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

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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.