Epigenetic Gene Regulation by the Ocular Environment

描述(申请人提供)：免疫豁免权是通过多种细胞机制在眼睛中维持的，这些机制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-黑素细胞刺激素，低MHC表达，缺乏淋巴引流，血眼屏障，Fas配体表达，以及前房相关免疫偏离。我们已经成功地确定了一个可能在维持眼睛免疫豁免方面发挥作用的分子机制，并为低风险患者在没有供体配型和配型的情况下进行角膜移植提供了一个基于基因的解释。根据强有力的证据，当外来细胞被引入眼睛时，它们会发生DNA甲基化，本研究项目的假设是，眼睛环境诱导表观遗传甲基化，下调眼睛内外来组织的基因表达，减轻破坏性的炎症性眼睛免疫反应。以下具体目标和方法针对这一假说的关键方面：(I)利用微阵列分析、实时荧光聚合酶链式反应和蛋白质印迹分析，确定眼睛环境上调的负责基因甲基化的甲基转移酶；ii)利用启动子-报告载体分析、甲基化特异性聚合酶链式反应和染色质免疫沉淀技术，测试眼睛环境启动的甲基化是通过特异性甲基化单个基因，还是通过诱导整体甲基化和染色质重塑来调节基因表达；(Iii)利用蛋白质分级、质谱分析和蛋白质组分析，分离和鉴定眼睛环境中负责触发表观遗传基因调控的因子(S)。更好地理解眼睛如何利用表观遗传调节的分子机制来维持眼睛免疫赦免，防止破坏性的先天和获得性免疫反应威胁到移植的角膜组织的生存，将为开发新的基于基因调控的策略来提高高危患者成功角膜移植的机会提供基础，这些高危患者由于持续性角膜炎症而接受移植的预后非常差。

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.