Dysregulation of the inflammatory response by Francisella tularensis

描述(由申请人提供)： 图拉热症是一种潜在的致命人畜共患病，由兼性细胞内细菌图拉氏菌引起，吸入10种微生物就足以导致严重的肺病、组织坏死和死亡。中性粒细胞(中性粒细胞，PMN)在肺部的积累对于感染这种病原体的动物的严重疾病的发展是必不可少的，随着更多的肺泡和细支气管会被感染的PMN和碎片堵塞，组织破坏稳步进行。中性粒细胞有助于图拉热病的进展和发病，而不是有效的宿主防御，这一直接证据表明，阻止PMN流入肺具有保护性，动物可以在否则会是致命感染的情况下存活下来。然而，大多数对图拉氏丝虫的研究都集中在这种微生物在巨噬细胞中的命运。我们的长期目标是详细确定人类中性粒细胞在图拉热症发病机制中的作用。为此，我们有了几个重要的发现。我们已经证明了天然的IgM对于图拉氏丝虫的调理作用是必需的，并确定了介导中性粒细胞和巨噬细胞感染的受体。我们发现土拉氏F菌通过在多个水平上破坏NADPH氧化酶的组装和活性来逃避氧化寄主防御，并确定了相关的毒力因子。我们还首次证明图拉氏原虫可以逃脱吞噬小体，并在中性粒细胞胞浆中复制，证实了其成功感染多种吞噬细胞类型的能力。这里特别相关的是我们发现了PMN毒力的特定方面。中性粒细胞是一种短命的细胞，它们被预先编程为通过结构性凋亡而死亡，这一过程通常被吞噬作用加速，对于消解炎症反应是必不可少的。与之形成鲜明对比的是，我们发现图拉氏丝菌抑制了PMN的凋亡并显著延长了细胞寿命，这在一定程度上是通过 对中性粒细胞基因表达的影响。这些数据值得注意，因为PMN周转缺陷表明炎症反应无效和失调。巨噬细胞及时清除濒临死亡的中性粒细胞是防止坏死和组织损伤的关键，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，我们假设图拉氏F菌通过影响一组特定的抗和促凋亡基因以及促生存因子的表达来抑制PMN的凋亡，并且巨噬细胞清除感染的PMN的缺陷有利于细胞坏死，持续感染，并阻止终止炎症反应所需的巨噬细胞的重新编程。为了验证这一点，我们提出了以下具体目标：1.阐明图拉夫酵母抑制细胞凋亡的分子机制，重点是Bax、XIAP和calastatin。2.寻找抑制中性粒细胞凋亡所需的细菌基因。3.阐明中性粒细胞寿命延长的功能后果。我们预计，拟议研究的完成将为解释以图拉热症为特征的失调炎症反应的分子机制提供基本的见解，我们的发现也将为其他疾病的研究提供参考，这些疾病的特征也是PMN周转缺陷，并更频繁地影响退伍军人，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.