Regulation of IFN-gamma production in human tuberculosis

描述（由申请方提供）：干扰素（IFN）-γ是抗结核分枝杆菌和其他细胞内病原体免疫的核心。免疫力低下的结核病患者对分枝杆菌抗原产生的IFN-γ mRNA和蛋白量减少，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IFN-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IFN-γ产生的机制。结核我们的具体目标是：1.在对M应答的CD 4+和CD 8 + T细胞中，鉴定体内与IFN-γ近端启动子结合的转录因子。结核病，使用微亲和纯化和染色质免疫沉淀; 2.评估CREB/ATF/AP-1等转录因子对M.使用针对转录因子的细胞内抗体递送和siRNA，通过CD 4+和CD 8 + T细胞的结核病诱导的IFN-γ基因表达; 3.使用免疫共沉淀和染色质免疫沉淀，然后进行Western印迹，表征与CD 4+和CD 8+细胞的IFN-γ近端启动子结合的转录复合物（增强体）的动力学和蛋白质-蛋白质相互作用。4.了解肺结核患者CREB/ATF/AP-1转录因子异常表达的机制。将在治疗期间测定结核病患者PBMC T细胞亚群中的转录因子水平。我们将确定是否暴露于免疫抑制细胞因子或M。结核感染的巨噬细胞影响CREB/ATF/AP-1的表达。将通过病毒表达载体和核转染增强结核病患者T细胞中的CREB/ATF/AP-1水平，并将确定对IFN-γ产生的影响。

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.