This R01 application explores the novel role the cytosolic RNA-sensing pattern-recognition receptor MDA5 plays in mediating host resistance against the human fungal pathogen Aspergillus fumigatus. Our understanding of how the immune system keeps fungal infections at bay in immune competent individuals remains ill-defined. Currently, there is a critical gap in understanding the early interactions between fungal conidia and tissue-resident phagocytes that are necessary for fungal clearance and host resistance. Our data demonstrate a novel role of fungal conidia in triggering the cytosolic RNA-sensing MDA5 receptor and initiating an IL28/IFNλ and CXCL10-CXCR3 inflammatory cascade which is necessary for host resistance in response to Aspergillus fumigatus across a wide array of fungal isolates. Thus, our central hypothesis is that host resistance pathways targeting A. fumigatus conidia serve as central hubs of inflammation providing protective immunity against the broadest range of A. fumigatus isolates. In SA1 we examine how fungal conidia growth dynamics and resistance to phagocyte-mediated killing enables triggering of the MDA5/MAVS receptor. Importantly, we also examine the molecular mechanism(s) of how A. fumigatus conidia trigger this cytosolic pattern-recognition receptor pathway. In SA2 we identify the specific leukocyte subsets which require MAVS for IL28/IFNλ expression and host resistance following A. fumigatus challenge. This will be done using novel Mavs conditional knock-out mouse lines, specifically examining the role of MDA5/MAVS signaling in the cellular cross-talk between CCR2+ monocyte and neutrophil, which has been shown to be critical for maintaining host resistance against A. fumigatus. Finally, in SA3 we elucidate the role of the CXCL10-CXCR3 inflammatory axis in mediating neutrophil-dependent host resistance against A. fumigatus. Overall, this research fills a critical knowledge gap regarding the mechanisms of protective mucosal immunity against A. fumigatus conidia through the activation of a novel MDA5/MAVS and CXCL10-CXCR3 inflammatory cascade. These data, together with other published fungal immune-mediated resistance pathways, could be used to risk stratify patients based on their potential susceptibility to developing invasive aspergillosis. This personalized medicine approach could be used to predict patients that should undergo early, aggressive monitoring and treatment for fungal infections in order to drive better clinical outcomes for patients.