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.
| Li, Zhuqing; Mahesh, Sankaranarayana P; Kim, Ben J et al. (2003) Expression of glucocorticoid induced TNF receptor family related protein (GITR) on peripheral T cells from normal human donors and patients with non-infectious uveitis. J Autoimmun 21:83-92 |
