This R01 renewal application seeks to extend our initial observation that the cytosolic RNA-sensing pattern- recognition receptor MDA5 plays a critical role in regulating the alveolar macrophage-mediated host resistance against the human fungal pathogen Aspergillus fumigatus. Mechanistic understanding of how alveolar macrophages keep fungal infections at bay in immune competent individuals remains ill-defined. Currently, there is a critical gap in understanding how the early host-pathogen interactions between fungi and alveolar macrophages work to drive fungal clearance and host resistance. Our data from the last funding period demonstrate a novel role of live fungal conidia in triggering the cytosolic RNA-sensing MDA5 receptor specifically in alveolar macrophages to initiate the host protective type I and type III interferon response. Thus, our central hypothesis is the lung specific programing of alveolar macrophages enables them to sense multiple vitality features of Aspergillus fumigatus isolates to serve as a central hub of inflammation and host resistance against this menacing fungal pathogen. In SA1 we identify features of alveolar macrophages that enable the effective type I and type III interferon response which are necessary for host resistance against A. fumigatus. This will be done using the adoptive transfer of a novel transgenic fetal-liver derived alveolar macrophage technology developed in our Co-I’s laboratory to our MavsCd11c conditional knock-out mouse line, which has been shown have a critical defect in host resistance against Aspergillus fumigatus. In SA2 we will mechanistically follow-up on our observation that there are fungal dsRNA-dependent and fungal dsRNA- independent mechanisms for triggering MDA5/MAVS activation. This will be done using novel isogenic pairs of Aspergillus fumigatus that are infected or not with a dsRNA mycovirus, as well as Aspergillus fumigatus mutants defective in mycotoxin production to identify the fungal effectors triggering MDA5/MAVS-dependent inflammatory responses. Overall, this research fills a critical knowledge gap regarding the molecular mechanisms of the host- pathogen interaction occurring in the lungs to drive the protective antifungal MDA5-dependent type I and type III interferon response induced by Aspergillus fumigatus in alveolar macrophages in both mice and humans.