Treatment with low-dose cytokines reduces oxidative-mediated injury in perilesional keratinocytes from vitiligo skin

Abstract

BackgroundVitiligo is a systemic dermatological disorder characterized by the loss of skin pigmentation due to melanocyte injury or aberrant functioning. Recent data underline its multifactorial etiology with significant involvement of autoimmune and redox alterations. The major role in vitiligo cellular immunity is displayed by augmented Th1 and Th17 and suppressed TREGs and Th2 lymphocyte populations. Our previous studies indicate a marked redox imbalance in perilesional (“PL”, i.e. obtained from visibly unaffected skin surrounding the depigmented area in vitiligo patients) keratinocytes where the massive infiltration of inflammatory cells takes place. No defined therapy exists for vitiligo. Although a number of approaches have been used for the induction of TREGs and Th2 cells, they may be associated with significant off-target effects. ObjectiveIn order to identify a targeted approach for vitiligo treatment we, first, aimed to investigate the possible source of ROS overproduction in PL keratinocytes. Second, we tested the effect of low-dose selected cytokines, on intra- and extracellular ROS production, cell viability and cell cycle of PL keratinocytes. MethodsThe in vitro study was conducted on primary PL keratinocytes obtained from the skin of vitiligo patients in our previous studies. The activity of NADPH oxidase was measured on intact PL and control keratinocytes, treated or not with cytokines, by luminometric assay. The following cytokines were selected for PL keratinocytes treatment: IL-10 and IL-4 (produced by TREGs and Th2, respectively), basic fibroblasts growth factor (bFGF) and neuropeptide β-endorphin (modulating the cellular resistance to oxidative stress and the immune response, respectively). All cytokines were used at concentration of 10fg/ml and were prepared by sequential-kinetic-activation (SKA). Intracellular ROS production and cell cycle were analyzed by flow cytometry using H2DCFDA and propidium iodide dyes, respectively. Cell viability was measured by fluorometric resazurin reduction method. ResultsOur results suggest that NADPH oxidase represents one of the main sources of ROS overproduction by PL keratinocytes. Further, SKA low-dose IL-10, β-endorphin and, particularly, IL-4 and bFGF display a positive effect on redox dyshomeostasis and viability and, in our experimental conditions, don’t affect the cell cycle of PL keratinocytes. ConclusionOur preliminary data suggest that low-dose IL-10, IL-4, β-endorphin and bFGF can be proposed as a new therapeutic tool for vitiligo treatment.