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

描述(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，分别促进产生干扰素-g的Th1细胞和产生IL-17的Th17细胞的扩增，在启动T细胞反应中起着关键作用。结核分枝杆菌早期分泌的6-kD(ESAT-6)蛋白是一种候选的疫苗抗原，但我们的初步数据表明，ESAT-6通过抑制p35和促进p19转录来抑制IL-12和增强IL-23的分泌。作为对结核分枝杆菌的反应，ESAT-6处理的DC更有利于T细胞产生IL-17而不是干扰素-g。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，并差异地调节DC产生IL-12和IL-23。为了确定这些重要作用的分子机制，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表达系统在人原代DC中确定在成熟过程中用ESAT-6处理未成熟DC是否降低p35和增加p19启动子控制的荧光素酶的表达(AIM 1.1)。接下来，我们将通过对ESAT-6有不同反应的潜在转录因子结合位点的序列缺失和点突变来鉴定p35和p19启动子中的ERE(目标1.2)。最后，我们将确定人类原代DC的p35和p19启动子中的ERE是否对结核分枝杆菌野生型和ESAT-6缺失突变株(AIM 1.3)有不同的反应。2.确定ESAT-6对p35和p19的差异调控蛋白。基于AIM 1中描述的ERE，我们将通过EMSA和ERE下拉试验(AIM 2.1)鉴定与ERE结合的蛋白质，并通过染色质免疫沉淀(AIM 2.2)验证ESAT-6介导的这些蛋白质与活DC中ERE的结合。我们将使用Western印迹(AIM 2.3)来确定ESAT-6如何影响DC中ERE结合蛋白的表达和激活。最后，我们将确定RNAi敲除ERE结合蛋白是否使DC对ESAT-6介导的p35和p19 mRNA的差异表达产生抗性(目标2.4)。了解ESAT-6抑制IL-12和刺激DC产生IL-23的机制将为调节Th1和Th17反应提供新的机制，有助于免疫保护和免疫病理。此外，这些研究将有助于设计更有效的抗结核病疫苗和确定免疫治疗的新治疗靶点。 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和结核分枝杆菌之间的相互作用，结核分枝杆菌是结核病的病原体。我们的初步数据表明，结核分枝杆菌的一种分泌蛋白ESAT-6启动人类树突状细胞刺激炎性Th17细胞，代价是保护Th1细胞，这可能导致广泛的组织损伤，这是结核病的特征。由于ESAT-6是候选疫苗抗原，了解它可能如何导致组织损伤和降低免疫保护是很重要的。这项提案的发现将揭示结核分枝杆菌如何通过ESAT-6降低保护性免疫反应并增加组织破坏效应。这些信息将导致改进和更安全的基于ESAT-6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.