PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) claims nearly 1.5 million lives each year and is one of the leading causes of death by an infectious agent worldwide. The lack of a protective TB vaccine for adult pulmonary disease, the length and toxicity of current antibiotic treatment regimens, and the rise in Mtb drug resistance all strongly motivate the pursuit of new host-directed therapies and strategies for vaccine design. Alveolar macrophages are the first cells in the lung to be infected with Mtb following aerosol transmission, performing a critical role as Mtb innate sentinels in the airway. They must initiate the host response that will recruit other innate cells into the lung and transfer bacteria to cells that can carry it to the draining lymph node for efficient immune priming. As tissue- resident myeloid cells, alveolar macrophages also perform a critical homeostatic function, clearing debris from the airway without triggering pulmonary inflammation. It is unknown how alveolar macrophages balance their innate sensing and tissue maintenance duties, while any delay in the initiation of the host response to Mtb provides the bacteria with additional time to replicate unchecked. Our previous results showed that Mtb-infected alveolar macrophages up-regulate a cell-protective signature, dependent on the transcription factor Nrf2. This in vivo response is distinct from the canonical pro-inflammatory response previously reported for Mtb-infected macrophages in vitro indicating that new information that can be gained from this in vivo approach. Our results showed that in the absence of Nrf2, Mtb-infected alveolar macrophages are more activated, less viable, and demonstrate enhanced control of bacteria within the first 10 days of infection, yet it is unknown how Nrf2 regulation of alveolar macrophage function early during infection impacts the subsequent stages of disease progression. The goal of this proposal is to develop diverse tools to modulate alveolar macrophage Nrf2 expression in order to define how Nrf2 impacts the timing and quality of the immune events following bacterial deposition in the airway, which ultimately lead to either disease progression or bacterial containment. First, we will characterize Nrf2 regulation of alveolar macrophage cell death and bacterial dissemination using myeloid- specific Nrf2 conditional knock-out strains (Aim 1). Second, we will develop an ex vivo system to study how Nrf2 interferes with alveolar macrophage innate sensing pathways and an shRNA lentiviral oropharyngeal delivery system to transiently block alveolar macrophage Nrf2 expression in vivo, allowing us to avoid any confounding effects of Nrf2 during development (Aim 2). The studies are driven by the hypothesis that the induction of a cell- protective program by Nrf2 prevents alveolar macrophages from mounting a rapid and effective pro-inflammatory response to Mtb infection, leading to delays in subsequent immune events that impair the host fr...