SELECTIVE DELETION OF NEUTROPHIL NADPH OXIDASE AND INNATE RESPONSES TO ASPERGILLUS FUMIGATUS

摘要 由于吸入机会霉菌烟曲霉(Aspergillusfumigatus，AF)的孢子而导致的肺炎仍然是一种生命- 慢性肉芽肿性疾病(CGD)和其他病情的威胁并发症 先天的抗真菌免疫力。CGD是一种遗传性免疫缺陷，由失活的 吞噬细胞NADPH氧化酶产生超氧化物(O2-)1，2缺乏杀菌活性O2-衍生的 氧物种(ROS)会导致反复的细菌和真菌感染。CGD患者也容易患上 过度和有害的炎症。CGD中的肺炎曲霉菌与脓性肉芽肿相关 使治疗复杂化的炎症。分生孢子被吸入后，真菌病原体的宿主受体- 相关分子模式(PAMP)通过以下方式触发炎症介质的产生和激活杀伤 巨噬细胞、中性粒细胞(中性粒细胞，PMN)和其他白细胞，迅速 消除正常宿主的房颤。如果不这样做，就会导致房颤肺炎。尽管患有CGD和 具有类似突变的基因工程小鼠(CGD小鼠)是最敏感的，我们的 了解NADPH氧化酶ROS如何控制房颤及其相关的肺部炎症 已定义。为了解决这个问题，我们开发了新的小鼠模型，在这些模型中，NADPH氧化酶是 在PMN或常驻肺泡巨噬细胞(AM)中选择性删除。我们的初步数据显示，老鼠 缺乏PMN NADPH氧化酶的ROS表现出与CGD小鼠相似的表型，并且对 挑战无菌真菌细胞壁后的房颤肺炎和炎症亢进，尽管完好无损 NADPH氧化酶在其他细胞中的活性。相反，小鼠AM中缺乏NADPH氧化酶，但有残留 在这些研究中，大多数中性粒细胞和单核细胞中的氧化酶活性与WT小鼠相似。我们建议确定 PMN NADPH氧化酶是如何在房颤的早期先天反应中起关键作用的，包括在 调节真菌PAMP引起的炎症。在目标1中，我们将评估PMN ROS是否扮演了非 在房颤初始反应中的多余作用，这对预防肺部感染至关重要，尽管有可能 在低接种量时，其他来源的氧化酶、ROS或非氧化机制就足够了。在目标2中，我们将 确定PMN NADPH氧化酶如何调节真菌PAMP诱导的炎症。我们假设缺乏 导致PMN ROS过度释放炎性细胞因子，包括PMN产生的趋化因子 PMN本身。我们将在体外检测缺乏氧化酶的PMN对无菌真菌PAMPs的反应 在肺中，以确定PMN ROS的丢失如何独立于主动感染而加剧炎症。 我们将研究阻断特定炎症介质在真菌细胞壁攻击后的影响。如果 有效，我们将在CGD小鼠身上评估这种方法作为一种减少过度炎症和 加强对房颤的控制。拟议的研究将为不完全表征的因素提供新的见解 导致危及生命的房颤感染，并指导新的治疗策略。

Summary Pneumonia resulting from inhaling spores of the opportunistic mold Aspergillus fumigatus (AF) remains a life- threatening complication of chronic granulomatous disease (CGD) and other conditions with compromised innate antifungal immunity. CGD is a heritable immunodeficiency arising from inactivating mutations in the phagocyte NADPH oxidase that generates superoxide (O2-).1, 2 Lack of microbicidal O2--derived reactive oxygen species (ROS) leads to recurrent bacterial and fungal infections. CGD patients are also prone to excessive and detrimental inflammation. Aspergillus pneumonia in CGD is associated with pyogranulomatous inflammation that complicates treatment. After conidia are inhaled, host receptors for fungal pathogen- associated molecular patterns (PAMPs) trigger inflammatory mediator production and activate killing by macrophages, neutrophils (polymorphonuclear leukocytes, PMN) and other leukocytes, which rapidly eliminates AF from normal hosts. Failure to do so results in AF pneumonia. Although patients with CGD and mice genetically engineered with similar mutations (CGD mice) are among the most susceptible, our understanding of how NADPH oxidase ROS control AF and its associated inflammation in the lung are ill- defined. To address this question, we developed new mouse models in which the NADPH oxidase is selectively deleted in PMN or resident lung alveolar macrophages (AM). Our preliminary data show that mice lacking only PMN NADPH oxidase ROS exhibited a phenotype similar to CGD mice, and were susceptible to both AF pneumonia and hyperinflammation following challenge with sterile fungal cell walls, despite intact NADPH oxidase activity in other cells. In contrast, mice lacking NADPH oxidase in AM, but with residual oxidase activity in most PMN and monocytes, resembled WT mice in these studies. We propose to determine how the PMN NADPH oxidase is crucial in the early innate responses to AF, including an unexplained role in regulating fungal PAMP-induced inflammation. In Aim 1, we will assess whether PMN ROS play a non- redundant role in the initial response to AF that is critical to prevent lung infection, although it is possible that other sources of oxidase ROS or non-oxidative mechanisms could suffice at low inocula. In Aim 2, we will determine how PMN NADPH oxidase regulates fungal PAMP-induced inflammation. We hypothesize that lack of PMN ROS leads to excessive release inflammatory cytokines, including PMN chemoattractants produced by PMN themselves. We will examine the response of oxidase-deficient PMN to sterile fungal PAMPs both in vitro and in the lung to determine how loss of PMN ROS exacerbates inflammation independent of active infection. We will investigate the impact of blocking specific inflammatory mediators after fungal cell wall challenge. If effective, we will evaluate this approach in CGD mice as a means to reduce excessive inflammation and improve control of AF. The proposed studies will provide novel insights into incompletely characterized factors that contribute to life-threatening infection with AF and guide new therapeutic strategies.