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

眼部炎症性疾病，包括葡萄膜炎，会导致严重的视力丧失。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，这些已经导致了人类患者的治疗方法。然而，关于这些步骤在炎症性疾病状态下的人类基因表达的信息是缺乏的。我们利用一种途径特异性基因芯片，对已知参与重点信号通路（如炎症和自身免疫途径）的基因进行了分析，分析了来自葡萄膜炎患者的50多份RNA样本和来自正常供体的40多份RNA样本，以确定基因表达谱，新的潜在靶基因，以了解葡萄膜炎的分子机制和潜在的治疗干预措施。我们发现葡萄膜炎患者与正常供体存在4种不同的分子基因表达谱。我们称这些特征为葡萄膜炎的分子特征。进一步分析表明，虽然分子标记与临床诊断之间缺乏相关性，但每个分子标记内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共享分子特征，但与正常供体不同，这表明遗传特征的重要性。令人惊讶的是，基因图谱显示，在一个经历了3个不同临床阶段（如活跃期、静止期和复发期）的病例中，基因表达模式几乎没有变化。此外，微阵列研究显示，当应用2倍的截止阈值时，在该通路特异性cDNA阵列芯片中的400个炎症和自身免疫性疾病相关基因中，葡萄膜炎患者与正常对照组相比，共有67个基因（16.7%）表达差异，其中56个基因上调，11个基因下调。其中28个基因通过real-time PCR阵列或real-time PCR终点法进一步验证，9个基因未报道与葡萄膜炎相关。特别令人感兴趣的是IL-22的鉴定。IL-22的表达最近与Th17细胞有关，Th17细胞是一种新发现的辅助性T细胞亚群，被认为主要参与一些Th1介导的自身免疫性疾病的发病机制，如多发性硬化症、牛皮癣、溃疡性结肠炎和小鼠葡萄膜炎模型。IL-22对免疫细胞的调节作用不大，主要作用于靶组织。此外，我们还与Egwuagus博士实验室合作，确定Th17细胞在结节病患者中的作用。在与miller博士实验室的合作中，我们进一步发现IL-22降低了人类原代胎儿RPE细胞的总组织阻力，这是RPE细胞维持组织完整性和血液-视网膜屏障稳态的重要生理特征。我们首次发现IL-22可能通过降低磷酸化bad水平导致培养的原代RPE细胞凋亡。Bad是一种众所周知的促细胞凋亡蛋白。最近的证据表明，Bad的磷酸化导致该蛋白的不作为，并被认为是调节Bad和细胞凋亡的机制之一。本研究将不再招募其他患者。

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.