INITIATION OF THE IMMUNE RESPONSE TO ASPERGILLUS FUMIGATUS

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PROJECT SUMMARY Invasive aspergillosis is devastating fungal infection and the most common form of mold pneumonia worldwide, with an estimated 200,000 cases annually. Aspergillus fumigatus, the most common etiologic agent of invasive aspergillosis, forms ubiquitous airborne spores that humans inhaled on daily basis. In immune competent individuals the respiratory innate immune system prevents the formation of tissue-invasive hyphae, a critical immunologic checkpoint. In patients with hematologic malignancies, in bone marrow and lung transplant recipients, and recently, in intensive care unit patients with COVID-19, numeric or functional defects in innate immune function lead to invasive disease. Despite contemporary antifungal drugs, mortality rates remain at 20- 40% in high risk groups, underscoring the need for improved understanding of the molecular and cellular basis of sterilizing immunity to advance immune-based adjunctive approaches. In the second funding period, we harnessed a fungal bioreporter that reports the mode of cell death to discover that neutrophils and monocyte-derived dendritic cells induce a regulated cell death in engulfed fungal cells. The concept that a higher eukaryote can exploit a regulated cell death machinery in a lower eukaryote is novel and, in the case of A. fumigatus, depends on host NADPH oxidase activity. NADPH oxidase-dependent fungal killing is modulated by two novel, essential intercellular crosstalk circuits that involves the early production of GM-CSF (GM-CSF circuit) and plasmacytoid dendritic cells (pDC circuit). During the next project period, we propose to gain a deeper understanding of the GM-CSF and pDC circuits. In Aim 1, we identify the essential cellular source of GM-CSF and, based on preliminary data, focus on pulmonary endothelial and epithelial cells as regulators of neutrophil-dependent fungal killing. In Aim 2, we define the pDC circuit and candidate transmitters and test models of direct or indirect activation by fungal cells or the lung inflammatory milieu. In Aim 3, we define the mechanisms by which the pDC circuit regulates neutrophils, and test its role in NAPDH oxidase assembly, activation, and neutrophil metabolism via the pentose phosphate pathway and its cooperativity with the GM-CSF circuit to mediate sterilizing immunity. The proposed studies are significant and innovative because they integrate innate immune crosstalk between the pulmonary endothelial, epithelial, and pDC compartments and infected myeloid phagocytes into a comprehensive model of respiratory immunity against mold pathogens. Understanding the induction, regulation, and participants of innate immune crosstalk addresses a critical knowledge gap that will inform immune-enhancing strategies in vulnerable patient groups.