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

描述(申请人提供)：由于结核病(TB)每年导致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干扰素-3。因此，这项建议的总体目标是了解控制结核病中干扰素-3分泌的信号通路。具体地说，我们建议在生理相关的模型中使用血液和胸液中的原代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可促进干扰素-3产生结核分枝杆菌。我们的合作者发现，CREB在结核病中的表达减少，但CREB对干扰素-3的正向调节。我们的初步数据显示，SLAM/IL-17影响结核患者CREB的激活和干扰素-3的产生。因此，我们假设：(1)SLAM诱导的干扰素-3涉及CREB的激活；(2)LR(低应答)结核病患者(对Mtb反应弱)产生的低干扰素-3是由SLAM信号和CREB激活异常引起的；(3)Th17细胞调节SLAM的表达，调节TB中的Th1反应。因此，我们提出了以下目标：1.确定SLAM/SAP(SLAM相关蛋白)通路如何调节健康个体的CREB。1.1.CREB通过SLAM信号激活。我们将确定Mtb刺激后的SLAM连接是否诱导CREB激活/与干扰素-3启动子结合。我们将使用CREB siRNA来检测Mtb和SLAM刺激的细胞中的干扰素-3mRNA/蛋白。1.2.SLAM激活的信号通路。我们将测定Mtb和SLAM刺激的细胞中PI3K、Akt、ERK和MAPK蛋白的活性。由于SAP抑制结核病中的干扰素-3，我们将在SAP缺陷患者中进行描述的研究。1.3.SLAM刺激的PI3K、Akt、MAPK、ERK信号通路是否激活CREB增强干扰素-3？通过对这些分子使用特异性的抑制剂和siRNA，我们将分析CREB在干扰素-3产生中的作用。2.明确结核患者SLAM/CREB通路异常的特征。2.1.确定SAP表达增加对结核病CREB/干扰素-3水平的影响。我们将在结核病患者的PBMC/PFMC中进行AIM 1.1的研究。我们将在LR的细胞中引入SAP siRNA，以确定CREB/干扰素-3的水平是否恢复。纵向研究将确定结核病治疗是否改善SAP/CREB表达的异常。2.2.SAP是否干扰SLAM介导的信号传递？我们将在LR患者的PBMC/PFMC中确定已识别的连接SLAM与CREB的分子的表达/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.探讨导致LR患者SAP升高的机制。我们将分析Ets的表达(控制SAP启动子的活性)以及AUF1/HUR和mRNA的稳定性。3.IL-17是否在结核的干扰素-3调节过程中调节SLAM/CREB的激活？我们将检测IL-17对结核分枝杆菌刺激的结核病患者PBMC/PFMC和对照组SLAM/IFN-3表达和CREB活化的影响。3.2.结核中IL-17的产生增加是否会减少SLAM诱导的CREB激活和干扰素-3的表达？我们将通过SLAM和MTB刺激患者和对照组的细胞来测量IL-17的产生。我们将中和IL-17的产生，并分析其对SLAM诱导的CREB激活、干扰素-3表达以及连接SLAM与CREB的分子的影响。这些研究将提供对结核病中控制干扰素-3的信号通路的洞察，这将对开发最大限度地提高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引起的传染病，每年在全世界造成近200万人死亡，这使得开发有效的疫苗成为紧迫的公共卫生优先事项。这一建议将为宿主对病原体的保护性反应的发展过程中涉及的免疫机制提供新的见解。因此，这些信息将加强对结核病免疫发病机制的了解，有助于提供关于控制干扰素-3产生的机制的新信息，干扰素-3是对包括病毒、真菌和寄生虫在内的细胞内病原体的免疫反应中的关键细胞因子。

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.