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）每年造成170万人死亡；它敦促更好地了解人类对结核分枝杆菌（MTB）的免疫反应。对结核病的保护性免疫需要IFN-3。因此，该提案的一般目标是了解控制结核病中IFN-3分泌的信号传导途径。具体而言，我们建议在与生理相关的模型中使用血液和胸膜流体的主要T细胞来研究这一过程。我们报道说，大满贯可以增强IFN-3对MTB的产生，尽管TB患者的大量表达异常。我们的合作者发现CREB在结核病中的表达降低，但CREB对IFN-3的积极调节。我们的初步数据表明，SLAM/IL-17会影响TB中的CREB激活和IFN-3产生。因此，我们假设：（1）猛击诱导的IFN-3涉及CREB激活； （2）LR（低响应者）结核病患者（对MTB反应较弱）产生的低IFN-3是由异常的猛击信号传导和CREB激活引起的； （3）TH17细胞调节大量表达调节TB中Th1反应。因此，我们提出以下目的：1。确定大满贯/SAP（大满贯相关蛋白）途径如何调节健康个体中的CREB。 1.1。通过猛击信号传导激活CREB。我们将确定MTB刺激后的结扎是否诱导CREB激活/与IFN-3启动子的结合。我们将使用CREB ​​siRNA测量用MTB和SLAM刺激的细胞中的IFN-3 mRNA/蛋白。 1.2。信号通路被猛击激活。我们将确定用MTB和SLAM刺激的细胞中PI3K，AKT，ERK和MAPK蛋白的活性。由于SAP在结核病中抑制IFN-3，因此我们将进行SAP缺乏患者中描述的研究。 1.3。猛击刺激的PI3K，AKT，MAPK，ERK途径是否激活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在IFN-3产生上的功能。 2。表征结核病患者的猛击/CREB途径中的异常。 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中对AIM 1.1进行研究。我们将在LR的细胞中引入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表达中的异常。 2.2。 SAP是否会干扰SLAM介导的信号传导？我们将在LR患者的PBMC/PFMC中确定将SLAM与CREB连接的已鉴定分子的表达/功能。将对结核病患者进行纵向研究，以研究结核病治疗对SLAM/SAP途径异常的影响。 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）和AUF1/hur和mRNA稳定性。 3。IL-17在IFN-3调节结核病期间是否调节SLAM/CREB激活？ 3.1。我们将确定IL-17对MTB刺激的TB患者的PBMC/PFMC和对照组中IL-17对SLAM/IFN-3表达和CREB激活的影响。 3.2。 TB中增强的IL-17产生是否会降低猛击诱导的CREB激活和IFN-3表达？我们将通过患者和对照组中的SLAM和MTB刺激的细胞来测量IL-17的产生。我们将中和IL-17的产生，并分析对大满贯诱导的CREB激活，IFN-3表达以及将SLAM与CREB联系起来的分子。这些研究将提供对控制结核病IFN-3的信号通路的见解，这对于最大化免疫反应的疫苗的开发至关重要。公共卫生相关性：结核病是细菌结核分枝杆菌产生的一种传染病，每年造成近20​​0万人死亡，这使得开发有效的疫苗成为紧急的公共健康优先事项。该提案将提供有关宿主对病原体的保护反应涉及的免疫机制的新见解。因此，该信息将增强有关结核病免疫发病发生的知识，并提供了有关控制IFN-3产生的机制的新信息，IFN-3是对细胞内病原体的免疫反应的关键细胞因子，包括病毒，真菌和寄生虫。