ESAT-6 primes dendritic cells with reduced IL-12 and increased IL-23 production

描述（由申请人提供）：结核病仍然是死亡的主要原因，部分原因是缺乏有效的疫苗。树突状细胞（DC）通过产生IL-12和IL-23在引发T细胞应答中是关键的，IL-12和IL-23分别有利于产生IFN-γ的Th 1细胞和产生IL-17的Th 17细胞的扩增。结核分枝杆菌（Mycobacterium tuberculosis，M.）tb）是一种候选疫苗抗原，但我们的初步数据显示，ESAT-6通过减少p35和增强p19转录，抑制IL-12并增强刺激的DC分泌IL-23。ESAT-6处理的DC响应于M. TB.基于这些数据，我们假设ESAT-6影响关键转录因子与p35和p19启动子的结合，并通过DC差异调节IL-12和IL-23的产生。为了确定这些重要影响的分子机制，我们建议：1。鉴定p35和p19启动子中的ESAT-6反应元件（ERE）。我们将首先使用p35和p19启动子控制的报告构建体表达系统在人原代DC中确定在成熟期间用ESAT-6处理未成熟DC是否降低p35并增加p19启动子控制的荧光素酶表达（目的1.1）。接下来，我们将通过对ESAT-6有差异反应的潜在转录因子结合位点的连续缺失和点突变来鉴定p35和p19启动子中的ERE（目的1.2）。最后，我们将确定原代人DC的p35和p19启动子中的ERE是否对M. tb野生型和ESAT-6缺失突变株（aim 1.3）。2.鉴定ESAT-6差异调节p35和p19的蛋白质。基于目标1中描述的ERE，我们将通过EMSA和ERE下拉试验鉴定与ERE结合的蛋白质（目标2.1），并通过染色质免疫沉淀验证ESAT-6介导的这些蛋白质与活DC中ERE的结合（目标2.2）。我们将使用蛋白质印迹法确定ESAT-6如何影响DC中ERE结合蛋白的表达和活化（目的2.3）。最后，我们将确定用RNAi敲低ERE结合蛋白是否使DC对ESAT-6介导的p35和p19 mRNA的差异表达具有抗性（目的2.4）。了解ESAT-6抑制DC产生IL-12和刺激DC产生IL-23的机制，将有助于深入了解Th 1和Th 17应答调节的新机制，这有助于免疫保护和免疫病理学。此外，这些研究将有助于设计更有效的抗结核疫苗和识别新的免疫治疗靶点。 公共卫生相关性：由于缺乏有效的疫苗，结核病仍然是世界范围内一个紧迫的公共卫生问题。开发这种疫苗需要详细了解宿主免疫细胞与结核分枝杆菌（结核病的病原体）之间的相互作用。我们的初步数据表明，ESAT-6，结核分枝杆菌的分泌蛋白，引发人类树突状细胞刺激炎性Th 17细胞的保护性Th 1细胞，这可能会导致广泛的组织损伤，这是结核病的特征。由于ESAT-6是一种候选疫苗抗原，因此了解它如何引起组织损伤并降低免疫保护作用非常重要。这项提案的发现将揭示结核分枝杆菌如何通过ESAT-6减少保护性免疫反应并增加组织损伤作用。这一信息将导致改进和更安全的ESAT-6为基础的结核病疫苗。

DESCRIPTION (provided by applicant): Tuberculosis remains a major cause of death, in part because of lack of an effective vaccine. Dendritic cells (DC) are critical in priming T cell responses through production of IL-12 and IL-23, which favor expansion of IFN-g-producing Th1 cells and IL-17-producing Th17 cells, respectively. The early secreted antigenic target of 6-kD (ESAT-6) protein of Mycobacterium tuberculosis (M. tb) is a candidate vaccine antigen, but our preliminary data show that ESAT-6 inhibits IL-12 and enhances IL-23 secretion by stimulated DC, by reducing p35 and enhancing p19 transcription. ESAT-6-treated DC favors T-cell production of IL-17 over IFN-g, in response to M. tb. Based on these data, we hypothesize that ESAT-6 affects binding of critical transcription factors to the p35 and p19 promoters, and differentially regulates production of IL-12 and IL-23 by DC. To identify the molecular mechanisms underlying these important effects, we propose to: 1. Identify ESAT-6 response elements (EREs) in the promoters of p35 and p19. We will first determine if treatment of immature DC with ESAT-6 during maturation reduces p35 and increases p19 promoter-controlled luciferase expression, using p35 and p19 promoter-controlled reporter construct expression systems in human primary DC (aim 1.1). Next, we will identify EREs in the promoters of p35 and p19 by serial deletion and point mutations of potential transcription factor binding sites that respond differentially to ESAT-6 (aim 1.2). Finally, we will determine if the EREs in the p35 and p19 promoters of primary human DC respond differentially to M. tb wildtype and an ESAT-6 deletion mutant strain (aim 1.3). 2. Identify proteins by which ESAT-6 differentially regulate p35 and p19. Based on the EREs delineated in aim 1, we will identify proteins that bind to ERE by EMSA and ERE-pulldown assays (aim 2.1) and verify ESAT-6-mediated binding of these proteins to the EREs in live DC by chromatin immunoprecipitation (aim 2.2). We will determine how ESAT-6 affects expression and activation of ERE-binding proteins in DC, using Western blot (aim 2.3). Finally, we will determine if knockdown of the ERE-binding proteins with RNAi make DC resistant to ESAT-6-mediated differential expression of p35 and p19 mRNA (aim 2.4). Understanding the mechanisms by which ESAT-6 inhibits IL-12 and stimulates IL-23 production by DC will provide insight into novel mechanisms for regulation of Th1 and Th17 responses, which contribute to immune protection and immunopathology. In addition, these studies will facilitate design of more effective antituberculosis vaccines and identification of novel therapeutic targets for immunotherapy. PUBLIC HEALTH RELEVANCE: Tuberculosis remains an urgent public health problem world wide due to lack of effective vaccine. Developing such vaccine requires detailed understanding of the interaction between host immune cells and Mycobacterium tuberculosis, the causative agent of tuberculosis. Our preliminary data demonstrate that ESAT-6, a secreted protein of Mycobacterium tuberculosis, primes human dendritic cells to stimulate inflammatory Th17 cells at the expense of protective Th1 cells, which can lead to extensive tissue damage that is characteristic of tuberculosis. Because ESAT-6 is a candidate vaccine antigen, it is important to understand how it may cause tissue damage and reduce immune protection. The findings from this proposal will uncover how Mycobacterium tuberculosis reduces protective immune responses and increases tissue-damaging effects through ESAT-6. This information will lead to improved and safer ESAT-6-based vaccines against tuberculosis.