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

描述（由申请人提供）：由于结核病（TB）每年导致170万人死亡，因此迫切需要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，该建议的总体目标是了解控制TB中IFN-3分泌的信号通路。具体而言，我们建议使用血液和胸腔积液中的生理相关模型的原代T细胞来研究这一过程。我们报道了SLAM增强Mtb的IFN-3产生，尽管SLAM在TB患者中表达异常。我们的合作者发现结核病中CREB表达减少，但CREB对IFN-3有正调节作用。我们的初步数据显示，SLAM/IL-17影响结核病中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活化;（2）由LR（低应答）TB患者（对Mtb具有弱应答）产生的低IFN-3由异常SLAM信号传导和CREB活化引起;（3）Th 17细胞调节SLAM表达，从而调节TB中的Th 1应答。因此，我们提出以下目标：1。确定SLAM/SAP（SLAM相关蛋白）通路如何调节健康个体的CREB。1.1.通过SLAM信号传导的CREB活化。我们将确定Mtb刺激后的SLAM连接是否会诱导CREB激活/与IFN-3启动子结合。我们将使用CREB siRNA来测量用Mtb和SLAM刺激的细胞中的IFN-3 mRNA/蛋白。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PI 3 K、Akt、ERK和MAPK蛋白的活性。由于SAP抑制TB中的IFN-3，我们将在SAP缺陷患者中进行所述研究。1.3. SLAM刺激的PI 3 K、Akt、MAPK、ERK通路是否激活CREB增强IFN-3？通过对这些分子使用特异性抑制剂和siRNA，我们将分析CREB对IFN-3产生的功能。2.描述结核病患者中SLAM/CREB通路的异常。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TB治疗是否改善SAP/CREB表达的异常。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连接的所鉴定的分子的表达/功能。将对TB患者进行纵向研究，以研究TB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）和AUF 1/HuR和mRNA稳定性。3. IL-17是否在TB中的IFN-3调节过程中调节SLAM/CREB活化？3.1.我们将确定IL-17对Mtb刺激的TB患者的PBMC/PFMC和对照中的SLAM/IFN-3表达和CREB活化的影响。3.2. TB中增强的IL-17产生是否减少SLAM诱导的CREB活化和IFN-3表达？我们将测量来自患者和对照的SLAM和Mtb刺激的细胞的IL-17产生。我们将中和IL-17的产生，并分析对SLAM诱导的CREB活化、IFN-3表达以及连接SLAM与CREB的分子的影响。这些研究将提供对控制结核病中IFN-3的信号通路的深入了解，这对于开发最大化免疫应答的疫苗至关重要。公共卫生相关性：结核病是一种由结核分枝杆菌（Mycobacterium tuberculosis）细菌产生的传染病，每年在全世界造成近200万人死亡，使得开发有效疫苗成为紧迫的公共卫生优先事项。这一建议将提供新的见解免疫机制参与发展的保护性反应的主机对病原体。因此，这一信息将增强有关结核病免疫发病机制的知识，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）的免疫应答中的关键细胞因子。

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.