SENESCENCE IN OCULAR INFLAMMATORY DISEASE Using unbiased bioinformatics analysis of deep immunophenotyping by flow cytometry, we have unraveled a phenotype in CD8 T cells, which exhibits features of senescence. Senescence is usually associated with aging, and leads to non-proliferative, but metabolically active cells. Like most autoimmune diseases, uveitis is believed to be mainly driven by CD4 T cells. Our observation opens the way to new prospect of uveitis and ocular inflammatory disease in general. Specifically, we found that the CD8+CD45RA+/-CD27-CCR7- T cells, corresponding roughly to the effector memory (TEM) subsets expressing, or not, CD45RA were increased in uveitis patients. Moreover, the relative abundance of this population was correlated with disease activity. Interestingly, those CD8 T cells exhibit features of senescence, such as upregulation of CD57, loss of proliferative ability, and aberrant cytokines production in response to TCR stimulation. Additionally we identified that the MAPK p38 was over-phosphorylated in patients CD8 T cells, especially TEM subset, and that in vitro treatment of uveitis patients' cells with a p38 inhibitor was sufficient to reverse the senescence functional defects in vitro. These findings raise the question of the role of these CD8 cells in uveitis, and more broadly the notion of senescence in immune cells. In collaboration with Dr. Arne Akbar (University College London) we are investigating the role of these cells in uveitis. In addition we are working with Dr. Richard Lee to start a pilot clinical study to test the effect of p38 inhibitor in uveitis patients. Additionally, we have performed whole transcriptome profiling of peripheral CD4+ T cells of patients with intermediate AMD and age-matched healthy control using RNA-Seq. These data showed moderate but significant differences of gene expression levels between AMD patients and controls, with dysregulation of multiple pathways and biological functions in CD4+ cells. Interestingly, these data support an immune dysregulation in AMD, with especially increased senescence-associated feature such as the reduction of CD28 expression or up-regulation of IL-6, in the AMD patients. We are currently extending these observations to a larger cohort of patient, to study the transcriptomes of both CD4 T cells and monocytes (CD14+ cells), isolated from peripheral blood of patients at various stages of AMD and age-matched healthy controls, as well as deep immunophenotyping of the peripheral blood by flow cytometry. EXTRACELLULAR VESICLES AND IMMUNOMODULATION Extracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated whether EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro. In collaboration with Dr. Leonid Margolis (NICHD), we have found that Retinal pigment epithelium cell line (ARPE-19) constitutively secrete extracellular vesicles (EV) in the size range of exosomes. Interestingly, EV release is increased in ARPE-19 cells stimulated with inflammatory cytokines. Extracellular vesicles from both non-stimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Moreover, EV from non-stimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, and exosomes from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce an inhibitory immune environment under homeostatic conditions. It is conceivable that, in an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes. This work was published in IOVS (PMID:27537259). APPLICATION OF EPIGENETIC CONTROL TO UVEITIS TREATMENT Epigenetic control of genes expression via reversible changes in DNA (acetylation, methylation) has been involved in many pathologies. Epigenetic drugs such as Histone DeAcetylase (HDAC) inhibitors are now used in treatment of some cancer. Furthermore, HDAC inhibitors have shown immunomodulatory effect in autoimmune settings such as juvenile Idiopathic arthritis (JIA). In collaboration with Dr. Richard Lee (University of Bristol), we started to study the potential relevance of epigenetic drugs in uveitis treatment. In a collaborative study we are looking both at human cells (T cells and monocytes) isolated from uveitis patients and healthy donors treated with an HDAC inhibitor, Givinostat. Interestingly, Givinostat showed differential effects on naive T cells compared to memory T cells, and additionally decreased the function of monocyte-derived DC. Currently we are assessing the effect of Givinostat in the murine experimental autoimmune Uveitis (EAU) model of IRBP-induced uveitis. DEVELOPMENT OF BIOINFORMATICS TOOLS TO IDENTIFY NEW DISEASE-RELEVANT CELL SUBSETS With the advent of high dimensional, high throughput data collections methods, such as multicolor flow cytometry, mass cytometry, or transcriptome analysis, the size of the datasets collected require development of new tools able to identify relevant information in a haystack of data. In collaboration Pr. Wolfgang Losert (University of Maryland), we have developed a new unbiased method to identify subtle cell subsets differences in comparative studies. This method allowed us to unraveled specific subsets, previously unknown, and linked them with specific disease state. Currently applied to flow cytometry and mass spectrometry datasets, we will also apply the method to transcriptomics analysis (e.g. our AMD study). A manuscript is under revision for PLOS One.
