ABSTRACT: Regulatory T cells (Tregs) are essential regulators of immune tolerance. In light of its potential therapeutic use, in conditions involving transplant rejection and autoimmune disorders, understanding the mechanisms involved in the polarization between inflammatory and regulatory cells is of great importance. Although several studies have shown that atRA and TGF-β can inhibit the polarization of naïve T cells into inflammatory Th17 cells, favoring the generation of stable FOXP3 expressing iTregs, the exact regulatory mechanisms involved are not fully understood. Increasing evidences have suggested several roles for microRNAs in the development and function of different types of T helper cells; however, the roles of individual microRNAs in Tregs function and development are largely unexplored. Here, we explored how atRA may post-transcriptionally regulate distinct signaling pathways, through the induction of specific microRNAs, controlling the balance between Th17 and T regulatory cells. For this, CD4+CD25-CD45RA+ naïve T cells were immunomagnetically isolated from umbilical cord blood and activated with anti-human CD2/CD3/CD28 beads in the presence of IL-2 alone (CD4Med) or with the addition of TGF-β and atRA (CD4TGF/atRA). Immunophenotyping by flow cytometry, whole genome mRNAs and microRNAs profiling and functional characterizations were performed. We report the generation of highly suppressive CD4+CD25hiCD127-FOXP3+ iTregs in the CD4TGF/atRA condition, that exclusively express a set of microRNAs predicted to target important signaling pathways, including PI3K/Akt, mTOR, Toll-Like Receptor, and IL-6/Jak/Stat. Accordingly, percentages of IL-17 and STAT3 expressing cells were strikingly reduced in CD4TGF/atRA condition. Microarray and qPCR results showed that the expression of IL6R, IL6ST, JAK1 and STAT3 (heavily targeted by identified miRs) are all downregulated in CD4TGF/atRA, as compared to CD4med and naïve T cells. Finally, we show that selected synthetic mimics (namely, miR-23a, -30a, -636 and -1299) are able to knockdown IL6R and IL6ST transcript levels, functionally inhibiting IL-17 expression and favoring the generation of FOXP3+ cells. Our work adds novel evidences supporting the central roles played by microRNAs in the post-transcriptional regulation of major pathways involved in the generation and function of regulatory T cells.
