Signaling pathways that regulate IFN-g production in human tuberculosis.

调节人类结核病中 IFN-g 产生的信号通路。

• 批准号: 8308264
• 负责人: Veronica Edith Garcia
• 金额: $ 7.08万
• 依托单位: FACULTAD DE CIENCIAS EXACTAS Y NATURALES DE LA UNIVERSIDAD DE BUENOS AIRES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-27 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308264
• 关键词: 3' Untranslated Regions; AUF1A protein; Affect; Antigens; Bacteria; Binding; Biological Assay; Biological Models; Blood; CREB1 gene; Cells; Cessation of life; Chemicals; Coculture Techniques; Communicable Diseases; Data; Defect; Development; Disorder by Site; Effector Cell; Frequencies; Goals; Health; HuR protein; Human; Immune; Immune response; Individual; Interferon Type II; Interferons; Interleukin-17; Knowledge; Lead; Ligation; Link; Liquid substance; Longitudinal Studies; MAP Kinase Gene; Measures; Mediating; Messenger RNA; Modeling; Mononuclear; Mycobacterium tuberculosis; Parasites; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Persons; Phosphorylation; Pleural; Process; Production; Proteins; Publishing; Regulation; Regulatory T-Lymphocyte; Reporting; Ribonucleases; Role; SLAM protein; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; T-Lymphocyte; Tuberculosis; Vaccines; Virus; Western Blotting; X-Linked lymphoproliferative disorders; base; cytokine; fungus; inhibitor/antagonist; insight; mRNA Expression; mRNA Stability; mRNA Transcript Degradation; microorganism antigen; neutralizing antibody; pathogen; promoter; protein expression; public health priorities; response; transcription factor; tuberculosis immunity; tuberculosis treatment; vaccine development

DESCRIPTION (provided by applicant): Because Tuberculosis (TB) causes 1.7 million deaths/year; it urges to better understand the human immune response to Mycobacterium tuberculosis (Mtb). Protective immunity to TB requires IFN-3. Thus, the general goal of this proposal is to understand the signaling pathways that control IFN-3 secretion in TB. Specifically, we propose to use primary T-cells from blood and pleural fluids in a physiologically relevant model to study this process. We reported that SLAM enhances IFN-3 production to Mtb, although SLAM expression is abnormal in TB patients. Our collaborators found reduced CREB expression in TB, but a positive regulation of IFN-3 by CREB. Our preliminary data shows that SLAM/IL-17 affects CREB activation and IFN-3 production in TB. Therefore, we hypothesize that: (1) SLAM-induced-IFN-3 involves CREB activation; (2) low IFN-3 produced by LR (low responder) TB patients (with weak response to Mtb) is caused by aberrant SLAM signaling and CREB activation; (3) Th17 cells regulate SLAM expression modulating Th1 responses in TB. Thus, we propose the following aims: 1. Determine how the SLAM/SAP (SLAM-associated protein) pathway modulates CREB in healthy individuals. 1.1. CREB activation by SLAM signaling. We will determine if SLAM ligation after Mtb-stimulation induces CREB activation/binding to the IFN-3 promoter. We will use CREB siRNA to measure IFN-3 mRNA/protein in cells stimulated with Mtb and SLAM. 1.2. Signaling pathways activated by SLAM. We will determine the activity of PI3K, Akt, ERK and MAPK proteins in cells stimulated with Mtb and SLAM. Because SAP inhibits IFN-3 in TB, we will perform the studies described in SAP deficient patients. 1.3. Do the SLAM-stimulated PI3K, Akt, MAPK, ERK pathways activate CREB enhancing IFN-3? By using specific inhibitors and siRNA to these molecules we will analyze CREB function on IFN-3 production. 2. Characterize the abnormalities in the SLAM/CREB pathway in TB patients. 2.1. Determine the effects of increased SAP expression on CREB/IFN-3 levels in TB. We will perform the studies of the aim 1.1 in TB patients' PBMC/PFMC. We will introduce SAP siRNA in LR's cells to determine if CREB/IFN-3 levels are restored. Longitudinal studies will determine if TB treatment ameliorates abnormalities in the SAP/CREB expression. 2.2. Does SAP interfere with the SLAM-mediated signaling? We will determine in LR patients' PBMC/PFMC the expression/function of the identified molecules linking SLAM to CREB. Longitudinal studies of TB patients will be performed to investigate the effect of TB treatment on abnormalities in the SLAM/SAP pathway. 2.3. Investigate the mechanisms leading to increased SAP in LR patients. We will analyze the Ets expression (controls SAP promoter activity) and AUF1/HuR and mRNA stability. 3. Does IL-17 modulate SLAM/CREB activation during IFN-3 regulation in TB? 3.1. We will determine the effect of IL-17 on SLAM/IFN-3 expression and CREB activation in Mtb-stimulated TB patients' PBMC/PFMC and controls. 3.2. Does enhanced IL-17 production in TB reduce SLAM-induced CREB activation and IFN-3 expression? We will measure IL-17 production by SLAM and Mtb-stimulated cells from patients and controls. We will neutralize IL-17 production and analyze the effects on SLAM-induced CREB activation, IFN-3 expression, and the molecules linking SLAM to CREB. These studies will provide insight into signaling pathways that control IFN-3 in TB, which will be critical for development of vaccines that maximize immune responses. PUBLIC HEALTH RELEVANCE: Tuberculosis, an infectious disease produced by the bacteria Mycobacterium tuberculosis, is responsible of almost 2 million deaths worldwide annually, making the development of an effective vaccine an urgent public health priority. This proposal will provide new insights about the immune mechanisms involved in the development of a protective response of the host against the pathogen. Therefore, this information will enhance the knowledge about the immunopathogenesis of tuberculosis, contributing with new information on the mechanisms that control the production of IFN-3, a key cytokine in the immune response to intracellular pathogens, including viruses, fungi and parasites.

描述（由申请人提供）：因为结核病 (TB) 每年导致 170 万人死亡；它敦促更好地了解人体对结核分枝杆菌 (Mtb) 的免疫反应。对结核病的保护性免疫需要 IFN-3。因此，该提案的总体目标是了解控制结核病中 IFN-3 分泌的信号通路。具体来说，我们建议在生理相关模型中使用来自血液和胸膜液的原代 T 细胞来研究这一过程。我们报道，尽管 SLAM 在结核病患者中表达异常，但 SLAM 增强了 Mtb 的 IFN-3 产生。我们的合作者发现 TB 中 CREB ​​表达减少，但 CREB ​​对 IFN-3 具有正向调节作用。我们的初步数据显示 SLAM/IL-17 影响 TB 中 CREB ​​的激活和 IFN-3 的产生。因此，我们假设：（1）SLAM诱导的IFN-3涉及CREB激活； (2) LR（低反应者）结核病患者（对 Mtb 反应较弱）产生的 IFN-3 水平较低，是由 SLAM 信号传导异常和 CREB ​​激活引起的； (3) Th17 细胞调节 SLAM 表达，从而调节 TB 中的 Th1 反应。因此，我们提出以下目标： 1. 确定 SLAM/SAP（SLAM 相关蛋白）途径如何调节健康个体中的 CREB。 1.1.通过 SLAM 信号激活 CREB。我们将确定 Mtb 刺激后的 SLAM 连接是否诱导 CREB ​​激活/与 IFN-3 启动子结合。我们将使用 CREB ​​siRNA 来测量 Mtb 和 SLAM 刺激的细胞中的 IFN-3 mRNA/蛋白质。 1.2. SLAM 激活的信号通路。我们将测定用 Mtb 和 SLAM 刺激的细胞中 PI3K、Akt、ERK 和 MAPK 蛋白的活性。由于 SAP 会抑制结核病中的 IFN-3，因此我们将在 SAP 缺陷患者中进行所述研究。 1.3. SLAM 刺激的 PI3K、Akt、MAPK、ERK 通路是否会激活 CREB ​​增强 IFN-3？通过对这些分子使用特定的抑制剂和 siRNA，我们将分析 CREB ​​对 IFN-3 产生的功能。 2. 描述结核病患者 SLAM/CREB ​​通路异常的特征。 2.1.确定 SAP 表达增加对 TB 中 CREB/IFN-3 水平的影响。我们将在结核病患者的PBMC/PFMC中进行目标1.1的研究。我们将在 LR 细胞中引入 SAP siRNA，以确定 CREB/IFN-3 水平是否恢复。纵向研究将确定结核病治疗是否可以改善 SAP/CREB ​​表达异常。 2.2. SAP 是否会干扰 SLAM 介导的信号传递？我们将确定 LR 患者的 PBMC/PFMC 中已识别的连接 SLAM 和 CREB ​​分子的表达/功能。将对结核病患者进行纵向研究，以调查结核病治疗对 SLAM/SAP 通路异常的影响。 2.3.研究导致 LR 患者 SAP 增加的机制。我们将分析 Ets 表达（控制 SAP 启动子活性）以及 AUF1/HuR 和 mRNA 稳定性。 3. IL-17 在 TB 的 IFN-3 调节过程中是否调节 SLAM/CREB ​​激活？ 3.1.我们将确定 IL-17 对 Mtb 刺激的结核病患者 PBMC/PFMC 和对照中 SLAM/IFN-3 表达和 CREB ​​激活的影响。 3.2.结核病中 IL-17 产生的增强是否会减少 SLAM 诱导的 CREB ​​激活和 IFN-3 表达？我们将测量来自患者和对照的 SLAM 和 Mtb 刺激细胞产生的 IL-17。我们将中和 IL-17 的产生，并分析对 SLAM 诱导的 CREB ​​激活、IFN-3 表达以及将 SLAM 与 CREB ​​连接的分子的影响。这些研究将深入了解结核病中控制 IFN-3 的信号通路，这对于开发最大化免疫反应的疫苗至关重要。公共卫生相关性：结核病是一种由结核分枝杆菌产生的传染病，每年导致全球近 200 万人死亡，因此开发有效的疫苗成为公共卫生领域的一项紧迫任务。该提案将为宿主针对病原体产生保护性反应所涉及的免疫机制提供新的见解。因此，这些信息将增强对结核病免疫发病机制的了解，有助于提供有关控制 IFN-3 产生的机制的新信息，IFN-3 是对细胞内病原体（包括病毒、真菌和寄生虫）免疫反应的关键细胞因子。