Regulation of IFN-gamma production in human tuberculosis

描述(申请人提供)：干扰素(干扰素)-伽马是抗结核分枝杆菌和其他细胞内病原体免疫的核心。免疫无效的结核病患者对分枝杆菌抗原的反应会产生较少的干扰素-γmRNA和蛋白质，但这些异常的机制尚不清楚。我们的初步数据表明，cAMP反应元件结合蛋白/激活转录因子/激活蛋白-1(CREB/ATF/AP-1)家族的转录因子通过与干扰素-γ近端启动子结合来调节IFN-γ的产生。我们的目标是了解CREB/ATF/AP-1转录因子调节干扰素-γ产生以应对结核分枝杆菌的机制。我们的具体目标是：1.利用微亲和纯化和染色质免疫沉淀，在体内鉴定与结核分枝杆菌应答的CD4+和CD8+T细胞中的干扰素-γ近端启动子结合的转录因子；2.利用细胞内抗体传递和转录因子的siRNA，评价CREB/ATF/AP-1和其他转录因子中和对结核分枝杆菌诱导的CD4+和CD8+T细胞表达干扰素-γ的影响；3.用免疫共沉淀和染色质免疫沉淀及Western blotting鉴定与CD4+和CD8+细胞的干扰素-γ近端启动子结合的转录复合体(增强体)的动力学和蛋白质-蛋白质相互作用。4.了解结核患者CREB/ATF/AP-1转录因子异常表达的机制。在治疗期间，将测定结核病患者PBMC T细胞亚群中的转录因子水平。我们将确定暴露于免疫抑制细胞因子或结核分枝杆菌感染的巨噬细胞是否会影响CREB/ATF/AP-1的表达。结核病人T细胞CREB/ATF/AP-1水平将通过病毒表达载体和核重组蛋白的表达而增加，其对干扰素-γ产生的影响将被确定。

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.