IBD: Mucosa Specific Regulation of IFN-gamma Production

IFN-γ在许多动物粘膜炎症的发生和持续中起重要作用 炎症性肠病（IBD）的模型，是克罗恩病的诱导和延续的核心 疾病（CD）。指示Th 1极化的细胞因子（IL-12、IL-18、TL 1A、IFN-γ）在炎症性 鼠和人粘膜。CD患者用抗IFN-γ单克隆抗体治疗显示出明显的临床改善 （CDAI降低）和CRP降低。我们发现IFNG调节区单倍型阳性 与炎症性肠病（IBD）呈负相关，进一步强调了 研究CD发病机制中IFN-γ调节的改变。全面抑制IFN-γ作为IBD的治疗可能 具有严重的缺点，因为Th 1细胞因子对于保护免受几种类型的免疫缺陷是必需的。 病原体，并且是维持免疫应答的适当平衡的核心。我们的总体目标， 因此，确定IFN-γ的表达是否可能被选择性地抑制在粘膜中， 鉴定参与IFNG表达调节的粘膜特异性分子机制， 干预的目标。我们建立了粘膜特异性IFNG启动子元件的存在， 鉴定了两个具有粘膜特异性增强子活性的区域。这些要素可以作为 实现粘膜IFN-γ产生的选择性减弱的几种方法。我们已经证明， 原则上，该策略可以起作用，因为将离散IFNG启动子元件转染到两个外周 固有层T细胞减弱IFN-γ蛋白的产生。此外，两个表观遗传分子 组蛋白乙酰化和IFNG甲基化机制，最近报道在 IFNG转录的调控。我们已经证明粘膜T细胞表现出改变的组蛋白乙酰化 和IFNG启动子的甲基化。这些数据提供了 我们的继续建议，基于存在粘膜特异性分子机制的假设， 顺式调节和表观遗传，特异性控制粘膜T细胞IFN-γ的产生， 为IFN-γ表达的区域定向衰减提供了机会。我们的研究旨在 鉴定IFNG启动子序列靶，并开发减弱粘膜的分子试剂， 消除系统性IFN-γ表达。我们的假设将通过以下文献中描述的研究来解决 以下具体目的：1）使用两个确定的IFNG区域的精细启动子分析来表征粘膜 特异性顺式调节。2)定义T细胞IFNG调节的粘膜特异性表观遗传机制。第三章 设计靶向调控区的竞争性寡核苷酸沉默和RNA干扰模式 和从目的1和2鉴定的甲基化位点，以减弱体外粘膜IFNG转录。 了解IBD中粘膜细胞因子产生的调节可以确定治疗IBD的机会。 这些疾病影响着多达100万美国人。

IFN-y plays an important role in the generation and perpetuation of mucosal inflammation in many animal models of inflammatory bowel disease (IBD) and is central to the induction and perpetuation of Crohn's disease (CD). Cytokines indicative of Th1 polarization (IL-12, IL-18, TL1A, IFN-y) are elevated in inflamed murine and human mucosa. CD patients treated with anti-IFN-y MAb showed marked clinical improvement (decreased CDAI) and reduction in CRP. We have discovered IFNG regulatory region haplotypes positively and negatively associated with inflammatory bowel disease (IBD), further stressing the importance of studying altered IFN-y regulation in CD pathogenesis. Global suppression of IFN-y as treatment for IBD may have serious drawbacks, because Th1 cytokines are essential for protection against several classes of pathogens, and are central to maintaining the appropriate balance of immune responses. Our overall goal, therefore, is to determine whether IFN-y expression might be selectively suppressed in the mucosa by identifying mucosa-specific molecular mechanisms involved in regulation of IFNG expression as potential targets of intervention. We established the existence of mucosa-specific IFNG promoter elements by identifying two regions with enhancer activity that was mucosa-specific. Such elements might be targeted in several ways to achieve selective attenuation of mucosal IFN-y production. We have demonstrated that this strategy can work, in principle, because transfection of discrete IFNG promoter elements into both peripheral and lamina propria T cells attenuates IFN-y protein production. Furthermore, two epigenetic molecular mechanisms, histone acetylation and IFNG methylation, have recently been reported to play critical roles in the regulation of IFNG transcription. We have shown that mucosal T cells exhibit altered histone acetylation and methylation of the IFNG promoter, compared to peripheral T cells. These data provide the foundation of our continuation proposal, based on the hypothesis that there are mucosa-specific molecular mechanisms, both cis-regulatory and epigenetic, which specifically control mucosal Tcell IFN-yproduction and which may present opportunities for regionally directed attenuation of IFN-y expression. Our studies are designed to identify IFNG promoter sequence targets and to develop molecular reagents that attenuate mucosal, without eliminating systemic, IFN-y expression. Our hypothesis will be addressed by studies described in the following Specific Aims: 1) Use fine promoter analysis of two defined IFNG regions to characterize mucosa specific cis-regulation. 2) Define mucosa specific epigenetic mechanisms of Tcell IFNG regulation. 3) Design competitive oligonucleotide silencing and RNA interference modalities targeted to regulatory regions and methylation sites identified from Aims 1 and 2, to attenuate in vitro mucosal IFNG transcription. Understanding regulation of mucosal cytokine production in IBD could identify opportunities for therapeutic development and potentially prevention for these diseases which affect as many as 1 million Americans.