Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus Figure 1. miRNAs miR-133b and miR-206 are syntenic to the IL-17A/IL-17F locus and are specifically expressed in Th17 cells polarized in vitro. (A) Schematic representation of the Il17a/f locus. Il17a and Il17f are syntenically linked in approx. 44 kB distance. The two miRNAs miR-133b and miR-206 are located in close proximity upstream to the Il17a/f locus. (B) VISTA plot of the mouse Il17a/f locus in which mouse sequence is shown on the x axis and percent similarity to human on the y axis. (C) Sequence comparison of mature miR-133b and miR-206 in H.s., M.m. R.n. and P.t. (D), (E) Spleen and peripheral lymph node cells were isolated from DO11.10 mice, co-cultured with sex-matched BALB/c irradiated feeder cells and polarized for the different T helper cell subset conditions (Th0, Th1, Th2, Th17 and Treg). Expression levels of miR-133b (D) and miR-206 (E) were analyzed by qRT-PCR. Values are plotted as fold difference compared to non-polarized cells (Th0). Error bars show 6SEM of n$3 experiments with 2–3 mice per experiment. doi:10.1371/journal.pone.0020171.g001 206. Expression levels of both miRNAs were increased in GFP- positive cells when compared to GFP-negative cells (both approximately 8-fold). Another marker used to distinguish subsets of cd T cells is CD27, and CD27 expression inversely correlates with the expression of IL-17A [28]. Thus, CD27 expression might serve as an additional tool to assess the correlation between miR- 206/133b expression and IL-17A expression. CD27-positive and Figure 2. IL-17 producing in vivo polarized CD4+ Th17 cells express elevated amounts of miR-133b and miR-206. (A) CD4+ T cells from C57BL/6 mice were sorted into IL-17A+ and IL-17A2 populations and analyzed for expression of miR-133b and miR-206 by qRT-PCR. Dot plots show post-sort analysis of one representative experiment. (B) CD4+ T cells were sorted into GFP+ (Rorct+) and GFP2 (Rorct2) populations from heterozygous Rorct reporter mice and analyzed as in (A). Dot plots show post-sort analysis of one representative experiment. Values are plotted as fold increase ( = ratio) compared to the respective negative population. The graphs show representative experiments from n = 3 independent experiments with similar results. Error bars represent SD values of triplicates from one experiment with 4–6 mice. doi:10.1371/journal.pone.0020171.g002 CD27-negative cd T cells were sorted from Tcrd-H2BeGFP reporter mice (Fig. 3C, right panels) and expression levels of miR-133b and miR-206 were assessed by qRT-PCR. In accordance with a less stringent correlation of CD27 expression and IL-17A expression, expression levels of miR-133b and miR- 206 were only slightly elevated (2-fold and 2.5-fold, respectively) in CD27-negative versus CD27-positive cd T cells (Fig. 3C, left panel). Taken together, these data indicate that co-expression of miR-206/133b and IL-17A is not restricted to CD4+ Th17 cells, but also holds true for IL-17A expressing innate lymphocytes, suggesting that this co-regulation is a general phenomenon shared between ab and cd T cells. IL-23 promotes expression of miR-206/133b Th17 cell polarization can be induced in vitro using a combination of various cytokines such as IL-23, TGF-b, IL-6 and IL-1b. Whereas IL-6 and low amounts of TGF-b are necessary to differentiate naive CD4+ T cells into Th17 cells, IL-23 appears to be essential for sustained differentiation of Th17 cells [29]. However, the extent of IL-17 induction by individual cytokines may vary and thus comparing induction of IL-17 with induction of miR-206/133b by individual Th17 polarizing cytokines might provide some mechanistic insight into miR-206/ 133b/IL-17 co-regulation. Therefore, DO11.10 TCR transgenic CD4+ T cells were polarized towards Th17 using individual polarizing cytokines as well as cytokine combinations. Notably, under these conditions, addition of IL-23 alone was able to induce low levels of miR-133b and miR-206 (Fig. 4A, B) as well as IL-17A as assessed by ELISA (Fig. 4C). Furthermore, combination of two cytokines induced marked up-regulation of the two miRNAs and IL-17A secretion only when IL-23 was part of the combination and no additive effect was observed, when two other cytokines were applied. Thus, of all cytokines tested IL-23 appears to be the most important for expression of miR-133b and miR-206 under standard conditions of in vitro polarization. Since in these experiments IL-4 and IFN-c but not TGF-b or other factors were blocked it is likely that traces of IL-23R inducing cytokines were derived from fetal bovine serum in the medium. This is consistent with the observation that combination of all four cytokines generally used for Th17 polarization, namely IL-23, TGF-b, IL-6 and IL-1b or the additional use of IL-21 and TNF-a resulted in a profound synergistic effect with respect to miR-133b and miR-206 upregulation when compared to IL-23 alone or a combination of two cytokines (Fig. 4A, B). IL-17A levels of secretion appeared to follow the same pattern (Fig. 4C). In order to more quantitatively assess the correlation between induction of miR-133b and miR- 206 versus secretion of IL-17A dependent on the different cytokine cocktails used for polarization, we determined the respective correlation coefficients. Both miR-133b induction (Fig. 4D) as well as miR-206 induction (Fig. 4E) correlated strongly with secretion of IL-17A with correlation coefficients R2 of 0.97 and 0.95, respectively. Taken together, these results point to an important role of IL-23 signaling in miR-206/133b induction in lymphocytes and strengthen the hypothesis of a strict co-regulation of miR- 206/133b and IL-17A. However, truly naive CD4+ T cells do not PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20171