Non-oxidative Resistance to A. fumigatus by Alveolar Macrophages

描述(由申请人提供)：由烟曲霉引起的感染是免疫功能受损的人死亡的主要原因。烟曲霉菌也对那些缺乏功能性NADPH氧化酶系统的人构成威胁，NADPH氧化酶系统是吞噬免疫细胞的防御性氧化剂生成系统。肺泡巨噬细胞(AM)是已知在抵抗烟曲霉分生孢子的早期天然防御中重要的吞噬细胞，已有报道称它能产生氧化剂来杀死参与其中的病原体。然而，关于AM杀死烟曲霉菌的分子机制的许多细节还不清楚。根据AM的抗真菌机制和NADPH氧化酶在这一过程中的作用的相互矛盾的数据，我们建议更好地定义AM保护免受烟曲霉菌感染的微生物杀菌机制。目前正在研究的总体假设是，AM对烟曲霉分生孢子的抗性是由NADPH酶不依赖的杀菌机制介导的，该机制涉及营养物质的隔离，导致分生孢子的死亡。为解决这一目标而提出的三个目标是：1)利用体外和体内分析，比较正常小鼠和缺乏功能性吞噬细胞氧化酶的gp91Phox-/-小鼠AM的分生孢子杀灭活性。关于AM的NADPH氧化酶在保护人类免受IPA侵袭中的作用的报道相互矛盾，我们的数据并未表明AM的呼吸爆发是抵抗曲霉病所必需的，正如我们已经证明中性粒细胞中的呼吸爆发一样。这些信息将有助于更好地理解NADPH氧化酶是如何应用于曲霉病免疫的。2)比较正常C57BL/6小鼠和gp91Phox-/-小鼠AM对NADPH氧化酶依赖的超氧化物歧化酶的释放，以描述这些小鼠AM产生氧化剂的能力。我们的初步数据表明，当由氧化剂触发的刺激剂或真菌颗粒的吞噬触发时，正常小鼠和gp91Phox-/-小鼠AM中的氧化剂产生没有显著差异。这些信息将提供更好的了解AM产生氧化剂的能力，以及3)比较正常C57BL/6小鼠AM暴露的分生孢子与低营养条件下的分生孢子的转录反应。这一目的将检验营养隔离是否是AM的一种分生孢子杀灭机制如果AM没有利用氧化杀灭分生孢子，那么应该通过内化分生孢子的转录反应来建议使用烟曲霉分生孢子作为AM分生孢子杀灭环境的生物探针。这一信息将有助于更好地了解AM杀死烟曲霉菌分生孢子的抗真菌机制。这些方法的长期目标是确定潜在的治疗靶点，以增强保护免受烟曲霉感染的自然免疫力。 公共卫生相关性：烟曲霉是一种吸入性真菌，如果免疫抑制，它可以感染各种类型的人类患者。目前，尽管进行了积极的医疗管理，但大多数感染了这种微生物的人都无法存活。这项工作的长期目标是确定使肺泡巨噬细胞能够杀死像孢子一样的烟曲霉分生孢子的分子机制，以便那些处于危险之中的人能够增强自然免疫力。

DESCRIPTION (provided by applicant): Infections caused by Aspergillus fumigatus are a leading cause of death in immune compromised individuals. A. fumigatus is also a threat to those lacking a functional NADPH oxidase system, a defensive oxidant-generating system of phagocytic immune cells. Alveolar macrophages (AM) are phagocytes known to be important in the early innate defense against A. fumigatus conidia, and have been reported to produce oxidants to kill engaged pathogens. However, many details about the molecular mechanisms by which AM kill A. fumigatus are not well understood. With conflicting data about the antifungal mechanisms of AM, and the role of NADPH oxidase in this process, we proposed to better define the microbicidal mechanism of AM that protects from infections by A. fumigatus. The overall hypothesis being investigated is that the AM-derived resistance to A. fumigatus conidia is mediated by NADPH oxidase-independent microbicidal mechanisms that involve nutrient sequestration, leading to conidial killing. Three aims proposed to address this goal are: 1) to compare the conidiacidal activity of AM from normal and gp91phox-/- mice that lack a functional phagocyte oxidase, using both in vitro and in vivo analyses. Reports on the role of NADPH oxidase of AM in protecting humans from IPA are conflicting, and our data do not indicate the respiratory burst in AM is necessary for a resistance to aspergillosis, as we have shown it is in neutrophils. This information will provide a better understanding of how the NADPH oxidase is applied in aspergillosis immunity. 2) to compare NADPH oxidase-dependent superoxide liberation by AM from normal C57Bl/6 and gp91phox-/- mice to describe the capabilities of oxidant production of AM in these mouse strains. Our preliminary data suggest that oxidant production in AM from normal and gp91phox-/- mice is not significantly different when triggered by oxidant-triggering stimulants, or by phagocytosis of fungal particles. This information will provide a better understanding of the capacity of oxidant generation by AM, and 3) to compare transcriptional responses of conidia exposed to AM from normal C57Bl/6 mice, to those in conidia subjected to low nutrient conditions. This aim will test whether nutrient sequestration is a conidiacidal mechanism of AM. If oxidative killing of conidia is not utilized by AM, then the use of A. fumigatus conidia as a bioprobe of the conidiacidal environment of the AM should be suggested by transcriptional responses of internalized conidia. This information will provide a better understanding of the antifungal mechanism of AM that kills A. fumigatus conidia. The long-term goal of these approaches is to identify potential therapeutic targets to augment the natural immunity that protects from infections by A. fumigatus. PUBLIC HEALTH RELEVANCE: Aspergillus fumigatus is an inhaled fungus that can infect human patients with various types if immune suppression. At this time, most individuals that become infected by this organism do not survive, despite aggressive medical management. The long- term goal of this work is to identify molecular mechanisms that enable alveolar macrophages to kill the spore-like conidial form of A. fumigatus, so that natural immunity can be augmented in those at risk.