Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in ab and cd T Cells Jan D. Haas1, Kiran Nistala2, Franziska Petermann 3 , Namita Saran1, Vijaykumar Chennupati1, Susanne Schmitz1, Thomas Korn3, Lucy R. Wedderburn2, Reinhold Fo¨ rster1, Andreas Krueger1., Immo Prinz1*. 1 Institute for Immunology, Hannover Medical School, Hannover, Germany, 2 Rheumatology Unit, Institute of Child Health, University College London, London, United Kingdom, 3 Klinikum rechts der Isar, Department of Neurology, Technical University Munich, Munich, Germany Abstract Differentiation of T helper 17 cells (Th17) is a multistep process that involves the cytokines IL-6, TGF-b, and IL-23 as well as IL-1b, IL-21, and TNF-a. Thereby, robust induction of the capacity to produce IL-17 involves epigenetic modifications of the syntenic Il17a/f locus. Using inbred mouse strains, we identified co-regulation of gene transcription at the Il17a/f locus with the nearby microRNAs miR-133b and miR-206 that are clustered approximately 45 kb upstream of Il17a/f. Expression of these microRNAs was specific for Th17 as compared to other CD4+ T cell subsets and this was equally valid for in vitro polarized and ex vivo derived cells. From all factors analyzed, IL-23 was the most important cytokine for the in vitro induction of miR-133b and miR-206 in naive CD4+ T cells of wild type mice. However, analysis of IL-23R deficient mice revealed that IL- 23R signaling was not essential for the induction of miR-133b and miR-206. Importantly, we found a similar co-regulation in CCR6+ and other cd T cell subsets that are predisposed to production of IL-17. Taken together, we discovered a novel feature of T cell differentiation towards an IL-17-producing phenotype that is shared between ab and cd T cells. Notably, the specific co-regulation of miR-133b and miR-206 with the Il17a/f locus also extended to human Th17 cells. This qualifies expression of miR-133b and miR-206 in T cells as novel biomarkers for Th17-type immune reactions. Citation: Haas JD, Nistala K, Petermann F, Saran N, Chennupati V, et al. (2011) Expression of miRNAs miR-133b and miR-206 in the Il17a/f Locus Is Co-Regulated with IL-17 Production in ab and cd T Cells. PLoS ONE 6(5): e20171. doi:10.1371/journal.pone.0020171 Editor: Alma Zernecke, Universita¨t Wu¨ rzburg, Germany Received February 10, 2011; Accepted April 14, 2011; Published May 26, 2011 Copyright: 2011 Haas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the German Research Foundation (DFG, www.dfg.de; Emmy-Noether Program, KR2320/2-1 and EXC62, ‘‘Rebirth,’’ A.K.; and SFB621-A14, I.P.). Open Access Publication supported by the DFG. JH, VC and NS are scholars of the Hannover Biomedical Research School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: prinz.immo@mh-hannover.de . These authors contributed equally to this work. Introduction microRNAs (miRNAs) are 21–24 nucleotide long non-coding RNAs which play a critical role in the regulation of gene expression. They usually target the 39 untranslated region (39- UTR) of their respective target gene(s) at the mRNA level – resulting in mRNA degradation, mRNA destabilization or inhibition of translation [1]. Gene regulation by miRNAs has recently emerged to be critical for both development and proper function of the immune system. Thus, various miRNAs such as miR-155, miR-223, miR-146, miR-150, miR-181a or the miR-17,92 cluster have been implicated in hematopoietic lineage decisions or in controlling different developmental checkpoints [2,3]. Furthermore, miRNAs contribute to the terminal differentiation of mature lymphocytes [4,5,6]. CD4+ T helper (Th) cells orchestrate the adaptive immune response so that appropriate effector mechanisms are elicited dependent on the nature of the invading pathogen. Th1 cells are induced to produce mainly IFN-c in order to fight viruses and intracellular bacteria, whereas Th2 cells produce IL-4/-5/-13 in response to infections by helminths and other parasites. More recently, Th17 cells have been described to secrete IL-17A, IL-17F and IL-22 to combat extracellular bacteria and fungi by stimulating epithelial cells to produce chemokines and cytokines, which drive the immune response e.g. by neutrophil influx [7,8]. Finally, CD4+ T cell differentiation can also lead to the development of tolerogenic induced regulatory T cells (iTreg). Dysregulation of Th cell differentiation may result in ineffective clearance of pathogens and/or in the development of autoimmune or allergic diseases. Therefore, Th cell differentiation needs to be tightly regulated. In this context, miRNAs have been shown to be involved in the development or function of all four main T helper subsets [9,10], i.e. miR-155 in Th1, miR-126 in Th2, miR-155 in Treg and miR-326 in Th17. For instance, deletion of a single miRNA, miR-155, can influence the fitness of Treg cells [11]. Furthermore, Ets-1, a negative regulator of Th17 differentiation, has been recently reported as a target of miR-326, the amount of which seems to be associated with disease relapse in multiple sclerosis patients [12]. However, these authors did not directly correlate the production of IL-17A, the key cytokine of Th17 cells, with disease onset and severity. In addition to CD4+ Th17 cells, cd T cells are major or even main producers of IL-17A depending on the physiological or PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20171