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Regnase-1 and Roquin Nonredundantly Regulate Th1 Differentiation Causing Cardiac Inflammation and Fibrosis

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Regnase-1 and Roquin Nonredundantly Regulate Th1 Differentiation Causing Cardiac Inflammation and Fibrosis Regnase-1 and Roquin Nonredundantly Regulate Th1 Differentiation Causing Cardiac Inflammation and Fibrosis This information is current as Xiaotong Cui, Takashi Mino, Masanori Yoshinaga, of October 2, 2021. Yoshinari Nakatsuka, Fabian Hia, Daichi Yamasoba, Tohru Tsujimura, Keizo Tomonaga, Yutaka Suzuki, Takuya Uehata and Osamu Takeuchi J Immunol published online 10 November 2017 http://www.jimmunol.org/content/early/2017/11/10/jimmun ol.1701211 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/11/10/jimmunol.170121 Material 1.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 10, 2017, doi:10.4049/jimmunol.1701211 The Journal of Immunology Regnase-1 and Roquin Nonredundantly Regulate Th1 Differentiation Causing Cardiac Inflammation and Fibrosis Xiaotong Cui,*,† Takashi Mino,* Masanori Yoshinaga,* Yoshinari Nakatsuka,* Fabian Hia,* Daichi Yamasoba,* Tohru Tsujimura,‡ Keizo Tomonaga,†,x Yutaka Suzuki,{ Takuya Uehata,* and Osamu Takeuchi* Regnase-1 and Roquin are RNA binding proteins that are essential for degradation of inflammatory mRNAs and maintenance of immune homeostasis. Although deficiency of either of the proteins leads to enhanced T cell activation, their functional relationship in T cells has yet to be clarified because of lethality upon mutation of both Regnase-1 and Roquin. By using a Regnase-1 conditional allele, we show that mutations of both Regnase-1 and Roquin in T cells leads to massive lymphocyte activation. In contrast, mutation of either Regnase-1 or Roquin affected T cell activation to a lesser extent than the double mutation, indicating that Regnase-1 and Roquin function nonredundantly in T cells. Interestingly, Regnase-1 and Roquin double-mutant mice suffered Downloaded from from severe inflammation and early formation of fibrosis, especially in the heart, along with the increased expression of Ifng, but not Il4 or Il17a. Consistently, mutation of both Regnase-1 and Roquin leads to a huge increase in the Th1, but not the Th2 or Th17, population in spleens compared with T cells with a single Regnase-1 or Roquin deficiency. Regnase-1 and Roquin are capable of repressing the expression of a group of mRNAs encoding factors involved in Th1 differentiation, such as Furin and Il12rb1, via their 39 untranslated regions. Moreover, Regnase-1 is capable of repressing Roquin mRNA. This cross-regulation may contribute to the synergistic control of T cell activation/polarization. Collectively, our results demonstrate that Regnase-1 and Roquin http://www.jimmunol.org/ maintain T cell immune homeostasis and regulate Th1 polarization synergistically. The Journal of Immunology, 2017, 199: 000–000. osttranscriptional regulation is critical for the control of induce inflammation (7, 8). These proinflammatory cytokine innate and adaptive immunity. Many immune-related mRNAs harbor AU-rich elements, as well as stem loops, and are P mRNAs have short half-lives because of conserved cis controlled by Regnase-1 and Roquin (9–11). These proteins reg- elements, including adenylate-uridylate (AU)-rich elements and ulate a set of common target mRNAs by recognizing common stem stem loop structures in their 39 untranslated regions (UTRs) (1, 2). loops (12). However, the mechanisms of regulation are different in Two major mRNA decay factors, Regnase-1 (also known as innate immune cells and embryonic fibroblasts. Regnase-1 degrades by guest on October 2, 2021 MCPIP1 or Zc3h12a) and Roquin (composed of Roquin-1 and the translationally active mRNA via its endoribonuclease activity by Roquin-2; also known as Rc3h1/2), are known to be immune associating with an RNA helicase, up-frameshift 1 (12). In contrast, regulators that affect inflammatory mRNA stability (3–6). Roquin controls translationally inactive mRNA independently of Regnase-1 and Roquin play essential roles in maintaining immune up-frameshift 1 activity by recruiting the CCR4-NOT deadenylase homeostasis. In innate immune cells, pattern recognition receptors, complex or the decapping enzyme EDC4 (6, 11). such as TLRs, initiate intracellular signaling pathways, leading to the Regnase-1 and Roquin are also important for the regulation of production of cytokines (e.g., TNF, IL-1b, and IL-6) that can further T cell activation. We have previously shown that mice lacking *Laboratory of Infection and Prevention, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; All authors participated in the discussion and interpretation of the study. X.C., T.M., †Department of Mammalian Regulatory Network, Graduate School of Biostudies, T.U., and O.T. designed the experiments. X.C. performed the majority of the exper- Kyoto University, Kyoto 606-8507, Japan; ‡Department of Pathology, Hyogo College iments and data analysis. T.M., T.U., M.Y., Y.N., D.Y., and K.T. helped with exper- of Medicine, Nishinomiya, Hyogo 663-8501, Japan; xLaboratory of RNA Viruses, iments. Y.S. performed RNA sequencing and the analysis of its data. F.H. performed Department of Virus Research, Institute for Frontier Life and Medical Sciences, detailed analyses of RNA sequence data. T.T. performed histological analyses. X.C. Kyoto University, Kyoto 606-8507, Japan; and {Laboratory of Functional Genomics, and O.T. wrote the manuscript. O.T. supervised the project. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The The RNA sequencing data presented in this article have been submitted to the DNA University of Tokyo, Kashiwa-shi, Chiba 277-8562, Japan Data Bank of Japan (http://www.ddbj.nig.ac.jp/index-e.html) under accession number ORCIDs: 0000-0002-7209-4312 (F.H.); 0000-0003-0405-7103 (K.T.). DRA006236. Received for publication August 23, 2017. Accepted for publication October 6, 2017. Address correspondence and reprint requests to Prof. Osamu Takeuchi, Laboratory of Infection and Prevention, Institute for Frontier Life and Medical Sciences, Kyoto This work was supported by AMED-CREST from the Japan Agency for Medical University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, Kyoto 606-8507, Japan. Research and Development and the Japan Society for the Promotion of Science E-mail address: [email protected] through the Core-to-Core Program. T.M. was funded by the Japan Society for the Promotion of Science KAKENHI (16K08832), Grants-in-Aid for Scientific Research The online version of this article contains supplemental material. on Innovative Areas “Genome Science” (221S0002 and 16H06279), the Takeda Abbreviations used in this article: ANA, antinuclear Ab; AU, adenylate-uridylate; Science Foundation, the Uehara Memorial Foundation, the Shimizu Foundation for CD4creReg-1fl/fl, CD4creRegnase-1fl/fl; CD4creReg-1fl/fl; RoqSan/San, CD4creRegnase-1fl/fl; Immunology and Neuroscience, the Naito Foundation, the Senri Life Science Foun- RoquinSan/San; KO, knockout; qPCR, quantitative PCR; Reg-1 KO, Regnase-1 KO; Roq dation, the Nakajima Foundation, and the Mochida Memorial Foundation for Medical DKO, Roquin-1/2 double KO; RoqSan/San, RoquinSan/San; TKO, triple KO; UTR, un- and Pharmaceutical Research. Y.N. was funded by a Japan Society for the Promotion translated region. of Science research fellowship for young scientists. T.U. was funded by the Shimizu Foundation for Immunology and Neuroscience. O.T. was funded by the Takeda Ó Science Foundation, the Daiichi Sankyo Foundation of Life Science, and the Uehara Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Memorial Foundation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701211 2 NONREDUNDANT ROLE OF REGNASE-1 AND ROQUIN IN T CELLS Regnase-1, specifically in T cells, showed spontaneous T cell Plasmids activation, leading to lymphocyte infiltration into various organs, The plasmids expressing Regnase-1 and Roquin have been described (3, 12, especially the lung and liver (9, 13). Regnase-1–deficient T cells 13). Full-length 39 UTRs of Tbx21, Furin, Il12rb1, Il18r, Roquin-1, highly produce Th-signature cytokines, including IFN-g, IL-17A, Roquin-2, and Regnase-1 sequences were inserted in the pGL3 vector. and IL-4 (13, 14). In contrast, mice harboring a M199R point Generation of Regnase-1–, Roquin-1–, and Roquin-2–deficient mutation in Roquin-1 (Sanroque), which has a dominant-negative Jurkat cell lines effect in suppressing the binding activity of Roquin-1 and its paralog, Roquin-2, developed autoimmunity that was accompa- Deletion of Regnase-1, Roquin-1, and Roquin-2 genes from Jurkat cells was nied by an increased follicular helper T cell population (5, 15, 16). performed using the CRISPR/Cas9 system. Briefly, pX330-GFP plasmid was digested with Bbs1 and ligated together with
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