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.
Showing the most recent 10 out of 13 publications
