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: 8556823
• 负责人: Robert Nussenblatt
• 金额: $ 37.5万
• 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/8556823
• 关键词: Acetylation; Adrenal Cortex Hormones; Age; Age related macular degeneration; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Autoimmune Process; Autoimmune Responses; Autoimmunity; B-Lymphocytes; Biological Markers; Blindness; Blood Cells; CD4 Positive T Lymphocytes; Cells; Characteristics; Chromatin; DNA Methylation; DNA Sequence; Data; Deacetylation; Development; Devices; Disease; Epigenetic Process; Exposure to; Eye; Functional RNA; Gene Chips; Gene Expression; Genes; Genetic Transcription; Germ Cells; Helper-Inducer T-Lymphocyte; Human; Immune; Immune response; Immune system; Inflammatory; Inflammatory Response; Interleukin-10; Interleukin-17; Investigation; Length; Leukocytes; Macular degeneration; Memory; Methylation; MicroRNAs; Modification; Molecular; Molecular Profiling; Natural Killer Cells; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Population; Production; Promoter Regions; Proteins; Protocols documentation; RNA; Recruitment Activity; Refractory; Refractory Disease; Reporting; Retinal; Role; Screening procedure; Siblings; Signal Pathway; Site; Small Interfering RNA; Steroids; Subgroup; T-Lymphocyte; Techniques; Testing; Th1 Cells; Th2 Cells; Therapeutic Intervention; Time; Tissues; Twin Multiple Birth; Uveitis; active control; cell type; cytokine; demethylation; genome wide association study; high risk; histone modification; interest; interleukin-22; monocyte; novel; pathogen; peripheral blood; promoter; receptor; response; transcriptomics

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.

包括葡萄膜炎在内的眼部炎症性疾病会导致严重的视力丧失。使用具有已知参与集中信号传导途径（例如炎症和自身免疫途径）的基因的途径特异性基因芯片。我们以前发现，存在4个不同的分子基因表达谱时，比较那些从葡萄膜炎患者和正常供体。我们将这些特征称为葡萄膜炎的分子标记。其中9个基因尚未报道与葡萄膜炎相关。特别感兴趣的是IL-22的鉴定。IL-22的表达最近与Th 17细胞相关。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的机制之一，因此，细胞凋亡。本研究将不再招募更多患者。此外，我们已经看到年龄相关性黄斑变性患者的免疫细胞中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变化。目前对表观遗传学的理解是研究控制体细胞遗传基因表达状态而不改变潜在DNA序列的机制，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）通过非编码RNA（siRNA，miRNA）控制转录。 我们已经启动了一项关于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性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造血细胞发育的控制。关于DNA甲基化在控制其他免疫细胞中细胞因子表达方面的综合研究，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基因缺失。我们已经获得了初步研究的初步数据。通过检测位于IL-10直接启动子区（转录起始位点上游1.4 kb）的4个CpG位点，我们发现CD 4 T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细胞主要是未甲基化的（小于25%）。我们的数据首次发现了IL-10启动子在免疫细胞的独特发育谱系中的差异甲基化。初始数据还表明，与整个CD 4 + T细胞（75%）和其他细胞类型相比，CD 4 + CD 45 RO+初始T细胞是最重甲基化的（90%），表明DNA甲基化在CD 4 + T细胞亚群中是不同的。我们还测试了在IL-10启动子区域中是否存在Th 0、Th 1和Th 2辅助性T细胞亚群的差异甲基化。已知T辅助细胞或CD 4 + T细胞在调节各种免疫应答中发挥核心作用。有一种教条认为，幼稚T细胞（Th 0细胞）分化为Th 1（促炎和抗病原体）型或Th 2（抗炎和抗寄生虫）型T细胞。Th 2细胞产生的IL-10比Th 1细胞产生的IL-10多得多。为了测试DNA甲基化是否可能有助于不同的IL-10产生，我们纯化了来自人供体的CD 4 + Cd 45 RA + CD 45 RO-幼稚T细胞，并通过按照建立的方案在不同极化条件下培养将那些幼稚T细胞（Th 0细胞）分化为Th 1或Th 2。 我们评估了年龄相关性黄斑变性患者，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患者人群。在外周血细胞和视网膜及脉络膜组织中，我们已经看到低甲基化与IL-17相关受体的强烈关联。我们已经看到IL-17受体和去甲基化之间存在很强的相关性。此外，这些变化在AMD患者的眼睛中被注意到。这项测试可以成为一种筛查设备，以确定患者在高风险的发展AMD。 此外，类固醇难治性葡萄膜炎患者在其外周血中具有类固醇难治性CD 4 + T细胞的特征性亚群。先前的研究已经证明，这种类固醇难治性表型仅限于具有产生IL-17能力的CD 4+细胞的中央记忆库。因此，我们比较了Th 1和Th 17细胞对皮质类固醇的转录组反应，以确定新的生物标志物和类固醇难治性疾病的治疗干预靶点。类固醇激素依赖患者比敏感患者有更大的倾向产生Th 17细胞，但与Th 1细胞相比，来自两组患者的Th 17细胞在暴露于Dex后的基因表达变化相似。 使用额外的技术，我们已经确定，一个亚组的葡萄膜炎患者有显着缩短端粒长度。