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。使用途径特定的基因芯片，其中包含已知参与集中信号途径的基因，例如炎症和自身免疫途径。我们以前发现，当比较葡萄膜炎患者和正常供者的基因表达谱时，有4个不同的分子基因表达谱。我们将这些图谱命名为葡萄膜炎的分子特征。在这些被评估的基因中，有9个基因尚未被报道与葡萄膜炎有关。特别令人感兴趣的是IL-22的鉴定。最近发现IL-22的表达与Th17细胞有关。我们首次发现IL-22可诱导原代培养的RPE细胞发生凋亡，可能是通过降低磷酸化Bad水平实现的。BAD是一种广为人知的促凋亡蛋白。最近的证据表明，Bad的磷酸化导致该蛋白的失活，并被认为是调节Bad从而导致细胞凋亡的机制之一。这项研究将不再招募更多的患者。此外，我们还看到老年性黄斑变性患者免疫细胞中IL-17活性增加。 在过去的一年里，我们特别关注与疾病相关的表观遗传学变化。目前对表观遗传学的理解是研究在不改变潜在DNA序列的情况下控制体细胞可遗传基因表达状态的机制，包括1)DNA甲基化/去甲基化2)组蛋白修饰(乙酰化/去乙酰化)3)。染色质结构修饰和4)非编码RNA(siRNA，miRNA)对转录的控制。 我们已经开始了一项关于DNA甲基化在免疫系统中的参与的长期研究，重点是细胞亚群和基因特异的DNA甲基化模式及其在自身免疫和眼内炎性疾病中的参与。DNA甲基化已被证明参与了造血细胞发育的控制。关于DNA甲基化在其他免疫细胞如单核细胞、NK细胞和B细胞以及具有抗炎作用的基因如IL-10基因中调控细胞因子表达的全面研究一直缺乏。我们初步研究的初步数据已经获得。通过检测位于IL-10直接启动子区(转录起始点上游1.4kb)的4个CpG位点，我们发现CD4T细胞高度甲基化(超过75%)，其次是NK细胞(约50%)，而单核细胞和B细胞主要是非甲基化(不到25%)。我们的数据首次在不同发育的免疫细胞系中发现了IL-10启动子的差异甲基化，初步数据还表明，与整个CD4+T细胞(75%)和其他细胞类型相比，CD4+CD45RO+初始T细胞的甲基化最严重(90%)，这表明在CD4+T细胞亚群中DNA甲基化是不同的。我们还测试了辅助性T细胞的Th0、Th1和Th2亚群的IL-10启动子区是否存在差异甲基化。T辅助细胞，或称CD4+T细胞，已知在调节各种免疫反应中发挥核心作用。有一种学说认为，NAVE T细胞(Th0细胞)可以分化为Th1(促炎和抗病原体)类型或Th2(抗炎和抗寄生虫)类型的T细胞。众所周知，Th2细胞产生的IL-10比Th1细胞产生的要多得多。为了测试DNA甲基化是否有助于产生不同的IL-10，我们从人类捐赠者那里纯化了CD4+CD45RA+CD45RO-NAVE T细胞，并按照既定的方案在不同的极化条件下培养，将这些NAVE T细胞(Th0细胞)分化为Th1或Th2。 我们评估了年龄相关性黄斑变性的患者，从患有不同疾病的双胞胎开始，然后是兄弟姐妹，然后是更广泛的AMD患者群体。我们已经看到外周血细胞以及视网膜和脉络膜组织中与IL-17相关的受体与低甲基化密切相关。我们已经看到IL-17受体和去甲基化之间有很强的联系。此外，在AMD患者的眼睛中也注意到了这些变化。这项测试可能成为一种筛查设备，以识别AMD发展的高危患者。 此外，类固醇难治性葡萄膜炎患者外周血中有一种特有的类固醇难治性CD4+T细胞亚群。以前的研究已经证明，这种类固醇耐药表型仅限于具有产生IL-17能力的中央记忆池的CD4+细胞。因此，我们比较了Th1和Th17细胞对糖皮质激素的转录反应，以确定新的生物标志物和激素难治性疾病治疗干预的靶点。类固醇难治性患者比敏感患者更容易产生Th17细胞，但与Th1细胞相比，接触地塞米松后，两组患者的Th17细胞在基因表达方面的变化都受到类似的限制。 使用其他技术，我们已经确定葡萄膜炎患者的一个亚组明显缩短了端粒长度。

Ocular inflammatory diseases, including uveitis, cause significant visual loss. 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 previously 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. Among those genes evaluatd 9 genes had 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. 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 inactivation 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. In addition, we have seen increased IL-17 activity in the immune cells of patients with age related macular degeneration. This past year we have particularly concentrated on epigenetic changes associated with disease. 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 structural modification and 4) Control of transcription by non-coding RNAs (siRNA, miRNA). 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. 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+ naive T cells are the most heavily methylated (90%) as compared to that of whole CD4+ T cells (75%) and other cell types, suggesting that DNA methylation is different in subsets of CD4+ T cells. We have also tested if there is a differential methylation in the IL-10 promoter region for Th0, Th1 and Th2 sub-sets of T helper cells. The T helper cells, or CD4+ T cells, are known to play central role in regulating various immune responses. There has been a dogma that nave T cells (Th0 cells) differentiate into either Th1 (pro-inflammatory and anti-pathogen) type or Th2 (anti-inflammatory and anti-parasitic) type of T cells. It is well established that Th2 cells produce much more IL-10 than what Th1 cells can produce. To test if DNA methylation may contribute to the differential IL-10 production, we purified CD4+Cd45RA+CD45RO- nave T cells from human donors and differentiate those nave T cells (Th0 cells) into either Th1 or Th2 by culturing under different polarization conditions following the established protocols. We have evaluated patients with age relatd macular degeneration, starting with twins who have disparate disease, then siblings and then a wider population of AMD patients. We have seen a strong association of hypomethylation with receptors associated with IL-17, in peripheral blood cells and in retinal and choroidal tissue. We have seen a strong association between an IL-17 receptor and demethylation. Further these changes were noted in the eye of AMD patients. This test could become a screening device to identify patients at high risk for the development of AMD. In addition, patients with steroid refractory uveitis have a characteristic subpopulation of steroid refractory CD4+ T cells in their peripheral blood. Previously studies have demonstrated that this steroid refractory phenotype is restricted to the central memory pool of CD4+ cells which have the capacity to generate IL-17. We therefore compared transcriptomic responses of Th1 and Th17 cells to corticosteroids in order to identify novel biomarkers and targets for therapeutic intervention in steroid refractory disease. Steroid referactory patients have a greater propensity than sensitive patients to generate Th17 cells, but Th17 cells from either group of patients have a similarly restricted change in gene expression following exposure to Dex compared with Th1 cells. Using additional techniques we have identified that a subgroup of uveitis patients have markedly shortened telomore length.