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). MICROBIOME IN UVEITIS (13-EI-0072) Intestinal microbiota emerges as a candidate responsible for priming the immune system in many immune mediated diseases as well as in animal models of uveitis2, however, it is not known whether (and how) gut dysbiosis might affect human uveitis. This study introduces the innovative concept of elucidating the role of gut microbiota in human uveitis. While animal models of uveitis indicate gut microbiome may play a role in induction or propagation of disease7 it is not clear whether or how gut microbiota may contribute to disease pathogenesis in human disease. This study has the potential to help identify patients with the highest risk of uveitis, improve our ability to identify subsets of patients suitable for microbial intervention strategies, predict treatment responders in advance and help devise personalized treatment regimens. The results of this study may help affect disease course in uveitis by manipulating the commensal microbiota and open a new avenue in therapeutics and diagnostics. To date, the study enrolled 51 participants (25% of recruitment goal in adult uveitis cohort). Based on our preliminary data, that indicated dysbiosis with expansion of the putative pathobionts in chronic uveitis patients, wethis project aims to: 1) identify and characterize microbiota signature associated with new onset treatment nave uveitis and chronic uveitis compared to healthy controls 2) To investigate the potential of gut microbiota as a biomarker of disease onset or responsiveness to treatment 3) To investigate how gut microbiota affects disease pathogenesis: We will investigate the immunoregulatory effect of disease relevant gut microbiota in in vitro and in vivo model systems by colonizing germ-free mice with disease relevant bacteria from uveitis patients. MOLECULAR CLASSIFICATION of UVEITIS (16-EI-0046) Uveitis is a heterogenous group of diseases where the common denominator is the inflammation in the uveal tissues. While less than half may have associated (other) systemic autoimmune diseases more than half remains undifferentiated. The disease course is unpredictable with remission and flares that can lead to blindness. There are no laboratory or reliable clinical markers of prognosis or predictors of disease activity. Current standard of care is corticosteroids followed by immunomodulatory drugs (non-biologic and biologic) regardless of the clinical subtypes. This approach disregards the possibility that the underlying mechanism or drivers of disease are different in different types of uveitis which is supported by the high failure rates of individual treatments in uveitis (up to 40-50%) and leads to undertreatment of some cases while exposing some patients to unnecessary treatment. Currently the disease is stratified based on anatomical locations (anterior, intermediate and posterior). While this has some implications for vision loss and may be partially helpful clinically its not informative of the underlying mechanism nor its a reliable indicator of disease remission, prognosis or treatment response.
The aim of this project is: 1)To identify molecular correlates of disease category and activity through clinical and transcriptional profiling of uveitis patients.2)To investigate the effects of therapy on uveitis molecular signatures.
