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。我们利用一种途径特异性基因芯片，分析了葡萄膜炎患者外周血淋巴细胞的60个RNA样本和正常供体的40多个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。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细胞是一种新特征的T辅助细胞亚群，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。与miller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细胞的总组织抵抗。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失活，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. 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 some 60 RNA samples isolated from peripheral blood leykocytes 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. 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 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 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. Based on microarray data that reveals a potentially critical role of IL-22 on retinal pigmented epithelium (RPE) cells, we further investigated the effect of RPE cells, and hence the surrounding environment of retina, on the development, differentiation and functions of human monocytes/macrophages. We initially established an ex vivo culturing system to test potential in vivo factors that might affect the development, differentiation and functions of monocytes/macrophages. In collaboration with Dr. Millers laboratory, we set up a culturing system that consists of either 1) freshly isolated CD14+ monocytes developed or differentiated under conditions that mimic a typical laboratory setting, e.g., grown on plastic surface; or 2) mimic intraocular environment, e.g., co-culturing CD14+ monocytes on the surface of primary human RPE cells. A series pro- or anti-inflammatory cytokines/chemokines, or their cocktails mimicking in vivo inflammatory signals, were also applied. By using this ex vivo culturing system, we were able to show that macrophages that differentiated from CD14+ monocytes either on an artificial plastic surface that mimics a laboratory setting or on the surface of the primary human RPE cells are both phenotypically and functionally different. Those differentiated on the plastic surface demonstrated a more M1-like type, or pro-inflammatory by nature, macrophages while those differentiated on the surface of RPE cells demonstrated a more M2-like type, or homeostatic by nature, macrophages, suggesting an active inductive effect of RPE cells on monocytes development. Furthermore, the macrophages that were differentiated on the plastic surface changed their behavior when later they were co-cultured with human RPE cells. They became a much less pro-inflammatory type, suggesting a suppressive effect of the RPE cells. We propose that the differences between the 2 groups of macrophages may reflect the true in vitro vs. in vivo scenarios. Our data further provide evidence that the intraocular environment is more anti-inflammatory which affect the functions of residential as well as migrating monocytes/macrophages. The retinal environment actively participates in the modulation of the biological activities of monocytes/macrophages. Our ex vivo data support our hypothesis that the retinal environment is primarily down-regulatory for inflammatory immune response. 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). 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+ 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.