The Orphan Nuclear 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

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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

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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. The Journal of Immunology

The Orphan 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 superfamily, which is activated in a - independent manner. To examine the role of NR4A3 in 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 of , 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 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 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 (AC-15; Sigma-Aldrich). sense, 59-CCUGGAGGCUGAGAUACAGAGUAAU-39, and anti-sense, 59-AU- UACUCUGUAUCUCAGCCUCCAGG-39 (Invitrogen). An electroporation T cell proliferation activity system, Nucleofector II (Lonza, Basel, Switzerland), was used to introduce BMDCs (C57BL/6) were subjected to syngeneic T cell proliferation assay siRNA into cells as previously described (13). 4 as follows. After introduction of siRNA, BMDCs (1 3 10 cells) generated Quantitative RT-PCR from C57BL/6 mice were pulsed with OVA323–339 peptide (Abgent, San Diego, CA), and then cocultured with 5 3 104 CD4+ T cells from OT-II The extraction of total RNA, reverse transcription for cDNA synthesis, and spleens for 72 h. T cell division was analyzed by monitoring CFSE (65- quantitative real-time PCR were performed using a ReliaPrep RNA Cell 08450; eBioscience, San Diego, CA) using flow cytometry. Miniprep System (Promega, Madison, WI), a ReverTra Ace qPCR RT kit

(TOYOBO, Osaka, Japan), and an Applied Biosystems StepOne real-time Microarray analysis Downloaded from PCR system (Applied Biosystems, Foster City, CA) with TaqMan Gene Global gene expression was analyzed by GeneChip Mouse Gene 1.0 ST Itgax Expression Assays (Applied Biosystems) #Mm00434455_m1 for , Array (Affymetrix) and GeneSpring 13.1 software (Agilent Technologies). Il6 Il10 #Mm00446190_m1 for , #Mm00439614_m1 for , #Mm00434174_m1 CEL files were summarized using an RMA16 algorithm with quantile for Il12b, #Mm00711660_m1 for Cd80, #Mm00444543_m1 for Cd86, normalization. The bottom 20% of values were considered background #Mm00516431_m1 for Irf4, #Mm00492567_m1 for Irf8, #4352339E for signals and thus excluded from analysis. Pathway analysis was performed Gapdh rodent , and a THUNDERBIRD probe qPCR Mix (TOYOBO), re- on QIAGEN’s Ingenuity Pathway Analysis (QIAGEN Redwood City, spectively. For the measurement of Nr4a1, Nr4a2, Nr4a3,andIkkb,the

www.qiagen.com/ingenuity). The microarray data of this study are avail- http://www.jimmunol.org/ following primer sets were used with THUNDERBIRD SYBR qPCR Mix: 9 9 able in Gene Expression Omnibus database (series accession no. GSE101831; NR4A1 forward, 5 -TCTGGTCCTCATCACTGATCGA-3 , NR4A1 reverse, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101831). 59-AATGCGATTCTGCAGCTCTTC-39, NR4A2 forward, 59-GCGCTTAGC- ATACAGGTCCAA-39,NR4A2reverse,59-GTGCAAGACCACCCCATTG-39, Expression plasmids NR4A3 forward, 59-CAGTGTCGGGATGGTTAAGGAA-39,NR4A3reverse, 59-CAGACGACCTCTCCTCCCTTT-39, IKKb forward, 59-TGGTGGTG- Expression plasmids, p33FLAG-NR4A3 and pIRES2-AcGFP1-33FLAG- CTCCCTGACA-39, IKKb reverse, CGTGAGGCCCTCGTTTGT-39. NR4A3, were generated as follows. A mouse NR4A3 cDNA fragment was amplified by PCR using a BMDC cDNA as a template and the following Determination of concentration oligonucleotides as primers: forward primer, 59-acgtgaattcACCCTGCGTG- CAAGCCCAGTA-39, and reverse primer, 59-acgtggatccGCTCAGAAAG- The concentrations of IL-2 (431001; BioLegend, San Diego, CA), IL-6 (M6000B), 9

GCAGGGTGTC-3 (NR4A3 cDNA and attached nucleotides are written by guest on September 27, 2021 IL-10 (M1000), and IL-12p40 (M1240; all from R&D Systems, Minneapolis, in capital letters and small letters, respectively; the EcoRI site introduced MN) in the culture media were each determined using an ELISA kit. into the forward primer and the BamHI site in reverse primer are under- Flow cytometry lined; TCA written in bold indicates the termination codon). After re- striction endonuclease digestion, the NR4A3 cDNA fragment was inserted Cell surface expression level of CD80 and CD86 was determined using a into EcoRI/BamHI-treated p33FLAG-CMV-7.1 (Sigma-Aldrich), and the MACS Quant (Miltenyi Biotec, Tubingen, Germany) with FITC-conjugated resulting plasmid was termed p33FLAG-NR4A3. The cDNA fragment of

FIGURE 1. Upregulation of NR4A3 expression in stimulated DCs. (A) Relative mRNA levels of CD11c and NR4A family members in BM cells cultured in the DC medium containing GM-CSF. Data are expressed as the ratio of the expression level in BM cells at day 0. (B) Relative mRNA level (left) and (right) levels of NR4A3 in BMDCs at 24 h after stimulation with 100 ng/ml LPS. Data are expressed as the ratio of the expression level of the respective control without stimulation. (C–E) NR4A3 protein level in BMDCs stimulated by TLR ligands (C), in BMDCs maintained in the presence of cycloheximide for 0–2 h (D), or in BMDCs maintained in the presence or absence of proteasome inhibitors (E). All results of quantitative RT-PCR of DCs in the current study are shown as mean 1 SD (n = 3), and similar results were obtained in three independent experiments. *p , 0.05. 2960 NR4A3 IN DENDRITIC CELLS

NR4A3 tagged with 33FLAG at the N terminus was amplified by PCR expression of IKKb was kindly provided by Dr. H. Nakano (Toho University using p33FLAG-NR4A3 as a template with the following forward primer, School of Medicine, Tokyo, Japan). Using Nucleofector II, 3 mg of expression 59-cactgctagcATTAACCATGGACTACAAAG-39 (NheI site and an initiation plasmid and 200 pmol of siRNA were cotransfected into BMDCs. codon are shown as underlined and bold, respectively) and the reverse primer Ubiquitination analysis used in the first cloning. The 33FLAG-NR4A3 cDNA was inserted into the NheI/BamHI site of pIRES-AcGFP (Clontech) to obtain pIRES-AcGFP- Expression plasmids, 1 mgofp33FLAG-NR4A3, and 1 mg of pME-HA- 33FLAG-NR4A3. The plasmid, pCR-Flag-IKKbeta (14), for the exogenous Ub (kindly provided by Dr. R. Sato, The University of Tokyo, Tokyo, Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Effects of NR4A3 knockdown on the activation and gene expression of DCs. BMDCs were transfected with either control siRNA (siCTRL) or NR4A3 siRNA (siNR4A3). At 24 h after transfection, the cells were left untreated or stimulated with LPS (A, B, D, and E) or TLR ligands (C) for 24 h. Quantitative RT-PCR (A, left, and B), Western blotting (A, right), ELISA (D), and FACS (E). Data of relative mRNA levels are expressed as the ratio of the expression level of the respective control siRNA-introduced nonstimulated cells. *p , 0.05. The Journal of Immunology 2961

Japan) were introduced into 293T cells (2 3 106) using 6 ml of FuGene6 CD80, CD86, IL-6, IL-10, and IL-12b (Fig. 4C, 4D). Interestingly, (Roche). After 24 h cultivation, cells were treated with 10 mM of MG132 approximately one third of upregulated by LPS stimulation for 12 h. Flag-tagged NR4A3 proteins were immunoprecipitated by M2 were downregulated by NR4A3 knockdown (Fig. 4C). Conversely, Affinity Gel (A2220; Sigma-Aldrich) and eluted by 33Flag peptide (F4799; Sigma-Aldrich). about one third of genes downregulated by LPS stimulation was overlapping genes upregulated by NR4A3 knockdown, suggesting a Statistical analysis counteracting effect of NR4A3 siRNA on LPS-responsive genes Statistical analysis was performed using a two-tailed Student t test, and (Fig. 4B–D). Consistent with this, Ingenuity Pathway Analysis de- p values , 0.05 were considered significant. tected the TLR signaling pathway as downregulated, as well as pathways involving DCs and T cells (Fig. 4E). These data suggest that NR4A3 knockdown suppressed the expression of a restricted Results number of genes relating to the stimulation and maturation of DCs, Effects of NR4A3 knockdown on activation and gene but did not lead to a global transcriptional downregulation. We expression of DCs excluded the possibility of an off-target effect of the NR4A3 siRNA In the DC generation from mouse BM cells by cultivation in the by using another NR4A3 siRNA encoding a different nucleotide presence of GM-CSF, we found that the expression level of NR4A3 sequence (Fig. 5A). Furthermore, we coexpressed NR4A3 using the mRNA was dramatically increased by upregulation of CD11c transient expression plasmid carrying NR4A3 cDNA without 39-UTR mRNA throughout development, whereas no apparent change was accompanied by NR4A3 siRNA designed at 39-UTR to eliminate the observed in mRNA levels of NR4A1 and NR4A2 (Fig. 1A). The side effects of siRNA, and found that the expression of CD86, which upregulating effect of GM-CSF on NR4A3 expression was con- was suppressed by the newly designed NR4A3 siRNA, was rescued Downloaded from sistent with data observed in a previous study showing that the by coexpression of NR4A3 (Fig. 5B). These results demonstrate that NR4A3 mRNA level in BM cells was transiently upregulated by pathogen-associated molecular pattern-induced stimulation upregu- GM-CSF stimulation, and peaked at 3 h after stimulation (5). When lates the NR4A3 protein level by accelerating gene transcription and LPS was added into the culture medium of BMDCs, the NR4A3 suppressing proteasome-mediated degradation, which enhances the mRNA level was raised 2-fold, and the protein level was dramati- gene expression and the function of activated BMDCs.

cally increased (Fig. 1B). Similarly, CpG or poly(I:C) stimulation http://www.jimmunol.org/ Effects of NR4A3 knockdown on NF-kB signaling increased the amount of NR4A3 protein (Fig. 1C), suggesting that NR4A3 was upregulated by TLR-mediated stimulation. Considering First, to evaluate the effect of NR4A3 knockdown on NF-kB that the increase in the NR4A3 band intensity was drastic in com- signaling, we determined the expression levels of IKK family pared with the increase in mRNA (Fig. 1B), and that maintenance of molecules based on a previous study indicating the transactivation BMDCs in the presence of protein synthesis inhibitor (Fig. 1D) and of the IKK family gene by NR4A3 (8), and found that the amount proteasome inhibitors (Fig. 1E) rapidly downregulated and markedly of IKKb transcript was significantly decreased in NR4A3 upregulated the NR4A3 protein level, respectively, NR4A3 expres- knocked-down BMDCs, whereas NR4A3 knockdown did not af- sion appears to be regulated not only in a transcriptional but also a fect the mRNA level of another IKK protein possessing phos- posttranslational manner. There is a possibility that ubiquitination is phatase activity, IKKa (Fig. 6A). Western blotting analysis by guest on September 27, 2021 involved in the degradation of NR4A3, because multiple bands were showed that the intensity of the IKKb protein band that was de- observed in Western blotting analysis of coexpression of NR4A3 tected in lysates prepared from immature BMDCs was increased and ubiquitin (Supplemental Fig. 1). It was also confirmed that by LPS stimulation, both of which were reduced under NR4A3 splenic DCs expressed markedly higher amounts of NR4A3 mRNA knocked-down conditions (Fig. 6B) in accordance with the mRNA compared with splenic lymphoid cells, and that the NR4A3 protein level (Fig. 6A). Under these conditions, the amount of phosphory- was detected in splenic DCs (data not shown). To investigate the role lated IkBa and RelA proteins was reduced in NR4A3 knocked-down of NR4A3 in the function of DCs, we analyzed gene expression BMDCs; in contrast, the total amount of RelA protein was increased of siRNA-introduced BMDCs. The expression levels of NR4A3 by NR4A3 knockdown (Fig. 6B), suggesting that decreased IKKb mRNA and protein were significantly reduced in NR4A3 siRNA- expression reduced the signal transduction degree downstream. introduced BMDCs compared with those of control siRNA- Next, to confirm whether reduced expression of cytokines and/or introduced BMDCs under both nonstimulated and stimulated costimulatory molecules in NR4A3 knocked-down activated BMDCs conditions (Fig. 2A). LPS stimulation markedly upregulated mRNA is dependent on a decrease of IKKb, BMDCs were transfected with levels of CD80, CD86, IL-6, IL-10, and IL-12b, which were sig- IKKb siRNA. As shown in Fig. 6C, introduction of IKKb siRNA nificantly suppressed by NR4A3 knockdown (Fig. 2B). NR4A3 significantly reduced the IKKb mRNA level in immature BMDCs knockdown also suppressed the CpG- or poly(I:C)-induced upreg- ulation of these cytokines and costimulatory molecules (Fig. 2C). The effect of NR4A3 knockdown on mRNA levels was also seen in the protein levels of released cytokines (Fig. 2D) and cell-surface costimulatory molecules (Fig. 2E). The T cell proliferation degree (Fig. 3A) and the amount of IL-2 released from T cells (Fig. 3B) were dramatically reduced when NR4A3 knocked-down BMDCs were used to stimulate T cells compared with those of control BMDCs, suggesting that the T cell proliferation activity of BMDCs was decreased by NR4A3 knockdown. To provide a more com- plete analysis of the genes affected by NR4A3 knockdown in BMDCs, we performed a microarray analysis. The numbers of FIGURE 3. Effects of NR4A3 knockdown in DCs on proliferation and upregulated and downregulated genes by NR4A3 knockdown IL-2 production levels of cocultured T cells. (A and B) At 48 h after siRNA were comparable when subtle (1.2–1.5-fold) changes are included transfection, BMDCs were pulsed with OVA peptide, and cocultured with (Fig. 4A). However, considerable expression changes ($1.5-fold) OT-II CD4+ T cells for 72 h. T cell division degree (A) and IL-2 released were accumulating in the downregulated genes (Fig. 4A–C), including from T cells (B) were determined. *p , 0.05. 2962 NR4A3 IN DENDRITIC CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Effects of NR4A3 knockdown on global gene expression. (A) The proportion of genes whose expressions were altered by NR4A3 siRNA compared with control siRNA. (B and C) Numbers of probe sets whose expressions were upregulated by $1.5-fold (B) and downregulated by $1.5-fold (C). Overlaps with probe sets whose expressions were altered by $2-fold in response to LPS stimulation are shown. The probe sets counteracted by NR4A3 siRNA are highlighted. (D) Relative mRNA levels quantified by microarray analysis. One sample for each condition. (E) Top 25 canonical pathways affected by NR4A3 siRNA were listed by Ingenuity Pathway Analysis. Pathways with coherent activity changes are shown with activity Z-scores. and canceled LPS-induced upregulation of IKKb mRNA. Under by a transient coexpression of IKKb, suggesting that IKKb’s func- these conditions, LPS-induced expression of IL-6 and IL-10 was tion is located downstream of NR4A3. These results indicate that significantly reduced by IKKb knockdown, even though the NR4A3 suppressed activation of NR4A3 knocked-down BMDCs is partly, at expression level in stimulated BMDCs was maintained (Fig. 6C). To least briefly, suppressed by upregulation of IL-6 and IL-10, due to assess the involvement of IKKb in the effects of NR4A3 knock- decreased IKKb expression. down, IKKb was exogenously expressed in NR4A3 knocked-down Although we showed that NR4A3 knockdown suppressed NF-kB BMDCs. As shown in Fig. 6D, suppression of LPS-induced up- signaling by downregulation of IKKb in DCs, a recent study re- regulation of IL-6 by NR4A3 knockdown was significantly restored ported NR4A3 to be an anti-inflammatory molecule because NR4A3 The Journal of Immunology 2963 Downloaded from

FIGURE 5. The effects of another siRNA clone and coexpression of NR4A3 resistant to siRNA. (A) The effect of another siRNA clone for NR4A3 on gene http://www.jimmunol.org/ expression in stimulated DCs. Clone #1 corresponds to NR4A3 siRNA used in Figs. 2–4. The nucleotide se- quence of clone #2 is different from that of clone #1. *p , 0.05. (B) Cell-surface expression of CD86 on BMDCs transfected with expression plasmid (pIRES2- AcGFP1-33FLAG-NR4A3) and NR4A3 siRNA designed at 39-UTR. by guest on September 27, 2021

overexpression caused downregulation of NF-kB signaling in smooth LPS stimulation on the expression of NR4A3 and IKKb was dif- muscle cells (15). In addition, NR4A3 was reported to be involved ferent between DCs and macrophages; briefly, LPS stimulation did in the development of human M2 macrophages (9). Therefore, to not upregulate mRNAs for IKKb and NR4A3 in macrophages confirm whether the role of NR4A3 in the expression of IKKb and (Fig. 7), whereas these expressions were increased in LPS-stimulated function of NF-kB signaling is specific to DCs or common in my- DCs (Fig. 6). In this condition, the expressions of IL-6 and IL-10 eloid lineages, we analyzed the effect of NR4A3 knockdown on gene were upregulated by LPS in macrophages, which were significantly expression in macrophages. As shown in Fig. 7, NR4A3 siRNA reduced by NR4A3 knockdown (Fig. 7). From this result, we con- significantly suppressed IKKb expression and LPS-induced upregu- clude that the involvement of NR4A3 in the expression of IKKb and lation of IL-6 and IL-10 in BM-derived macrophages. The effect of cytokines is commonly observed in DCs and macrophages. 2964 NR4A3 IN DENDRITIC CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Effects of NR4A3 knockdown on the expression and function of IKKb in DCs. (A) Quantitative RT-PCR to determine mRNA levels in NR4A3 siRNA-introduced BMDCs. (B) Aliquots of whole proteins (15 mg per lane) of the indicated cells were subjected to SDS-PAGE and immunoblot analysis using the indicated Abs. (C) Relative mRNA levels in IKKb siRNA-introduced BMDCs. (D) Relative mRNA levels of IL-6 in BMDCs where NR4A3 siRNA and IKKb expression plasmids were cotransfected. *p , 0.05.

Involvement of NR4A3 in IRF-dependent expression of measured mRNA levels of hematopoietic cell–specific transcrip- IL-12p40 tion factors PU.1 and IRF8, which transactivate the IL-12b pro- As mentioned in the above results, IKKb knockdown reduced LPS- moter with forming heterodimer, and of IRF4, which is also a induced expression of IL-6 and IL-10 (Fig. 6C). In contrast, LPS- hematopoietic cell–specific potentially form- induced expression of IL-12 was reduced in NR4A3 knocked-down ing a heterodimer with PU.1. The mRNA expression levels of IRF4 BMDCsaswellasIL-6andIL-10(Figs.2B,8A,left)butwas and IRF8 were significantly decreased in NR4A3 knocked-down completely retained in IKKb knocked-down BMDCs (Fig. 8A, BMDCs (Fig. 8C), whereas the PU.1 mRNA level was not af- right). To identify other factors involved in IL-12b expression, we fected by NR4A3 siRNA (data not shown). The protein levels of The Journal of Immunology 2965

FIGURE 7. Effects of NR4A3 knockdown on the expression and function of IKKb in macrophages. Quantitative RT-PCR to determine mRNA levels in NR4A3 siRNA-introduced macrophages. Data repre- sents mean 1 SD of three independent experiments with triplicate samples. *p , 0.05. Downloaded from http://www.jimmunol.org/

IRF4 and IRF8 were also reduced in NR4A3 siRNA-introduced mRNA. These results suggest that the positive role of NR4A3 in cells (Fig. 8B), suggesting that decreased IRF4 and/or IRF8 in LPS-dependent cytokine production and in the expression of IRF4 NR4A3 knocked-down BMDCs is the cause of the suppressed and IRF8 is not common in all of the NR4A family, but rather induction of IL-12b. Next, to evaluate the effect of IRF4 and/or specific for NR4A3. by guest on September 27, 2021 IRF8 levels on the magnitude of the IL-12b induction, we determined the mRNA level of IL-12b in IRF4 and/or IRF8 siRNA-introduced Discussion BMDCs. As shown in Fig. 8D, single knockdown of either IRF4 or NRs are transcription factors, which are composed of a DNA- IRF8 and double knockdown of IRF4 and IRF8 significantly re- binding domain and an LBD, of which 49, 47, and 48 members have duced the IL-12b mRNA level in nonstimulated and stimulated been found in the mouse, rat, and human, respectively (16). Several BMDCs. Although there is cross-regulation between NR4A3 and small hydrophobic compounds have been identified as endogenous IR4/IRF8, as the downregulation of NR4A3 was observed in IRF4 ligands binding to NRs via the LBD, which results in activation of and/or IRF8 siRNA-introduced activated BMDCs, the finding that NRs, including retinoid for NR1B/RAR members, estrogen for the IL-12b mRNA level declined in IRF8 knocked-down nonstimulated NR3A/ER members, and glucocorticoid for NR3C1/GR. In contrast, BMDCs, despite the NR4A3 level remaining unchanged, indicates NR4A members are termed orphan NRs, because they lack identi- that downregulation of IRF8 causes a decrease in IL-12b transcription. fied ligands and the LBD was judged to contain no cavity based on The IRF8 mRNA level significantly declined in IRF4 knocked-down the three-dimensional structure of NR4A2/Nurr1 (1). BMDCs, whereas IRF8 knockdown did not affect the IRF4 mRNA We have investigated the mechanism of gene expression in DCs, level, suggesting the possibility that IRF4 is indirectly involved in and found that a hematopoietic cell–specific transcription factor, IL-12b expression by regulating IRF8 transcription, and/or that it PU.1, positively regulates the expression of molecules playing directly transactivates the IL-12b promoter. important roles in DC function including CD80, CD86 (13), OX40L To evaluate the specific effect of NR4A3 on the expression of (17), TNF-a (18), cofactor CIITA, and subsequently MHC class II IRF4, IRF8, and cytokines, we analyzed mRNA levels of these (10). To find other target molecules of PU.1 in DCs that are involved molecules in NR4A1 or NR4A2 knocked-down BMDCs (Fig. 8E). in DC function as transcription factors, we performed a DNA micro- First, the effects of LPS stimulation on mRNA levels of NR4A1 array analysis and found that NR4A3 mRNA was dramatically de- and NR4A2 were opposite. Briefly, the NR4A2 mRNA level was creased in PU.1 siRNA-introduced BMDCs. The findings of our significantly increased by LPS stimulation, whereas the NR4A1 mRNA experiments, which showed that the mRNA level of NR4A3, but not was decreased by the stimulation. The NR4A2 siRNA-introduced NR4A1 and NR4A2, was dramatically increased in parallel with the DCs, in which NR4A2 mRNA levels were effectively reduced both CD11c mRNA level during DC development from BM cells (Fig. in the presence and absence of LPS stimulation, expressed compa- 1A), and that the NR4A3 mRNA level in splenic DCs was signifi- rable amounts of IRF4 and IRF8 mRNAs as those of control siRNA- cantly higher than that in other splenic cells (data not shown), pro- introduced DCs and exhibited a normal degree of upregulation of mpted us to analyze the role of NR4A3 in DC function. IL-12b and IL-10 mRNAs. In contrast, mRNA levels of IRF4 and In the current study, we demonstrated that NR4A3 is involved in LPS-induced upregulation of IL-12b and IL-10 were increased in the gene expression of activated DCs by altering NF-kB signaling. NR4A1 knocked-down DCs, with increased expression of NR4A3 In a recent study, overexpression of NR4A3 in vascular smooth 2966 NR4A3 IN DENDRITIC CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. Involvement of NR4A3 in the IRF-dependent expression of IL-12p40. (A) IL-12b mRNA levels in BMDCs transfected with NR4A3 siRNA (left) or IKKb siRNA (right). (B) The protein levels of IRF4 (left) and IRF8 (right) in siRNA-introduced BMDCs analyzed by Western blotting. (C and D) Relative mRNA levels of transcription factors (C) and IL-12b (D) in NR4A3 siRNA-, IRF4 siRNA-, and/or IRF8 siRNA-introduced BMDCs. (E) Relative mRNA levels of transcription factors and cytokines in NR4A1 siRNA- (top) or NR4A2 siRNA- (bottom) introduced BMDCs. *p , 0.05. muscle cells reduced LPS-, TNF-a–, or oxLDL-mediated stimu- NR4A3 does not form a heterodimer with RXR (19, 20), we could lation (15). Therefore, it was concluded that NR4A3 exhibited an not exclude the possibility that NR4A3 may function with forming anti-inflammatory function with preventing the activation of NF-kB a complex with other transcription factors. In the Western blotting signaling, which is opposite to the findings of our present study. This analyses of the current study, multiple bands appeared in the staining discrepancy may be due to the difference in cell types, as hemato- for anti-NR4A3 Ab. As the intensity of all bands was reduced poietic cell–specific KO of NR4A3 accelerated atherosclerosis (5), by NR4A3 knockdown (Fig. 2A), we suggest that these molecules whereas full KO of NR4A3 prevented this disease (6). Alterna- recognized by anti-NR4A3 Ab are translated from NR4A3 mRNAs. tively, considering that there is possible cooperation with other mol- When NR4A3 was exogenously expressed using full-length NR4A3 ecules, overexpression experiments are not simple gain-of-function cDNA, the molecular mass of this protein was ∼75 kDa, suggesting studies. In the current study, we confirmed that the gene expression of that a major band at 75 kDa is translated from full-length mRNA activated macrophages was reduced by NR4A3 knockdown. There- and that others may be produced by translation from splice variants fore, we conclude that the suppression of stimulation-induced sig- and/or posttranslational modification. In the Western blotting analysis naling by NR4A3 knockdown is a common observation in myeloid using phos-tag gel, several extra bands were detected (data not lineages. Although it was reported that unlike NR4A1 and NR4A2, shown), suggesting that NR4A3 is phosphorylated in some condi- The Journal of Immunology 2967 tions. However, we think that the marked difference of molecular 2. Ponnio, T., Q. Burton, F. A. Pereira, D. K. Wu, and O. M. Conneely. 2002. The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of weights cannot be explained by phosphorylation. The Nr4a3 gene the mouse inner ear. Mol. Cell. Biol. 22: 935–945. possesses at least two promoters, which may also affect the ex- 3. Po¨nnio¨, T., and O. M. Conneely. 2004. nor-1 regulates hippocampal axon pression pattern of NR4A3 variants in various cells. The discrepancy guidance, pyramidal cell survival, and seizure susceptibility. Mol. Cell. Biol. 24: k 9070–9078. of the role of NR4A3 in NF- B signaling may be explained by the 4. Arkenbout, E. K., V. de Waard, M. van Bragt, T. A. van Achterberg, variation in NR4A3 proteins. Regardless, to clarify this issue, further J. M. Grimbergen, B. Pichon, H. Pannekoek, and C. J. de Vries. 2002. Protective detailed experiments are required. function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Cir- The reduced level of IKKb was identified to be a cause of the culation 106: 1530–1535. suppressed expression of IL-6 and IL-10 in LPS-stimulated DCs. 5. Qing, H., Y. Liu, Y. Zhao, J. Aono, K. L. Jones, E. B. Heywood, D. Howatt, C. M. Binkley, A. Daugherty, Y. Liang, and D. Bruemmer. 2014. Deficiency of A previous study reported that NR4A1, 2, and 3 transactivate the the NR4A orphan nuclear receptor NOR1 in hematopoietic stem cells accelerates IKKi promoter, which contains a functional NR4A binding site, in- atherosclerosis. Stem Cells 32: 2419–2429. dicating that the NR4A family is involved in the NF-kB–dependent 6. Zhao, Y., D. A. Howatt, F. Gizard, T. Nomiyama, H. M. Findeisen, E. B. Heywood, K. L. Jones, O. M. Conneely, A. Daugherty, and D. Bruemmer. expression of inflammatory genes in macrophages (9). In the current 2010. Deficiency of the NR4A orphan nuclear receptor NOR1 decreases study the IKKi transcript level was not affected by NR4A3 knock- monocyte adhesion and atherosclerosis. Circ. Res. 107: 501–511. down (data not shown) and the NR4A family has 7. Sekiya, T., I. Kashiwagi, R. Yoshida, T. Fukaya, R. Morita, A. Kimura, H. Ichinose, D. Metzger, P. Chambon, and A. Yoshimura. 2013. Nr4a receptors not been found on the IKKb promoter so far. Further detailed are essential for thymic regulatory T cell development and immune homeostasis. analysis is required to clarify the role of NR4A3 in the expression of Nat. Immunol. 14: 230–237. b 8. Pei, L., A. Castrillo, M. Chen, A. Hoffmann, and P. Tontonoz. 2005. Induction of IKK . NR4A orphan nuclear receptor expression in macrophages in response to in- NR4A3 knockdown suppressed the expression of IRF4 and flammatory stimuli. J. Biol. Chem. 280: 29256–29262. Downloaded from IRF8. IRF4 was recently identified to be important for in vivo 9. De Paoli, F., J. Eeckhoute, C. Copin, J. Vanhoutte, C. Duhem, B. Derudas, J. Dubois-Chevalier, S. Colin, C. Zawadzki, B. Jude, et al. 2015. The neuron- development of a DC subpopulation specialized to regulate Th2 derived orphan receptor 1 (NOR1) is induced upon human alternative macrophage differentiation (21, 22). Therefore, NR4A3 activity might not only polarization and stimulates the expression of markers of the M2 phenotype. affect the maturation of DCs but also the population balance of Atherosclerosis 241: 18–26. 10. Kitamura, N., H. Yokoyama, T. Yashiro, N. Nakano, M. Nishiyama, S. Kanada, DCs. It was shown that IL-4–induced polarization of human M2 T. Fukai, M. Hara, S. Ikeda, H. Ogawa, K. Okumura, and C. Nishiyama. 2012. macrophages induced upregulation of NR4A3, whose knockdown Role of PU.1 in MHC class II expression through transcriptional regulation of http://www.jimmunol.org/ class II transactivator pI in dendritic cells. J. Allergy Clin. Immunol. 129: 814– suppressed the expression of several M2 markers (9). NR4A3 may 824.e816. be a key molecule for the regulation of immune responses via modu- 11. Yashiro, T., M. Kubo, H. Ogawa, K. Okumura, and C. Nishiyama. 2015. PU.1 lation of the subpopulation balance of DCs and macrophages. suppresses Th2 cytokine expression via silencing of GATA3 transcription in dendritic cells. PLoS One 10: e0137699. Detailed analysis is required to evaluate the effect of NR4A3 activity 12. Weischenfeldt, J., and B. Porse. 2008. Bone marrow-derived macrophages in various immune-related cells. Although IRF4 and IRF8 mRNAs (BMM): isolation and applications. CSH Protoc. 2008: pdb.prot5080. were decreased by NR4A3 knockdown, the role of NR4A3 in the 13. Kanada, S., C. Nishiyama, N. Nakano, R. Suzuki, K. Maeda, M. Hara, N. Kitamura, H. Ogawa, and K. Okumura. 2011. Critical role of transcription expression of IRF4 and IRF8 is still unknown. A chromatin immu- factor PU.1 in the expression of CD80 and CD86 on dendritic cells. Blood 117: noprecipitation assay using an anti-NR4A3 Ab suitable for significant 2211–2222.

14. Nakano, H., M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, and by guest on September 27, 2021 interaction with intact NR4A3 may be useful to clarify the - K. Okumura. 1998. Differential regulation of IkappaB kinase alpha and beta by tionship between NR4A3 and IRFs. two upstream kinases, NF-kappaB-inducing kinase and mitogen-activated pro- In the current study, we demonstrated that NR4A3 knockdown tein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95: 3537–3542. 15. Calvayrac, O., R. Rodrı´guez-Calvo, I. Martı´-Pamies, J. Alonso, B. Ferra´n, dramatically affected the function of DCs and the subsequent S. Aguilo´, J. Crespo, A. Rodrı´guez-Sinovas, C. Rodrı´guez, and J. Martı´nez- + proliferation of CD4 T cells. We found that the function of a Gonza´lez. 2015. NOR-1 modulates the inflammatory response of vascular certain population of DCs and the activation of T cells are impaired smooth muscle cells by preventing NFkB activation. J. Mol. Cell. Cardiol. 80: 34–44. in NR4A3 KO mice (T. Yashiro and C. Nishiyama, unpublished ob- 16. Zhang, Z., P. E. Burch, A. J. Cooney, R. B. Lanz, F. A. Pereira, J. Wu, servations), and a recent report demonstrated that NR4A3 KO mice R. A. Gibbs, G. Weinstock, and D. A. Wheeler. 2004. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution are susceptible to infection due to the migration defect of DCs (23), from the rat genome. Genome Res. 14: 580–590. suggesting that NR4A3 plays an important role in DC-mediated 17. Yashiro, T., M. Hara, H. Ogawa, K. Okumura, and C. Nishiyama. 2016. Critical responses in vivo. We plan to further investigate the role of NR4A3 role of transcription factor PU.1 in the function of the OX40L/TNFSF4 promoter in dendritic cells. Sci. Rep. 6: 34825. in DCs in adaptive immunity, to clarify its efficacy as a therapeutic 18. Fukai, T., C. Nishiyama, S. Kanada, N. Nakano, M. Hara, T. Tokura, S. Ikeda, target for immune-related diseases. H. Ogawa, and K. Okumura. 2009. Involvement of PU.1 in the transcriptional regulation of TNF-alpha. Biochem. Biophys. Res. Commun. 388: 102–106. 19. Perlmann, T., and L. Jansson. 1995. A novel pathway for vitamin A signaling Acknowledgments mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev. 9: We are grateful to the members of the Laboratory of Molecular Biology and 769–782. 20. Zetterstro¨m, R. H., L. Solomin, T. Mitsiadis, L. Olson, and T. Perlmann. 1996. Immunology, Department of Biological Science and Technology, Tokyo heterodimerization and developmental expression distin- University of Science, for constructive discussions and technical support. guish the orphan nuclear receptors NGFI-B, Nurr1, and Nor1. Mol. Endocrinol. 10: 1656–1666. 21. Gao, Y., S. A. Nish, R. Jiang, L. Hou, P. Licona-Limo´n, J. S. Weinstein, H. Zhao, Disclosures and R. Medzhitov. 2013. Control of T helper 2 responses by transcription factor The authors have no financial conflicts of interest. IRF4-dependent dendritic cells. Immunity 39: 722–732. 22. Williams, J. W., M. Y. Tjota, B. S. Clay, B. Vander Lugt, H. S. Bandukwala, C. L. Hrusch, D. C. Decker, K. M. Blaine, B. R. Fixsen, H. Singh, et al. 2013. Transcription factor IRF4 drives dendritic cells to promote Th2 differentiation. References Nat. Commun. 4: 2990. 1. Wang, Z., G. Benoit, J. Liu, S. Prasad, P. Aarnisalo, X. Liu, H. Xu, N. P. Walker, 23. Park, K., Z. Mikulski, G. Y. Seo, A. Y. Andreyev, P. Marcovecchio, A. Blatchley, and T. Perlmann. 2003. Structure and function of Nurr1 identifies a class of M. Kronenberg, and C. C. Hedrick. 2016. The transcription factor NR4A3 ligand-independent nuclear receptors. Nature 423: 555–560. controls CD103+ dendritic cell migration. J. Clin. Invest. 126: 4603–4615.