Use Of Microarrays and Epigenetics In Gene Expression Of Uveitis & AMD Patients

微阵列和表观遗传学在葡萄膜炎基因表达中的应用

• 批准号: 7734617
• 负责人: ROBERT B. NUSSENBLATT
• 金额: $ 14.88万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734617
• 关键词: Acetylation; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Area; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; Blindness; Blocking Antibodies; Blood-Retinal Barrier; CD4 Positive T Lymphocytes; Cells; Chromatin; Clinical; Collaborations; DNA; DNA Methylation; DNA Sequence; Daclizumab; Data; Deacetylation; Development; Disease; End Point Assay; Epigenetic Process; Functional RNA; Future; Gene Chips; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Germ Cells; Helper-Inducer T-Lymphocyte; Histones; Homeostasis; Human; Human Genetics; IL10 gene; Immune; Immune system; Inflammatory; Interleukin 2 Receptor; Interleukin-10; Investigation; Laboratories; Mediating; Metabolic; Methylation; MicroRNAs; Modeling; Modification; Molecular; Molecular Profiling; Multiple Sclerosis; Mus; NCAM1 gene; Natural Killer Cells; Nature; Numbers; Pathogenesis; Pathway interactions; Patients; Pattern; Peripheral; Pharmaceutical Preparations; Phase; Phosphorylation; Physiological; Polymerase Chain Reaction; Population; Production; Promoter Regions; Proteins; Psoriasis; RNA; Recruitment Activity; Recurrence; Relative (related person); Reporting; Resistance; Sampling; Sarcoidosis; Schools; Siblings; Signal Pathway; Site; Small Interfering RNA; Subgroup; System; Systemic Lupus Erythematosus; T-Lymphocyte; Th2 Cells; Therapeutic Intervention; Thinking; Time; Tissues; Transcript; Ulcerative Colitis; Uveitis; base; bisulfite; cDNA Arrays; cell type; clinical Diagnosis; cytokine; demethylation; fetal; genetic profiling; interest; interleukin-22; intravenous injection; monocyte; promoter; prospective; receptor; trait

Ocular inflammatory diseases, including uveitis, cause significant visual loss. Previous non-human investigations have identified several cell types, receptor systems and metabolic intermediates that have led to treatment approaches for human patients. However,information on the human genetic expression of these steps in defined inflammatory disease states is lacking.Using a pathway specific gene chip with genes which are known to be involved in focused signaling pathways, e.g. inflammatory and autoimmune pathways, we have analyzed more than 50 RNA samples from uveitis patients and more than 40 RNA samples from normal donors to identify gene expression profiles, new potential target genes for understanding molecular mechanisms and potential therapeutic interventions for uveitis. We have found that there exist 4 distinct molecular gene expression profiles when comparing those from uveitis patients to those from normal donors. We termed those profiles molecular signatures for uveitis. Further analysis showed that, although there is lack of correlation between the molecular signatures and the clinical diagnoses, the genes within each molecular signature are functionally related. We also found that siblings with similar clinical diagnosis shared molecular signatures but differ to those of normal donors, suggesting the importance of genetic traits. Surprisingly, genetic profiling indicated that the gene expression patterns changed very little in one case who underwent 3 distinct clinical phases, e.g., active, quiescent and recurrent phase. In addition, the microarray study revealed that, when a 2-fold cut-off threshold was applied, there were a total of 67 genes (16.7%) that were differentially expressed among uveitis patients when compared to normal controls with 56 genes up-regulated and 11 genes down regulated among the 400 inflammatory and autoimmune diseases associated genes in this pathway-specific cDNA array chip. Among those genes, 28 genes were further validated either by real-time PCR array or real-time PCR endpoint assay, with 9 genes that have not been reported to be involved in uveitis. Of particular interest is the identification of IL-22. The expression of IL-22 has been recently associated with Th17 cells, a newly characterized T helper cell sub-population that are believed to primarily contribute to the pathogenesis of some Th1 mediated autoimmune diseases such as multiple sclerosis, psoriasis, ulcerative colitis, and the mouse uveitis model. IL-22 has little regulatory effect on immune cells but has primarily an effect on target tissues. In addition we have collaborated with Dr. Egwuagus laboratory in identifying the involvement of Th17 cells in sarcoidosis patients. In collaboration with Dr. Millers lab, we further discovered that IL-22 decreased the total tissue resistance of human primary fetal RPE cells, an important physiological feature of RPE cells to maintain tissue integrity as well as homeostasis of the blood-retinal barrier. We showed for the first time that IL-22 resulted in apoptosis in cultured primary RPE cells, possibly by decreasing the phosphorylated-Bad level. Bad is a well known pro-apoptosis protein. Recent evidence suggests that phosphorylation of Bad results in inaction of this protein and is considered one of the mechanisms in regulating Bad and hence, apoptosis.No further patients will be recruited into this study. The current understanding of epigenetics is the study of mechanisms that control somatically heritable gene expression status without changes in the underlying DNA sequence, including 1) DNA methylation/demethylation 2) Histone modification (Acetylation/deacetylation) 3). Chromatin modification and 4) Control of transcription by non-coding RNAs (siRNA, miRNA). Prospective: We have initiated a long term investigation on the involvement of DNA methylation in the immune system, focusing on cell subpopulations and gene specific DNA methylation patterns and its involvement in autoimmunity and intraocular inflammatory disease. DNA methylation has been shown to participate in the control of hematopoeitic cell development. Comprehensive studies on DNA methylation in controlling cytokine expression in other immune cells, e.g., monocytes, NK cells and B cells, and genes with anti-inflammatory effect, e.g., IL-10 gene, have been lacking. In collaboration with Dr. Hejtmancik in the OGVFB branch, we have established a reproducible strategy to study DNA methylation, including bisulfite treatment based DNA conversion, PCR amplification of bisulfite converted DNA and sequencing based confirmation of CpG methylation. Preliminary data from our initial studies have been obtained. By examining 4 CpG sites located in IL-10 immediate promoter region (1.4 kb upstream of transcriptional starting site), we found that CD4 T cells are heavily methylated (more than 75%), followed by NK cells (about 50%), while monocytes and B cells are predominantly unmethylated (less than 25%). Our data for the first time discovered differential methylation of the IL-10 promoter in distinctively developed lineage of immune cells, Initial data also suggest that CD4+CD45RO+ nave T cells are the most heavily methylated (90%) as compared to that of CD4+ T cells (75%) and other cell types, suggesting that DNA methylation is different in subsets of CD4+ T cells. Preliminary data also indicated that there is no difference in DNA methylation of IL-10 promoter region among Th0, Th1 and Th2 cells despite differences of IL-10 production capacity among those cells. We suspect that 1) DNA methylation may not necessarily contribute to the differential gene regulation of IL-10 production in Th0, Th1 and Th2 cells and 2) DNA methylation in other CpG sites of IL-10 genes may have not been identified. Future plans for DNA methylation studies: 1. Expand our initial observation to CD56bright cells. We have reported that a subgroup of CD56 cells, termed CD56bright cells, expanded in the peripheral after the donors receive intravenous injection of daclizumab (an IL2 receptor blocking antibody for treating uveitis). We also noticed that those CD56bright cells make much more IL-10 than Cd56dim cells. However, there are 2 schools of hypothesis regarding the nature of those CD56bright cells. One hypothesis is that CD56bright cells are immature NK cells while the others suggested that those CD56bright cells are regulatory NK cells. We thought that our initial observation of differential methylation might be able to help answer that question. 2. Expand to patients with uveitis and AMD. Several studies have previously suggested that DNA methylation is involved in the pathogenesis of autoimmune diseases such as SLE and MS. It will be very interesting to compare the methylation status of IL-10 promoter region of those patients to normal controls. We also plan to use a similar strategy to examine DNA methylation status in AMD patients because we suspect that AMD may represent one kind of intraocular inflammatory disease which may share epigenetic regulatory mechanisms with uveitis patients. 3. Whole genome promoter methylation studies. We will use NimbleGens whole genome promoter tiling array which represents some 30,000 transcripts and covers 56 Mbp region to screen differential methylation status on other areas of the genome relative to the immune system rich in areas that can be potential sites for methylation . We will begin comparing disease (uveitis and AMD) to controls.

包括葡萄膜炎在内的眼部炎症性疾病会导致明显的视觉丧失。以前的非人类研究已经确定了几种细胞类型，受体系统和代谢中间体，这些细胞类型导致了针对人类患者的治疗方法。然而，缺乏有关这些步骤在定义的炎症性疾病状态下的人类遗传表达的信息。使用途径特定的基因芯片，其基因已知与聚焦信号通路有关，例如炎症和自身免疫性途径，我们已经分析了来自葡萄膜炎患者的50多个RNA样品和正常供体的40多个RNA样品，以鉴定基因表达谱，新的潜在靶基因，用​​于了解分子机制和潜在的葡萄膜炎治疗干预措施。我们发现，将葡萄膜炎患者与正常供体的患者进行比较时，存在4个不同的分子基因表达谱。我们称这些葡萄膜炎的分子特征。进一步的分析表明，尽管分子特征与临床诊断之间缺乏相关性，但每个分子特征中的基因在功能上是相关的。 我们还发现，具有相似临床诊断的兄弟姐妹具有分子特征，但与正常供体的兄弟姐妹不同，表明遗传特征的重要性。令人惊讶的是，基因分析表明，在一个情况下，基因表达模式发生了很小的变化，他们经历了3个不同的临床阶段，例如活动，静止和经常性阶段。 此外，这项微阵列研究表明，当应用2倍的截止阈值时，与56个基因上调的正常对照相比，在葡萄膜炎患者中，总共有67个基因（16.7％）在葡萄膜炎患者中差异表达，而在这一速度cdna中，在400个炎症和自身免疫性疾病的基因中，有400个炎症和自身免疫性疾病的基因在400个炎症和自身免疫性疾病中的基因。在这些基因中，通过实时PCR阵列或实时PCR终点测定进一步验证了28个基因，据报道没有涉及葡萄膜炎的9个基因。特别有趣的是IL-22的识别。 IL-22的表达最近与Th17细胞有关，Th17细胞是一种新表征的T辅助细胞亚构造，据信主要有助于某些TH1介导的自身免疫性疾病的发病机理，例如多发性硬化症，牛皮癣，溃疡性结肠炎和小鼠葡萄膜模型。 IL-22对免疫细胞几乎没有调节作用，但主要对靶组织产生影响。此外，我们还与Egwuagus博士实验室合作，以确定Th17细胞在结节病患者中的参与。与Millers博士实验室合作，我们进一步发现，IL-22降低了人类原代胎儿RPE细胞的总组织耐药性，这是RPE细胞维持组织完整性以及血液视网膜屏障的稳态的重要生理特征。我们首次表明IL-22导致培养的原代RPE细胞凋亡，这可能是通过降低磷酸化的-BAD水平。坏是一种众所周知的促凋亡蛋白。最近的证据表明，这种蛋白质不可能的磷酸化结果不适，被认为是调节不良和细胞凋亡的机制之一。不再将患者招募到这项研究中。 当前对表观遗传学的理解是对控制体面可遗传基因表达状态的机制的研究，而不会改变潜在的DNA序列，包括1）DNA甲基化/脱甲基化2）组蛋白修饰（乙酰化/脱乙酰化）3）。染色质修饰和4）通过非编码RNA（siRNA，miRNA）控制转录。 前瞻性：我们已经对DNA甲基化参与免疫系统进行了长期研究，重点是细胞亚群和基因特异性DNA甲基化模式及其参与自身免疫性和眼内炎症性疾病。 DNA甲基化已被证明参与了血肿细胞发育的控制。缺乏对其他免疫细胞中细胞因子表达的DNA甲基化的全面研究，例如单核细胞，NK细胞和B细胞以及具有抗炎作用的基因，例如IL-10基因，缺乏抗炎作用。通过与OGVFB分支机构的Hejtmancik博士合作，我们建立了一种可再现的策略来研究DNA甲基化，包括基于硫磺的DNA转化，Bisulfite转化DNA的PCR扩增以及基于测序的CPG甲基化确认。我们的初步研究的初步数据已获得。通过检查位于IL-10直接启动子区域（转录起始位点上游1.4 kb）中的4个CpG位点，我们发现CD4 T细胞被大量甲基化（超过75％）（超过75％），其次是NK细胞（大约50％），而单核细胞和B细胞则是不甲基化的（少于25％）。我们的数据首次在免疫细胞的独特谱系中首次发现IL-10启动子的差异甲基化，初始数据还表明，与CD4+ T细胞（75％）和其他细胞类型相比，CD4+ CD45RO+ NAVE T细胞是最重的甲基化（90％），这表明DNA甲基化在CD4中是不同的CD4+ T4+ T4。初步数据还表明，尽管这些细胞之间IL-10的生产能力差异，但TH0，Th1和Th2细胞之间IL-10启动子区域的DNA甲基化却没有差异。我们怀疑1）DNA甲基化可能不一定有助于尚未鉴定出IL-10基因的其他CpG位点中Th0，Th1和Th2细胞中IL-10产生的差异基因调节。 DNA甲基化研究的未来计划：1。将我们的初始观察结果扩展到CD56Bright细胞。我们报道说，在捐助者接受静脉注射daclizumab（一种用于治疗葡萄膜炎的IL2受体阻断抗体）后，称为CD56Bright细胞的CD56细胞的亚组在外围扩张。我们还注意到，这些CD56Bright细胞比CD56DIM细胞产生的IL-10要多得多。但是，关于这些CD56Bright细胞的性质，有2个假设。一个假设是CD56褐色细胞是未成熟的NK细胞，而其他CD56褐色细胞是调节性NK细胞。我们认为，我们对差异甲基化的最初观察可能能够帮助回答这个问题。 2。扩展到葡萄膜炎和AMD患者。先前的几项研究表明，DNA甲基化参与自身免疫性疾病（例如SLE和MS）的发病机理。将这些患者的IL-10启动子区域的甲基化状态与正常对照组进行比较将非常有趣。我们还计划使用类似的策略检查AMD患者的DNA甲基化状态，因为我们怀疑AMD可能代表一种眼内炎症性疾病，可能与葡萄膜炎患者共享表观遗传调节机制。 3。整个基因组启动子甲基化研究。我们将使用敏捷的整个基因组启动子瓷砖阵列，该阵列代表约30,000个转录本，并覆盖56 MBP区域，以筛选基因组其他区域的差异甲基化状态，相对于富含甲基化潜在部位的免疫系统，相对于富含甲基化部位的免疫系统。我们将开始将疾病（葡萄膜炎和AMD）与对照进行比较。