express IL-23R. In order to clarify the role of IL-23 for the induction of miR-206/133b in vivo we sorted ab or cd T cells from either heterozygous or homozygous IL-23R-GFP knock-out/ reporter-knock-in mice [30]. We found that IL-23R expression was not required for miR-206/133b expression in IL-23R- reporter GFP+ cells (Fig. 5). We thus propose that IL-23 chiefly promotes the expansion and maintenance of IL-17 producing cells. Primary human Th17 cells, but not Th1 cells, express miR- 133b and miR-206 Human Th17 cells have been suggested to be involved in diseases such as multiple sclerosis [31] or juvenile idiopathic arthritis [32], but have been described to differ in certain aspects from their murine counterparts, e.g. human Th17 cells co-express IFN-c more often than murine Th17 cells [33]. Given these apparent differences, we tested whether miR-206/133b and IL-17 were co-regulated in human Th17 cells as well. Therefore, we isolated human primary Th17, Th1 and Th0 cells from healthy donors using a combination of IL-17 and IFN-c cytokine secretion assays as described [34]. Although a certain degree of donor variability was observed, expression levels of miR-133b and miR- 206 were consistently higher in IL-17A secreting human T cells (2 to 4-fold and 15 to 60-fold, respectively) when compared to Th0 cells (Fig. 6). In contrast, miR-206/133b levels were largely identical between human Th1 and Th0 cells. Thus, our data indicate that the miR-206/133b cluster is co-regulated with IL-17 Figure 5. IL-23R signalling is not essential for the induction of miR-133b and mir-206. IL-23R+ (GFP+) or IL-23R2 (GFP2) cd and ab T cells from either homozygous (IL-23R2/2) or heterozygous IL-23Rgfp.KI mice were compared for their miR-133b and miR-206 expression in (A) and (B). GFP+ cd and ab T cells from heterozygous or GFP+ cd and ab T cells from homozygous IL-23Rgfp.KI mice were compared for their miR- 133b and miR-206 expression in (C) and (D). Error bars show 6SEM of n = 3 experiments with cells pooled from 10 mice per group. doi:10.1371/journal.pone.0020171.g005 Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus Figure 6. Primary human Th17 cells, but not Th1 cells, express miR-133b and miR-206. CD4+ T cells were isolated from peripheral blood of 3 human healthy donors, enriched using magnetic beads and then FACS sorted into 3 different populations: IL-17A+, IL-17A2 IFNc+ and IL-17A2IFN-c2. Expression levels of miR-133b and miR-206 were analyzed by qRT-PCR. Values are plotted as fold increase compared to the respective IL-17A2 IFN-c2 population. P-value for miR-206 in IL-17A+ compared to IFN-c+ expressing cells was 0.0645. Error bars show 6SEM for the 3 different donors. doi:10.1371/journal.pone.0020171.g006 in human T cells as well. In conclusion, we provide evidence that the two miRNAs of the miR-206/133b cluster neighboring the Il17a/f locus are co-regulated in murine and human lymphocytes. Little impact of ectopic miR-206/133b expression on Th17 differentiation Finally, to examine a potential functional role of the miR-206/ 133b cluster in Th17 polarization, we undertook a series of efforts to manipulate the expression of the two investigated miRNAs in T cells both in vivo and in vitro. To this end, we employed retroviral vectors for miRNAs miR-133b and miR-206 based on MDH1- PGK-GFP_2.0 [35]. The respective constructs were used for stable transduction of ovalbumin-specific T cells from DO11.10 mice that were previously stimulated with their cognate OVA-peptide for 24 h under non-polarizing conditions. After transduction, T cells were cultured under Th17 or other conditions for an additional 7 days. After restimulation with PMA/ionomycin, cells were fixed and stained for intracellular IL-17. IL-17 producing cells were equally frequent among transduced GFP+ and non- transduced GFP2 DO11.10 cells (Fig. 7A). Furthermore, to test a potential role of miR-133b and miR-206 in the development of IL- 17 producing cells in vivo, we retrovirally transduced bone marrow PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20171