The overall goal of this project is to define molecular pathways involved in the activation of innate immunity to airborne infection with gram negative bacteria that are potential biological weapons, including the agents of plague (Yersinia pestis), tularemia (Francisella tularensis) and melioidosis (Burkholderia pseudomallei). We will use aerosol challenge models in genetically modified mice to explore the roles of Toll-like receptors (TLRs) and cell populations in mediating inflammatory and immune responses to live bacteria and selected bacterial ligands. The central hypothesis is that TLR-mediated signaling is essential for the activation of innate immunity to Y. pestis and other Gram negative bacteria in the lungs. The specific aims are: Specific Aim 1. Determine the role of MyD88 in mediating innate immunity to aerosolized Y. pestis, F. tularensis, and B. pseudomallei. This aim will test the hypothesis that MyD88-deficient animals will fail to activate resident and recruited defenses, leading to accelerated bacterial replication in the lungs and early dissemination of infection in comparison with wild-type controls. Caspase 1 deficient animals also will be tested to evaluate the role of IL-1- and IL-18-mediated activation pathways that are signaled via MyD88 independently of TLRs. Specific Aim 2. Determine the role of TLR4 and TLR2 in mediating innate immunity to aerosolizcd Y. pestis. The first part of this aim will test the hypotheses that recognition of Y. pestis LPS is mediated by TLR4; that TLR4 has a role in activating host resistance to live Y pestis; that the LPS of Y. pestis grown at 37 degrees C is poorly recognized by human MD2/TLR4 in vivo; and that defective LPS recognition by human TLR4/MD2 contributes to blunted innate immune responses to aerosolized Y. pestis. The second part of this aim will test the hypotheses that the low calcium response virulence antigen (LcrV) of Y. pestis induces a TLR-2-dependent IL-10 response in vivo that suppresses innate immunity and permits accelerated bacterial replication and dissemination. Specific Aim 3. Determine the role of bone marrow-derived cells and respiratory epithelial cells in the activation of innate immune responses to Y. pestis. This aim will use bone marrow chimeras of MyD88-deficient and wild type mice and transgenic mice expressing a dominant negative IkB under the surfactant protein C promoter to test the hypothesis that both marrow-derived and parenchymal cells are involved in the activation of innate resistance to Y. pestis in the lungs.