一过程也可能受到损害。鉴于这些数据，我们假设F。土拉热通过影响抗凋亡基因和促凋亡基因以及促存活因子的特定子集的表达来抑制PMN凋亡，并且巨噬细胞对受感染的PMN的清除缺陷有利于细胞坏死、维持感染并阻止终止炎症反应所需的巨噬细胞的重编程。为了验证这一点，我们提出了以下具体目标：1。探讨F.土拉菌，重点是BAX，XIAP和钙蛋白酶抑制蛋白。2.目的：鉴定细菌中抑制中性粒细胞凋亡所需的基因。3.阐明中性粒细胞寿命延长的功能后果。我们希望完成拟议的研究将提供基本的洞察到的分子机制，占失调的炎症反应是兔热病的特点，我们的研究结果也将通知其他疾病的研究，也是由PMN周转缺陷的特点，并影响退伍军人更频繁，如慢性阻塞性肺疾病。

DESCRIPTION (provided by applicant): Tularemia is a potentially fatal zoonosis of humans caused by the facultative intracellular bacterium, Francisella tularensis, and inhalation of as few as ten organisms is sufficient to cause severe pneumonic disease, tissue necrosis, and death. Accumulation of neutrophils (polymorphonuclear leukocytes, PMNs) in the lungs is essential for development of severe disease in animals infected with this pathogen, and tissue destruction progresses steadily as more alveoli and bronchioles become clogged with infected PMNs and debris. Direct evidence that neutrophils contribute to tularemia progression and pathogenesis rather than effective host defense is demonstrated by the fact that blockade of PMN influx into the lung is protective, and animals survive what would otherwise be a lethal infection. Nevertheless, most studies of F. tularensis have focused on the fate of this organism in macrophages. Our long-term goal is to define in detail the role of human neutrophils in the pathogenesis of tularemia. To this end we have made several important discoveries. We have shown that natural IgM is required for opsonization of F. tularensis and identified receptors that mediate infection of both neutrophils and macrophages. We discovered that evasion of oxidative host defense is achieved via the ability of F. tularensis to act at multiple levels to disrupt NADPH oxidase assembly and activity, and identified relevant virulence factors. We were also the first to show that F. tularensis can escape the phagosome and replicate in neutrophil cytosol, confirming its ability to successfully infect multiple phagocytes types. Particularly relevant here is our discovery of PMN-specific aspects of virulence. Neutrophils are short-lived cells that are preprogrammed to die by constitutive apoptosis, a process that is typically accelerated by phagocytosis and is essential for resolution of the inflammatory response. In marked contrast, we find that F. tularensis inhibits PMN apoptosis and significantly prolongs cell lifespan, and this is achieved, in part, via effects on neutrophil gene expression. These data are noteworthy as defects in PMN turnover are indicative of an ineffective and dysregulated inflammatory response. Timely clearance of dying PMNs by macrophages is essential to prevent necrosis and tissue damage, and our preliminary data suggest that this process may also be impaired. In view of these data we hypothesize that F. tularensis inhibits PMN apoptosis by affecting expression of a specific subset of anti- and proapoptotic genes and pro-survival factors, and that defects in clearance of infected PMNs by macrophages favors cell necrosis, sustains infection, and prevents the reprogramming of macrophages that is required for termination of the inflammatory response. To test this, we propose the following Specific Aims: 1. To elucidate the molecular mechanisms of apoptosis inhibition by F. tularensis with a focus on the BAX, XIAP and calpastatin. 2. To identify bacterial genes required for PMN apoptosis inhibition. 3. To elucidate the functional consequences of prolonged neutrophil lifespan. We expect that completion of the proposed studies will provide fundamental insight into the molecular mechanisms that account for the dysregulated inflammatory response that is characteristic of tularemia, and that our findings will also inform studies of other diseases that are also characterized by defects in PMN turnover, and affect Veterans more frequently, such as chromic obstructive pulmonary disease.