pathological context [13]. Recently, we and others identified a subset of cd T cells expressing the CC chemokine receptor (CCR) 6 as potent IL-17A producers upon cytokine stimulation, encounter with pathogen products or environmental cues [14,15]. Genome-wide gene expression analysis has recently revealed that eukaryotic gene regulation is not only dependent on the direct action of transcription factors on promoter regions, but is also subject to higher order regulation dependent on the location of genes within the genome [16]. Thus, co-expression of genes depends in part on their relative distance with neighboring genes being frequently co-expressed [17], either because of shared regulatory elements or because of extended chromatin opening. Likewise, the gene for two miRNAs miR-133b and miR-206, which are probably expressed from a bicistronic pri-miRNA [18], is located directly upstream of the Il17a and Il17f (Il17a/f) gene locus. So far, miR-206 and miR-133b have been reported to be specifically important for muscle regeneration and development [18,19,20] and their expression has been suggested to be largely restricted to skeletal muscle and osteoblasts. However, based on their close proximity to the Il17a/f gene locus we hypothesized that expression of these two miRNAs and secretion of the main Th17 cytokine IL-17A might correlate. Here, we demonstrate elevated expression of miR-206/133b in in vitro polarized Th17 cells as well as in freshly isolated murine and human Th17 cells and in IL-17A producing innate lymphocytes such as CCR6- expressing cd T cells. Furthermore, we show that amongst multiple Th17 polarizing cytokines, IL-23 was the most important one for miR-133b and miR-206 expression. Taken together, our data reveal a previously unrecognized expression of miR-206/ 133b in lymphocytes, which is tightly coordinated with the expression of IL-17A. Results miRNAs miR-133b and miR-206 are syntenic to the Il17a/f locus and are specifically expressed in Th17 cells polarized in vitro Many miRNAs have been found to be clustered and are likely to be co-expressed when less than 50 kb apart [21]. miR-133b and miR-206 form such a cluster with a distance of approximately 4 kb between the coding sequences of the two mature miRNAs. We found that in the murine genome the miR-206/133b cluster lies in close proximity (less than 50 kb upstream) to the genes coding for the cytokines IL-17A and IL-17F (Fig. 1A), which themselves are organized syntenically in a head-to-head direction and probably arose through gene duplication. The miR-206/133b cluster is present in all vertebrate genomes analyzed so far [22]. Performing whole genome alignment using VISTA [23] we tested whether the proximity of miR-206/133b to the Il17a/f locus is similarly conserved. This analysis revealed that indeed miR-206/133b is syntenic to the Il17a/f locus in mouse and humans (Fig. 1B) as well as in rat and chimpanzee (data not shown). Furthermore, the mature miRNA sequences of miR-133b and miR-206 are identical between mouse, human, rat and chimpanzee (Fig. 1C). Genome-wide expression analysis has revealed that neighboring genes are frequently co-expressed [17] suggesting that the miR- 206/133b cluster and IL-17A/F might be co-expressed as well. To test this hypothesis, we isolated CD4+ T cells from transgenic mice expressing the DO11.10 T cell receptor specific for ovalbumin (amino acids 323–339). Cells were stimulated under neutral conditions (Th0) or polarized in vitro towards the Th1, Th2, Th17 and Treg lineages. Expression of miR-133b and miR-206 was assessed using qRT-PCR. Polarization towards Th17 resulted in a Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus 13-fold increase of miR-133b expression when compared to Th0 cells, whereas polarization into other CD4+ T cell lineages did not alter miR-133b expression (Fig. 1D). Similarly, elevated miR-206 expression (78-fold relative to Th0) was only detected in Th17, but not in Th1, Th2 or Treg polarized cells (Fig. 1E). However, additional stimulation of Th17-polarized cells with PMA/ ionomycin did not influence the expression of miR-133b and miR-206 (Fig. S1). Together, these results indicate that the two miRNAs of the miR-206/133b cluster are indeed co-regulated together and with the neighboring IL-17 locus. Th17 cells express elevated amounts of miR-133b and miR-206 in vivo Although in vitro polarization recapitulates the phenotypes of various Th lineages, in vitro and in vivo polarized cells may not necessarily be identical. Therefore, we assessed miR-133b and miR-206 expression levels in freshly isolated T cells. To this end, CD4+ T cells were sorted ex vivo based on IL-17A expression as assessed using an IL-17A secretion assay (Fig. 2A, right panels). Consistent with data obtained from in vitro polarized cells, IL-17A secreting cells expressed 16-fold higher levels of miR-133b and 26- fold higher levels of miR-206 when compared to IL-17A negative CD4+ T cells (Fig. 2A, left panel). Since miR-133b and, especially, miR-206 are important for muscle cell proliferation and differentiation [24], we sought to exclude the possibility that cell activation and proliferation correlated with heightened expression of these miRNAs. However, when comparing their expression in sorted activated/memory CD4+ T cells versus naive CD4+ T cells we found only a marginal upregulation of miR-206/133b in the former (,2 fold), which may reflect the few Th17 cells contained in the activated/memory CD4+ T cell population (Fig. S2). Furthermore, RORct has been described as signature transcrip- tion factor for Th17 cells. Therefore, we extended our analyses of miR-206/133b expression to CD4+ T cells positive or negative for RORct expression as assessed by expression of a GFP reporter gene knocked into the Rorc locus [25]. Low levels of GFP fluorescence intensity precluded the isolation of pure GFP-positive and GFP-negative populations (Fig. 2B, right panels). Neverthe- less, both miR-133b and miR-206 expression was elevated 7-fold and 8-fold, respectively, in cells enriched for RORct (GFP) expression when compared to RORct (GFP)-negative cells (Fig. 2B left panel). Thus, in CD4+ T cells miR-133b and miR-206 are co- expressed with IL-17A in vivo. Co-expression of IL-17A and miR-206/133b in cd T cells Recently, evidence has emerged that innate lymphocytes such as cd T cells constitute a major source of IL-17A [13,26]. We and others have shown that IL-17A production in cd T cells is largely restricted to a CC chemokine receptor (CCR) 6 expressing subpopulation [14,15]. It was thus instrumental to test whether co- expression of miR-133b and miR-206 is restricted to CD4+ Th17 cells or whether it is a more general phenomenon. To this end, we isolated cd T cells based on reporter gene expression from C57BL/ 6-Tcrd-H2BeGFP mice [27], in which all cd T cells express high levels of GFP, and separated these cells into CCR6-positive and CCR6-negative subsets (Fig. 3A, right panels). qRT-PCR revealed that CCR6-positive cd T cells expressed 15-fold and 21-fold higher levels of miR-133b and miR-206, respectively, when compared to CCR6-negative cd T cells (Fig. 3A, left panel). Expression of RORct is not only indicative of CD4+ Th17 cells, but also characterizes cd T cells expressing IL-17A [26]. Therefore, we isolated GFP-positive and GFP-negative cd T cells from RORct reporter mice as described for Figure 2B (Fig. 3B, right panels) and assessed the expression of miR-133b and miR- PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20171