ARTICLE Received 17 Feb 2015 | Accepted 27 May 2015 | Published 13 Jul 2015 DOI: 10.1038/ncomms8639 OPEN MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3 Lingyun Zhang1, Fang Ke1, Zhaoyuan Liu1, Jing Bai1, Jinlin Liu1, Sha Yan1, Zhenyao Xu1, Fangzhou Lou1, Hong Wang1, Huiyuan Zhu1, Yang Sun1, Wei Cai1, Yuanyuan Gao1, Qun Li2, Xue-Zhong Yu3, Youcun Qian4, Zichun Hua5, Jiong Deng6, Qi-Jing Li7 & Honglin Wang1,8 Peripherally derived regulatory T (pTreg) cell generation requires T-cell receptor (TCR) signalling and the cytokines TGF-b1 and IL-2. Here we show that TCR signalling induces the microRNA miR-31, which negatively regulates pTreg-cell generation. miR-31 conditional deletion results in enhanced induction of pTreg cells, and decreased severity of experimental autoimmune encephalomyelitis (EAE). Unexpectedly, we identify Gprc5a as a direct target of miR-31. Gprc5a is known as retinoic acid-inducible protein 3, and its deficiency leads to impaired pTreg-cell induction and increased EAE severity. By generating miR-31 and Gprc5a double knockout mice, we show that miR-31 promotes the development of EAE through inhibiting Gprc5a. Thus, our data identify miR-31 and its target Gprc5a as critical regulators for pTreg-cell generation, suggesting a previously unrecognized epigenetic mechanism for dysfunctional Treg cells in autoimmune diseases. 1 Shanghai Institute of Immunology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 2 Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 3 Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425, USA. 4 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 5 State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, China. 6 Key Laboratory of Cell Differentiation and Apoptosis of Minister of Education, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 7 Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710, USA. 8 Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. Correspondence and requests for materials should be addressed to H.W. (email: [email protected]). NATURE COMMUNICATIONS | 6:7639 | DOI: 10.1038/ncomms8639 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8639 cells serve as a central cellular player in adaptive promoter24. Gprc5a was targeted by miR-31 through direct immunity, and their activation and differentiation binding to its 30-untranslated regions (30-UTR), and its deficiency Tare elicited by signals from T-cell receptor (TCR), resulted in the impairment of pTreg-cell induction and increased co-stimulatory receptors and various cytokines1. Once activated EAE severity. Thus, our findings demonstrated that miR-31 þ by an antigen, naive CD4 T cells proliferate and differentiate negatively regulated pTreg-cell generation by targeting Gprc5a, into various T helper (TH) cell subsets, including TH1, TH2, TH17 suggesting a novel epigenetic mechanism for impaired pTreg-cell and regulatory T (Treg) cells, that release different cytokines and induction in autoimmunity. exhibit distinct effector functions2. Besides their critical role in driving immune responses against infections, TH1 and TH17 cells participate in the pathogenesis of autoimmune inflammatory Results diseases, such as experimental autoimmune encephalomyelitis miR-31 expression is triggered by TCR signalling. Report of 3 (EAE) . Moreover, naive T cells differentiate into Treg cells FoxP3 mRNA harbouring the target sequence of miR-31 exhibiting immunosuppressive capacity, and the transcriptional promoted us to investigate its role in the induction and/or 4,5 factor FoxP3 controls their development and fucntion . function of Treg cells which are vital for preventing autoimmune 21 According to their origins, Treg cells are divided into disease . We induced EAE, an animal model of MS, with myelin thymus-derived Treg (tTreg) cells derived from the thymus, oligodendrocyte glycoprotein peptide (MOG35–55) in mice to peripherally derived regulatory T (pTreg) cells generated out investigate expression pattern of miR-31 in pathogenic T cells in of the thymus under various inductive signals, and in vitro- the tissue-specific autoimmune inflammation. miR-31 expression 6,7 þ induced regulatory T (iTreg) cells . It is now clear that naive was assessed in splenocytes and sorted CD4 T cells at day 10 CD4 þ T cells sorted as FoxP3 À in the thymus possess full post immunization. We found that the expression of miR-31 was þ potential to differentiate into pTreg cells, thus are potential targets significantly increased in both splenocytes and pathogenic CD4 for therapeutic interventions for chronic inflammatory T cells in EAE mice compared with healthy controls (Fig. 1a). We 7,8 þ À high diseases . Independent on thymus, pTreg cells differentiate in next stimulated the TCR of naive T (CD4 CD25 CD62L ) secondary lymphoid organs and tissues, and require TCR cells with plate-coated anti-CD3- and soluble anti-CD28-specific signalling and the cytokines TGF-b and IL-2 (ref. 7), and only antibodies, and we detected that the miR-31 expression was a low antigen dose of a high-affinity TCR ligand is optimal to increased B125-fold in activated CD4 þ T cells compared with 9 generate a persistent population of pTreg cells in vivo . untreated naive T cells (Fig. 1b). Together, these data suggest that þ So far, dysfunctional Treg cells are identified in several TCR signalling induces miR-31 expression in CD4 T cells. autoimmune disorders including mutiple sclerosis (MS)10–12. Because the TCR signal coordinating with lineage-specific One of the failures of Treg-cell-mediated immunoregulation is cytokines triggers naive T cells to differentiate into specialized inadequate numbers of Treg cells that may be due to defective effector cells, we sought to examine miR-31 expression in 13 induction of pTreg cells in the periphery . Thus, understanding different T-cell subsets. We differentiated naive T cells in vitro of molecular mechanisms underlying pTreg-cell generation might under polarizing conditions for the generation of TH1, TH17 and provide deeper insights into physiological and pathological iTreg cells in cultures as these T-cell subsets are critical in the immune responses in autoimmune inflammatory diseases. pathology of EAE25–27. At 4 days after activation, miR-31 MicroRNAs (miRNAs) are single-stranded, small noncoding expression was 29.5-fold higher in TH1 cells, 47.4-fold higher in RNAs located in introns or exons of protein-coding genes TH17 cells, but there was 5.6-fold reduction in iTreg cells than that as well as in non-coding genes14. miRNAs have been implicated of naive T cells (Fig. 1c), which suggested a possible regulatory in maintaining immune homeostasis during stress, such as role for miR-31 in CD4 þ T-cell lineage differentiation. Because inflammation, by regulating gene expression at post- miR-31 has been implicated to negatively regulate FoxP3 15 21 transcriptional level . Several studies have reported that expression in human Treg cells , we sought to investigate specific miRNA signatures were observed for specialized whether upregulation of miR-31 coincides with downregulation T-cell subsets, and these miRNAs are dynamically regulated of FoxP3 during iTreg-cell induction. We polarized naive T cells 16,17 gfp during T-cell maturation . Dicer and Drosha are two derived from FoxP3 reporter mice into iTreg cells, and essential components for the generation of miRNA, and loss of examined miR-31 expression in sorted CD25 þ FoxP3 À and these factors leads to defects in lymphocyte differentiation and CD25 þ FoxP3 þ cells. The miR-31 expression in CD25 þ FoxP3 þ autoimmune inflammation18,19. Recently, accumulating evidence cells was B90-fold lower than that in CD25 þ FoxP3 À has demonstrated that miRNAs are also crucial for Treg-cell population (Fig. 1d,e). These data demonstrate that miR-31 is 17–20 development, function and stability .Treg cells display a set preferentially diminished in iTreg cells. Although the expression 21 of miRNAs that is distinct from conventional T cells . However, of miR-31 was slightly increased in tTreg and pTreg cells intrinsic miRNAs involved in the polarization of Treg cells compared with iTreg cells, its expression in either tTreg or pTreg from naive T cells in vitro and in vivo settings are largely was not significantly different between control and EAE mice, undetermined. suggesting that Treg cells maintain baseline miR-31 expression In this study, we showed that miR-31 expression was triggered in vivo (Supplementary Fig. 1a). To further identify why iTreg cells by TCR signalling, and downregulated by TGF-b1-induced exhibit diminished levels of miR-31, we activated naive T cells FoxP3. The conditional deletion of miR-31 in CD4 þ T cells with CD3- and CD28-specific antibodies in the absence or led to enhanced induction of pTreg cells in the periphery, and presence of TGF-b1, and measured the time-dependent decreased severity of EAE. Retinoic acid (RA) regulates the appearance of miR-31. miR-31 abundance was gradually expression of genes required for cell proliferation, differentiation increased during the stimulation with CD3 and CD28 and survival by binding its nuclear retinoic acid receptors (RARs) antibodies in the absence of TGF-b1, however, decreased at and retinoid X receptors (RXRs)22. Although RA has been shown 12 h when FoxP3 was induced by adding TGF-b1 (Fig. 1f). 23 to enforce pTreg-cell generation , the mechanism by which RA Moreover, TGF-b1 dose-dependently decreased miR-31 promotes pTreg-cell induction is ill-defined.