Legionella pneumophila and Mycobacterium tuberculosis, the etiologic agents of Legionnaires' disease and tuberculosis respectively, are facultative intra-cellular pathogens which parasitize human alveolar macrophages and blood monocytes recruited to the site of lung infection. An effective host response to these infections depends on cell-mediated immunity, and cytokine networks modulate this response. The overall hypothesis driving this proposal is that L. pneumophia and M. tuberculosis subvert the human alveolar macrophage cytokine response to facilitate their own survival by inducing permissive host cytokines which disarm the protective antimicrobial mechanisms of the phagocyte.
The specific aims are: 1) to define the role of cytokine activation of human alveolar macrophages in determining the tolerance or elimination of intracellular infection by L. pneumophila and M. tuberculosis; 2) to determine whether apoptosis of infected alveolar macrophages diminishes the viability of intra-cellular L. pneumophila and M. tuberculosis; 3) to develop and use a virulent L. pneumophila strain transduced with the A victoria green fluorescent protein gene to co-localize intracellular bacteria and specific components of the human alveolar macrophage host response in infected cells. The experimental approach to the specific aims will use an established in vitro model in which human alveolar macrophages are infected with L. pneumophila or M. tuberculosis. Effects of cytokines on intracellular growth will be measured using selected combinations of recombinant cytokines and blocking antibodies. Specific interactions signaling cytokine induction by live bacteria and cell wall glycolipids will be assessed by blocking complement receptors, CD14, and LBP. Macrophage apoptosis will be assessed by cellular morphology, Annexin V binding, and by DNA fragmentation assays, and associated changes in bacterial viability will be quantitated. Individual cell response by infected cells will be assessed by flow cytometry and fluorescent microscopy co-localization of green fluorescent protein expressing bacterial strains with selected markers of host defense. These studies will provide important insights into the human host responses to L. pneumophila and M. tuberculosis infections. A thorough understanding of these responses will be necessary for the development of rational immunotherapeutic approaches to the treatment of Legionnaires' disease and tuberculosis, which will be increasingly important for the treatment of immunocompromised patients and infections with drug-resistant strains.
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