The Impact of SARS-CoV-2 Immune Dysregulation on Antifungal Immunity

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Project summary Unlike most invasive mold infections, COVID Associated Pulmonary Aspergillosis (CAPA) occurs in individuals with otherwise intact immune systems suggesting novel biological mechanisms mediating susceptibility to fungal infection. This proposal seeks to evaluate the novel conceptual advancement that immune responses targeting intracellular viral pathogens compromise essential human neutrophil antifungal effector functions required to kill large extracellular fungal hyphae. In Aim 1, we will utilize BAL samples and autopsy formalin-fixed paraffin embedded (FFPE) lung tissues to characterize the expression of human neutrophil antifungal effectors, epithelial cell necroptosis, iron content, and fungi in the respiratory tract during SARS-CoV-2 and CAPA infection. In Aim 2, we will use primary bronchiolar epithelial cell air-liquid interface (ALI) cultures, CRISPR knockout cell lines (MLKL, RIPK1, RIPK3), chemical inhibitors of cell death pathways, and neutrophil:fungal killing assays to determine the mechanism by which SARS-CoV-2 mediates epithelial cell death, iron release, mold growth, and human neutrophil antifungal effector functions. In Aim 3, we will utilize a novel CAPA infection mouse model, cre-lox knockdown of human ACE2 expression in neutrophils (MRP8-Cre) and lung epithelial cells (CC10-Cre) along with iron-deficient fungal mutant strains (ΔsidA, ΔftrA), to evaluate the in vivo impact of SARS-CoV-2 infection on lung epithelial cell necroptosis, iron release, siderophore-dependent fungal growth, and neutrophil recruitment/activation. We believe that the results of the proposed study will shed new light on the fundamental biology mediating viral-associated secondary mold infection which may enable the development of improved therapeutic regimens that mitigate the risk of developing CAPA.