The long-term goal of this project is to understand the role of type I, semi-invariant natural killer T (NKT) cells in generating mucosal immunity against respiratory infectious diseases. NKT cells are innate-like lymphocytes that, by recognizing microbial glycolipids or microbe-induced self lipids, assert a role in infectious diseases. NKT cells are disproportionately enriched in the lung mucosa, but the role of these T cells in respiratory infectious diseases is not fully understood. The lung mucosa is a major site for Francisella tularensis (Ft) invasion that oft-time causes a fatal infectious disease known as pulmonary tularemia. Ft infections of the skin and intestine can spread systemically, especially when left untreated, and cause pulmonary tularemia. Tularemia is an inflammatory, sepsis-like disease. Consistent with a role for NKT cells in pro-inflammatory diseases and sepsis, we discovered that Ft subspecies holarctica-derived live vaccine strain (LVS) quickly activated lung interstitial NKT cells and induced runaway inflammation. Thus, LVS-infected immune competent mice became severely morbid and succumbed to a tularemia-like disease. Accordingly, NKT cell-deficient mice recovered from disease and lived despite similar bacterial burden in the lungs of both mutant and wild type mice. Survival was likely because NKT cell-deficient mice had developed the protective induced bronchus- associated lymphoid tissue (iBALT) at the peak of infection. Further, the iBALT-suppressing regulatory T cells are enriched in wild type mice, whilst the iBALT-inducing IL-17-producing cells, potentially mucosa-associated invariant T cells (MAIT), are enriched in NKT cell-deficient mice, and vice versa. These new data suggest that a Treg to IL-17-producing MAIT cell imbalance underlie tularemia-like disease caused by LVS infection. Guided by these findings, we hypothesize that an Ft-derived glycolipid agonist(s) activates NKT cells in the lungs, stirring up an inflammatory milieu that prevents iBALT formation and, thereby, causing fatal tularemia- like disease in mice. To test this central hypothesis, we will elucidate the cellular and molecular mechanisms that underlie the presentation of the cytoplasmic glycolipid agonists to NKT cells and cause tularemia-like disease during a natural Ft infection (Aims 1 and 2). Further, we will elucidate and validate the chemical structure of the LVS and the type A Ft-derived glycolipid agonist(s) (Aim 3). These proposed studies will rigorously test the prevailing assumption that DCs are critical for presenting CD1d-restricted glycolipid agonists and activating NKT cells during a natural bacterial infection. We expect to ...