Epigenetic Gene Regulation by the Ocular Environment

描述（由申请人提供）：眼部的免疫特权是通过多种细胞机制来维持的，这些细胞机制可以防止眼睛中针对病原体或自身抗原的破坏性炎症免疫反应。这些细胞机制包括在眼环境中产生免疫抑制细胞因子和神经肽，如转化生长因子- β和α -黑素细胞刺激激素，MHC表达低，淋巴引流缺乏，血眼屏障，Fas配体表达和前房相关免疫偏差。我们已经成功地确定了一种可能在维持眼免疫特权中发挥作用的分子机制，并为低风险患者在没有供体分型和匹配的情况下角膜移植的异常高成功率提供了基于基因的解释。根据强有力的证据表明，当外源细胞被引入眼睛时，DNA甲基化会发生，本研究项目的假设是，眼环境诱导表观遗传甲基化，从而下调眼内外源组织中的基因表达，减轻破坏性炎症性眼部免疫反应。以下具体目标和方法解决了这一假设的关键方面：(1)利用微阵列分析、实时PCR和Western blot分析，鉴定眼环境上调的甲基转移酶对基因甲基化负责；(2)利用启动子报告载体测定、甲基化特异性PCR和染色质免疫沉淀技术，检测眼环境引发的甲基化是否通过特异性甲基化单个基因或通过诱导整体甲基化和染色质重塑来调节基因表达；（iii）利用蛋白质分离、质谱和蛋白质组学分析，分离并鉴定眼部环境中触发表观遗传基因调控的因素。更好地了解眼睛如何利用表观遗传调控的分子机制来维持眼部免疫特权，并防止威胁移植角膜组织生存的破坏性先天和适应性免疫反应，将为开发新的基于基因调控的策略提供基础，以提高因持续性角膜而接受移植预后极差的高风险患者成功移植角膜的机会炎症。

DESCRIPTION (provided by applicant): Immune privilege is maintained in the eye by multiple cellular mechanisms that prevent damaging inflammatory immune responses against pathogens or self-antigens in the eye. These cellular mechanisms include production of immunosuppressive cytokines and neuropeptides in the ocular environment such as transforming growth factor-beta, and alpha-melanocyte stimulating hormone, low MHC expression, lack of lymphatic drainage, a blood-ocular barrier, Fas Ligand expression, and anterior chamber associated immune deviation. We have successfully identified a molecular mechanism that may play a role in maintaining ocular immune privilege and provides a gene-based explanation for the exceptionally high success rate of corneal transplants in low risk patients without donor-typing and matching. Based on strong evidence that DNA methylation occurs in foreign cells when they are introduced into the eye, the hypothesis of this research project is that the ocular environment induces epigenetic methylation that down regulates gene expression in foreign tissues within the eye and mitigates destructive inflammatory ocular immune responses. The following specific aims and methods address key aspects of this hypothesis: (i) Identify the methyltransferases up regulated by ocular environment responsible for gene methylation, using micro array analysis, real-time PCR, and Western blot analysis, ii) Test whether methylation initiated by the ocular environment regulates gene expression by specifically methylating individual genes or by inducing global methylation and chromatin remodeling, using promoter-reporter vector assays, methylation specific PCR and chromatin immunoprecipitation techniques, (iii) Isolate and identify the factor(s) in the ocular environment responsible for triggering epigenetic gene regulation, using protein fractionation, mass spectroscopy, and proteome analysis. A better understanding of how the eye utilizes the molecular mechanisms of epigenetic regulation to maintain ocular immune privilege and prevent destructive innate and adaptive immune responses that threaten the survival of transplanted corneal tissue will provide the basis for development of new gene regulation-based strategies to improve chances of successful corneal transplants in high-risk patients whose prognosis for accepting a transplant are extremely poor due to persistent corneal inflammation.