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在启动T细胞反应中起关键作用，这两种细胞分别有利于产生ifn -g的Th1细胞和产生il -17的Th17细胞的扩增。结核分枝杆菌（M. tb） 6-kD （ESAT-6）蛋白的早期分泌抗原靶点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的转录，通过刺激DC抑制IL-12并增强IL-23的分泌。esat -6处理的DC对结核分枝杆菌的反应更有利于t细胞产生IL-17，而不是IFN-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，并通过DC差异调节IL-12和IL-23的产生。为了确定这些重要作用背后的分子机制，我们建议：确定p35和p19启动子中的ESAT-6响应元件（EREs）。我们将首先确定在未成熟DC成熟期间用ESAT-6处理是否会降低p35并增加p19启动子控制的荧光素酶表达，在人类原代DC中使用p35和p19启动子控制的报告构建表达系统（aim 1.1）。接下来，我们将通过序列删除和对ESAT-6有不同反应的潜在转录因子结合位点的点突变来鉴定p35和p19启动子中的EREs（目的1.2）。最后，我们将确定原发性人DC的p35和p19启动子中的EREs是否对结核分枝杆菌野生型和ESAT-6缺失突变株有不同的反应（aim 1.3）。2. 鉴定ESAT-6差异调节p35和p19的蛋白。基于目标1中描述的EREs，我们将通过EMSA和ERE-pull - down试验（目标2.1）鉴定与ERE结合的蛋白，并通过染色质免疫沉淀验证esat -6介导的这些蛋白与活DC中EREs的结合（目标2.2）。我们将使用Western blot（目的2.3）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结合蛋白是否会使DC对esat -6介导的p35和p19 mRNA差异表达产生抗性（aim 2.4）。了解ESAT-6通过DC抑制IL-12和刺激IL-23产生的机制，将有助于了解Th1和Th17反应调控的新机制，从而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疫苗和确定新的免疫治疗靶点。

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.