IBD: Mucosa Specific Regulation of IFN-gamma Production

干扰素-γ在许多动物黏膜炎症的发生和延续中起着重要作用。 炎症性肠病(IBD)模型，在克罗恩病的诱导和持续发展中起中心作用 疾病(CD)。炎症中Th1极化的细胞因子(IL-12、IL-18、TL1a、干扰素-γ)升高 小鼠和人的粘膜。接受抗干扰素-γ单抗治疗的CD患者临床症状明显改善。 (CDAI降低)和CRP降低。我们已经积极地发现了IFNG调节区的单倍型 与炎症性肠病(IBD)呈负相关，进一步强调了 探讨干扰素-γ在CD发病机制中的调控变化。全球抑制干扰素-γ治疗IBD可能 有严重的缺陷，因为Th1细胞因子对于预防几种类型的 病原体，是维持适当的免疫反应平衡的中心。我们的总体目标是， 因此，确定干扰素-γ在粘膜中的表达是否可能被选择性地抑制。 识别可能参与调控IFNG表达的粘膜特异性分子机制 干预的目标。我们通过以下方法证实了黏膜特异性IFNG启动子元件的存在 确定两个具有粘膜特异性增强子活性的区域。这些元素可能会成为 实现选择性抑制粘膜干扰素-γ产生的几种方法。我们已经证明了这一点 原则上，策略是可行的，因为将离散的IFNG启动子元件导入两边的外周 固有层T细胞可抑制干扰素-γ蛋白的产生。此外，两个表观遗传分子 组蛋白乙酰化和IFNG甲基化的机制最近被报道在 IFNG转录的调控。我们已经证明，粘膜T细胞表现出组蛋白乙酰化的改变 与外周T细胞相比，IFNG启动子的甲基化。这些数据为以下工作提供了基础 我们的延续建议，基于存在黏膜特有的分子机制的假设， 顺式调节和表观遗传，它特异性地控制粘膜T细胞干扰素的产生，并可能 目前存在区域性的干扰素-γ表达减弱的机会。我们的研究旨在 鉴定IFNG启动子序列靶点并开发减弱黏膜的分子试剂，而不需要 消除系统性、干扰素-γ的表达。我们的假设将通过 具体目标如下：1)使用两个定义的IFNG区域的精细启动子分析来表征粘膜 特定的顺式调控。2)明确T细胞IFNG调节的黏膜特异性表观遗传学机制。3) 设计针对调控区域的竞争性寡核苷酸沉默和RNA干扰模式 以及AIMS 1和AIMS 2中发现的甲基化位点，以减弱体外粘膜IFNG转录。 了解IBD黏膜细胞因子产生的调节可以发现治疗机会 这些疾病的发展和潜在的预防影响了多达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.