MiR-146a and ADAPTIVE IMMUNITY: A Novel Player in the Regulation of Normal Immune function and Inflammation

MicroRNAs (miRNAs) have been shown to affect haematopoietic differentiation, acting as important determinants of cellular fate specification. The aim of this work is to investigate the role of miRNAs in the activation of T lymphocytes. By microarray analysis, we observed differential expression of miRNAs in distinct subclasses of T lymphocytes: CD8+ naïve, CD8+ memory effector, CD8+central memory; CD4+ naïve, CD4+ memory effector and CD4+ central memory. We found that miR-146a is expressed in separated primary T cells, in both CD4+ and CD8+ cells, but not in naïve T cells. Furthermore, a significative up-regulation of miR-146a expression levels was also obtained in the Jurkat T cell line, stimulated with αCD3-αCD28 antibodies, or with PMA and ionomycin, which are stimuli that mimic antigen recognition by TCR. Therefore, we attempted to discover the mechanisms of miR-146a transcriptional regulation. Some indications about the pathways possibly involved were obtained by treatment of induced Jurkat cells with specific pharmacological inhibitors of pathways triggered by TCR signalling.. We found that Cyclosporine (CsA), a Calcineurin inhibitor, significantly impaired miR-146a up-regulation, thus suggesting that the pathways affected by CsA play a key role in the induction of miR-146a upon TCR engagement. This observation prompted us to further investigate with molecular tools the transcription factors which are responsible for miR-146a expression in T lymphocytes. A deeper investigation to carefully characterize the cis regulatory elements involved in miR-146a regulation was performed through a bioinformatic analysis of miR-146a promoter and a luciferase assay. These approaches allowed us to identify conserved consensus sites for T lymphocytes-specific transcription factors, which are involved in pathways triggered by TCR signalling and are required for miR-146a induction. The results obtained indicate that NF-κB and c- ETS are involved in miR-146a induction in T cell activation pathway. Moreover, we also demonstrated that miR-146a is also induced in Jurkat cells by an inflammatory stimulus, TNF-α treatment, and NF-κB is the main transcription factor responsible of miR-146a induction. Another intriguing aspect of miRNAs study is the identification of miRNAs biological role in a cell. We identified miR-146a targets 5 taking advantage of a 3’-untranslated region (UTR) luciferase reporter assay. We tested a number of predicted miR-146a targets, relevant for T cell physiology. In particular, we report here that miR-146a downmodulates the apoptosis process via translational inhibition of FADD and it reduces AP-1 transcriptional activity, through the downregulation of c-FOS Altogether, our findings reveal miR-146a involvement in T cell activation process, significantly affecting key properties of lymphocytes.