Tuberculosis (TB) is the leading cause of death from infection globally. Our knowledge of the cellular and molecular events that link inhalation of the causative bacterium, Mycobacterium tuberculosis (Mtb), with either clearance or productive infection remains limited. The type I interferon (IFN) response is among the first innate immune responses triggered in Mtb-infected macrophages. Growing evidence suggests that type I IFNs, and specifically cross-talk between the type I IFN response and the IL-1 axis, drive pathogenesis in TB. However, our understanding of the cascade of events that initiate the type I IFN response in Mtb-infected host cells is incomplete. The literature increasingly supports a model in which mitochondrial damage is a key driver of type I IFN production in Mtb-infected macrophages. In preliminary work, we have uncovered a set of previously unappreciated additional cellular pathways that contribute to Mtb-induced type I IFNs, including the ER stress response (ESR), lipid droplet (LD) formation, and eicosanoid production. In the proposed work, we will build upon our preliminary results to define molecular relationships between ER stress, LD formation, eicosanoid production, and mitochondrial damage in type I IFN response to Mtb. We will then use a murine model of infection to test the impact of modulating the ESR on infection outcomes. In Aim 1, we will use CRISPR technology to build genetic tools to study the pathways of interest. Using these tools and small molecule inhibitors, we will then test links between arms of the ESR, LD formation, eicosanoid production, and type I IFNs. To more completely characterize the role of the ESR and individual response pathways in the macrophage response to Mtb, we will additionally perform multiplexed cytokine analysis, transcriptional profiling, and metabolomics using our genetic and small molecule tools that perturb the ESR. In Aim 2, we will test which of the identified contributors to type I IFNs drive mitochondrial damage. In Aim 3, we will use small molecule inhibitors in two murine models of TB infection to determine how modulating the ESR in the context of TB infection changes bacterial burden, immune cell recruitment to the lung compartment, histopathology, cytokine responses, and the transcriptional response. Upon achieving our aims, we anticipate having developed a new, more complex model for induction of type I IFNs in Mtb-infected macrophages. Further, we anticipate having determined how the ESR shapes the macrophage response to Mtb infection and contributes to infection outcomes in vivo. We anticipate these results will ultimately inform the development of novel host directed therapies for TB.