Project Summary Intracellular bacterial pathogens such as Legionella pneumophila, the causative agent of Legionnaires’ Disease, are responsible for significant disease burden in the United States every year. Successful control of these pathogens is reliant on the inflammatory cytokine Tumor Necrosis Factor (TNF). TNF is responsible for the upregulation of innate immune activity, and directs the inflammatory response. Therapeutics targeting TNF signaling are common in treatment of inflammatory disorders such as rheumatoid arthritis or ulcerative colitis; however, these therapeutics carry with them an increased susceptibility to intracellular bacterial infection. TNF has proven a critical factor in defense against Legionella and other intracellular pathogens, and the molecular details of TNF signaling are well characterized. The downstream effects of TNF, however, are diverse – ranging from promoting cell survival to triggering programmed cell death – and thus the precise mechanisms through which TNF mediates anti-bacterial defense are still unclear. Our new preliminary data indicate that, upon Legionella infection, TNF is required for optimal inflammatory cytokine production in bone marrow-derived macrophage (BMDM) culture. The mechanism by which TNF promotes cytokine production during Legionella infection is unknown. Furthermore, we present new additional data showing that mice and cells deficient for the apoptotic caspase, Caspase-8, possess a defect in control of Legionella infection that cannot be rescued by exogenous rTNF treatment, suggesting that TNF’s downstream mechanism may be related to the induction of apoptosis of infected host cells. Thus, Aim 1 seeks to test the hypothesis that pro-survival and inflammatory signaling serves to upregulate the production of protective cytokines by infected cells. Aim 2 seeks to test the hypothesis that TNF-dependent apoptotic signaling in infected cells acts to restrict the replicative niche of the bacteria and thus limit growth and spread. This research will provide fundamental insight into how TNF mediates immune control of intracellular bacterial infection, and may provide a basis for the development of improved therapeutics that dampen pathological inflammation without increased risk of infection.