Regulation of IFN-gamma production in human tuberculosis

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DESCRIPTION (provided by applicant): Interferon (IFN)-gamma is central to immunity against Mycobacterium tuberculosis and other intracellular pathogens. Tuberculosis patients with ineffective immunity produce reduced amounts of IFN-gamma mRNA and protein in response to mycobacterial antigens, but the mechanisms underlying these abnormalities are unknown. Our preliminary data indicate that transcription factors of the cAMP response element-binding protein/activation transcription factor/activator protein-1 (CREB/ATF/AP-1) family regulate production of IFNgamma by binding to the IFN-gamma proximal promoter. Our goal is to understand the mechanisms by which CREB/ATF/AP-1 transcription factors regulate IFN-gamma production in response to M. tuberculosis. Our specific aims are: 1. Identify transcription factors that bind to the IFN-gamma proximal promoter in vivo in CD4+ and CD8+ T-cells that respond to M. tuberculosis, using microaffinity purification and chromatin immunoprecipitation; 2. Evaluate the effects of neutralization of CREB/ATF/AP-1 and other transcription factors on M. tuberculosis-induced IFN-gamma gene expression by CD4+ and CD8+ T-cells, using intracellular antibody delivery and siRNAs to transcription factors; 3. Characterize the kinetics and protein-protein interactions of the transcriptional complex (enhanceosome) that binds to the IFN-gamma proximal promoter of CD4+ and CD8+ cells, using coimmunoprecipitation and chromatin immunoprecipitation, followed by Western blotting. 4. Understand the mechanisms that mediate aberrant expression of CREB/ATF/AP-1 transcription factors in tuberculosis patients. Transcription factor levels will be determined in PBMC T-cell subpopulations in tuberculosis patients during treatment. We will determine if exposure to immunosuppressive cytokines or to M. tuberculosis-infected macrophages affects CREB/ATF/AP-1 expression. CREB/ATF/AP-1 levels in T-cells of tuberculosis patients will be enhanced by viral expression vectors and nucleofection, and the effects on IFN-gamma production will be determined.