The Orphan Nuclear Receptor NR4A3 Is Involved in the Function of Dendritic Cells Masanori Nagaoka, Takuya Yashiro, Yuna Uchida, Tomoaki Ando, Mutsuko Hara, Hajime Arai, Hideoki Ogawa, Ko This information is current as Okumura, Kazumi Kasakura and Chiharu Nishiyama of September 27, 2021. J Immunol 2017; 199:2958-2967; Prepublished online 11 September 2017; doi: 10.4049/jimmunol.1601911 http://www.jimmunol.org/content/199/8/2958 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/09/09/jimmunol.160191 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 23 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/199/8/2958.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! 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The Journal of Immunology The Orphan Nuclear Receptor NR4A3 Is Involved in the Function of Dendritic Cells Masanori Nagaoka,*,1 Takuya Yashiro,*,1 Yuna Uchida,* Tomoaki Ando,† Mutsuko Hara,† Hajime Arai,† Hideoki Ogawa,† Ko Okumura,† Kazumi Kasakura,* and Chiharu Nishiyama*,† NR4A3/NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, which is activated in a ligand- independent manner. To examine the role of NR4A3 in gene expression of dendritic cells (DCs), we introduced NR4A3 small interfering RNA (siRNA) into bone marrow–derived DCs and determined the expression levels of mRNA and proteins of cytokines, cell surface molecules, NF-kB signaling–related proteins, and transcription factors. The expression level of NR4A3 was markedly upregulated by TLR-mediated stimulation in DCs. NR4A3 knockdown significantly suppressed LPS, CpG, or poly(I:C)- mediated upregulation of CD80, CD86, IL-10, IL-6, and IL-12. Proliferation and IL-2 production levels of T cells cocultured with Downloaded from NR4A3 knocked-down DCs were significantly lower than that of T cells cocultured with control DCs. Furthermore, the expression of IKKb, IRF4, and IRF8 was significantly decreased in NR4A3 siRNA-introduced bone marrow–derived DCs. The knockdown exper- iments using siRNAs for IKKb, IRF4, and/or IRF8 indicated that LPS-induced upregulation of IL-10 and IL-6 was reduced in IKKb knocked-down cells, and that the upregulation of IL-12 was suppressed by the knockdown of IRF4 and IRF8. Taken together, these results indicate that NR4A3 is involved in TLR-mediated activation and gene expression of DCs. The Journal of Immunology, 2017, 199: 2958–2967. http://www.jimmunol.org/ he NR4A subfamily of the nuclear hormone receptor super- orphan receptors, which function as constitutively active (ligand family consists of NR4A3/NOR1 along with NR4A1/Nur77 independent) transcription factors, based on the identified three- T and NR4A2/Nurr1. These molecules are recognized to be dimensional structure of the ligand-binding domain (LBD) of NR4A2 (1). NR4A3 was first focused on in neurons, because NR4A3-deficient mice exhibit defects in the inner ear and in hippocampal devel- *Laboratory of Molecular Biology and Immunology, Department of Biological Science opment (2, 3). In subsequent studies, the role of NR4A3 in athero- and Technology, Faculty of Industrial Science and Technology, Tokyo University of sclerosis has been analyzed, because high expression of NR4A3 by guest on September 27, 2021 Science, Tokyo 125-8585, Japan; and †Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan together with NR4A1 and NR4A2 was detected in atherosclerotic 1M.N. and T.Y. contributed equally to this work. lesions (4). Hematopoietic cell–specific NR4A3 knockout (KO) ac- ORCID: 0000-0002-8548-4424 (T.A.). celerates atherosclerosis with monocytosis (5), whereas full NR4A3 KO mice show decreased atherosclerosis (6), suggesting that NR4A3 Received for publication November 9, 2016. Accepted for publication August 12, 2017. possesses hematopoietic cell–specific functions, especially those that This work was supported by the Funding Program for Next Generation World-Leading are monocyte lineage–specific, although NR4A3 is expressed in Researchers through Ministry of Education, Culture, Sports, Science and Technology various cell types including smooth muscle cells and neuronal of Japan (MEXT) Grant LS111 (to C.N.), the MEXT-Supported Program for the cells. A recent study investigating NR4A in acquired immunity Strategic Research Foundation at Private Universities (Translational Research Center, Tokyo University of Science), and the Tokyo Biochemical Research Foundation. T.Y. showed that triple KO mice lacking all NR4A members do not has been supported by Research Fellowship 10792 (2012–2014) from the Japanese produce regulatory T cells, and develop systemic autoimmunity Society for the Promotion of Science for Young Scientists. K.K. is supported by postdoctoral fellowships (2014–2015) from the Tokyo University of Science and (7). In this study, it was revealed that NR4A is involved in regulatory Research Fellowship 3241 (2016) from the Japanese Society for the Promotion of T cell development by directly transactivating the Foxp3 promoter. Science for Young Scientists. However, the role of NR4A3 in the development and function of a The microarray data presented in this article have been submitted to the Gene Expression typical professional Ag-presenting cell, the dendritic cell (DC), is Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101831) under accession number GSE101831. largely unknown, even though several studies indicated that NR4A3 plays critical roles in the development and inflammatory response of M.N. performed experiments and analyzed data; T.Y. designed research, performed experiments, analyzed data, and wrote the paper; Y.U. performed experiments and monocyte/macrophage lineages (5, 8, 9). In the current study, we analyzed data; T.A. analyzed data and wrote the paper; M.H. performed experiments analyzed the involvement of NR4A3 in the specific gene expression and analyzed data; H.A., H.O., and K.O. provided experimental tools; K.K. per- formed experiments; and C.N. designed research and wrote the paper. and maturation of DCs. Address correspondence and reprint requests to Prof. Chiharu Nishiyama, Laboratory of Molecular Biology and Immunology, Department of Biological Science and Tech- Materials and Methods nology, Faculty of Industrial Science and Technology, Tokyo University of Science, Mice and cells 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan. E-mail address: chinishi@rs. tus.ac.jp BALB/c and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, The online version of this article contains supplemental material. Japan), and OT-II mice were from the Jackson Laboratory (Bar Harbor, Abbreviations used in this article: BM, bone marrow; BMDC, bone marrow–derived ME). Bone marrow–derived DCs (BMDCs), splenic DCs, and BM-derived DC; DC, dendritic cell; KO, knockout; LBD, ligand-binding domain; siRNA, small macrophages were prepared as previously described (10–12). BMDCs interfering RNA; UTR, untranslated region. from C57BL/6 mice were used for a syngeneic T cell proliferation assay, and other experiments were performed with BALB/c cells. All animal Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 experiments were performed according to the approved guidelines of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601911 The Journal of Immunology 2959 Institutional Review Board of Tokyo University of Science, and this study anti-CD80 Ab (16-10A1; TONBO Biosciences, San Diego, CA) and PE- was specifically approved by the Animal Care and Use Committee of Tokyo conjugated anti-CD86 Ab (GL-1, TONBO). University of Science. Western blot analysis Knockdown experiments using small interfering RNA Western blotting was performed as described previously (11). Abs used in NR4A3 small interfering RNA (siRNA) [Stealth Select RNAi, MSS207089 (#1), this study are as follows: anti-NR4A3 (ab94507; Abcam), anti–IKK-b and MSS207090 (#2)], IRF4 siRNA (MSS205501), IRF8 siRNA (MSS236847), (D30C6), anti-phosphorylated IKK a/b (16A6), anti-IkBa (L35A5), anti- IKKb siRNA (MSS205392), NR4A1 siRNA (MSS205160), NR4A2 siRNA phosphorylated IkBa (14D4), anti-p65 (D14E12), anti-phosphorylated p65 (MSS276105), and control siRNA (Stealth siRNA negative control) were pur- (93H1; all from Cell Signaling Technology, Danvers, MA), anti-IRF4 (sc-6059; chased from Invitrogen (Carlsbad, CA). NR4A3 siRNA targeting the 39- Santa Cruz Biotechnology), anti-IRF8 (sc-6058; Santa Cruz Biotechnology), untranslated region (UTR) used for the NR4A3 coexpression experiment was anti-Flag (F1804; Sigma-Aldrich, St. Louis, MO), anti-hemagglutinin (S1827; synthesized using a custom service as follows: NM_015743.3_stealth_3911, Clontech), and anti–b-actin