BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3 dephosphorylation

BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3... Abstract Growing evidence has revealed that the transcription factor basic leucine zipper transcription factor ATF-like 2 (BATF2) has unique transcriptional activities, including regulating cytokines via TLR signals in macrophages, which affect mortality due to infection and cancer. On the basis of genome-wide analyses using the chromatin immunoprecipitation-sequencing technique, we found that dual-specificity phosphatase 2 (Dusp2) had a significantly lower acetyl-histone status in Batf2−/− bone marrow-derived macrophages (BMDMs) compared with wild-type (WT) BMDMs. The phosphatase DUSP2 has been reported to play a critical role in inflammatory responses. Therefore, we evaluated the BATF2 transcriptional activities on the Dusp2 promoter. We found that the DUSP2 and IL-12 p40 expression levels were significantly lower in Batf2−/− BMDMs than in WT controls following their stimulation with TLR7 ligands. Further in vitro studies revealed that phospho-STAT3 was up-regulated and NF-κB p50/p65 were down-regulated in Batf2−/− BMDMs compared with their levels in WT controls. Additionally, Th1 immunity was impaired in Batf2−/− mice following their stimulation with TLR7 ligands. We also found that BATF2 interacts with NF-κB p65 and promotes DUSP2 expression through the NF-κB-binding site in the Dusp2 promoter at −203 to −121. Collectively, our findings suggest that BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3 dephosphorylation. IL-12, inflammation, macrophage, Th1, TLR7 Introduction The basic leucine zipper transcription factor ATF-like 2 (BATF2) is a member of the BATF family. Along with BATF2, the BATF family includes BATF (SFA2) and BATF3 (JDP1; p21SNFT), and its members also belong to the AP-1 basic leucine zipper transcription factor family. Although BATF family members were initially thought to function only as inhibitors of AP-1 (1), recent studies have suggested that these factors additionally have unique and positive transcriptional activities (2). It was recently reported that the IL-12 p40 expression was significantly lower in Batf2−/− macrophages following their stimulation by TLR7 ligands, leading to the impairment of Th1 immunity and the attenuation of the anti-tumor effect in Batf2−/− mice (3). Additionally, Batf2-deficient mice have a higher mortality from Toxoplasma gondii infection compared with wild-type (WT) mice (4). On the basis of these findings, we analyzed the genome-wide status of histone acetylation (H3K27Ac) in WT and Batf2−/− bone marrow-derived macrophages (BMDMs) that had been stimulated with R848, a TLR7 ligand, by using the chromatin immunoprecipitation-sequencing (ChIP-Seq) technique. The results indicate that dual-specificity phosphatase 2 (Dusp2) had a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs. DUSP2, also called phosphatase of activated cells 1 (PAC-1), is a member of the DUSP family. Unlike other DUSP family members, the expression of DUSP2 is restricted to immune cells (5) and has been mostly associated with immune tissues, such as the spleen and thymus (6, 7). As with Batf2−/− macrophages, Dusp2−/− macrophages have impaired effector responses, including inflammatory mediator production (5). Additionally, the IL-12 p40 expression level is lower in Dusp2-deficient macrophages following their stimulation by LPS (5). Therefore, in this study, we further assessed the role of BATF2, particularly its positive transcriptional activities via TLR signals, and evaluated its transcriptional activities on the Dusp2 promoter. Methods Mice Batf2−/− mice were generated as previously described (3). Mice were maintained in our specific pathogen-free animal facility according to the institutional guidelines. Experiments were generally performed at 6–12 weeks of age. All animal experiments were performed with approval from the Animal Research Committee of the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). Cells RAW 264.7 cells, which were described previously (8), were grown in RPMI-1640 containing 10% FCS. BMDMs were generated as described previously (9). Briefly, mouse bone marrow cells were obtained from femurs and differentiated into macrophages in RPMI-1640 with 10% FCS containing 3 ng ml−1 macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA). Medium was changed on day 3. Macrophages were harvested and stimulated with R848 (100 ng ml−1; InvivoGen, San Diego, CA, USA) on day 5. Plasmids Batf2 cDNA was obtained via PCR from a mouse cDNA library as described previously (3). Dusp2 and Myc-tagged Batf2 were purchased from OriGene Technologies (MC204715 and MR203728, respectively; Rockville, MD, USA). Dusp2 pro-Luc plasmid was produced by cloning. The mouse Dusp2 promoter was obtained via PCR from normal genomic DNA and subsequently cloned into the Kpn I–Bgl II sites of the restricted Il12b pro-Luc construct as previously described (10). The Il12b 400-bp pro-Luc, RelA cFlag pcDNA3 and c-Rel cFlag pcDNA3 were gifts from Stephen Smale; Howard Hughes Medical Institute, Chevy Chase, MD, USA; and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA, USA, respectively (Addgene plasmid 20020, 20012 and 20013, respectively; Cambridge, MA, USA) (11, 12). ChIP-Seq analysis ChIP analysis was performed using ChIP-IT Express Enzymatic (Active Motif, Carlsbad, CA, USA). First, 2 × 107 BMDMs were stimulated with R848 (100 ng ml−1). Six hours later, the cells were fixed with 20 ml of 1% formaldehyde for 10 min, followed by a wash with 10 ml of PBS, and the fixation reaction was stopped by adding 10 ml of glycine stop fix solution for 5 min. Cell extracts were prepared in lysis buffer (10 mM HEPES-KOH [pH 7.8], 10 mM KCl, 0.1 mM EDTA [pH 8.0], protease inhibitor cocktail [Roche, Basel, Switzerland], 0.1% Nonidet P-40) and were homogenized using a Dounce homogenizer (BioMasherII; Nippi, Tokyo, Japan) on ice. Enzymatic shearing to produce DNA fragments was performed for 30 min. Immunoprecipitation then was performed on these fragments using 8 µg of one of the following antibodies: anti-BATF2 (L-24) (sc-130972; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-histone H3 (acetyl K27) (ab4729; Abcam, Cambridge, UK). After the samples had been incubated overnight with 50 µl of protein G magnetic beads, the beads were washed. DNA was eluted in 40 µl of elution buffer AM2, and 40 µl of reverse cross-linking buffer was then added. After centrifugation, the resulting supernatants were collected and incubated at 95°C for 15 min. After samples were returned to room temperature, 2 µg of proteinase K was added, and the samples were incubated for 1 h at 37°C. DNA was then purified via ethanol precipitation. The ChIP-Seq was performed by Filgen (Aichi, Japan), and the resulting data were analyzed by CLC Genomics Workbench 10 (Qiagen, Hilden, Germany). Topical application of imiquimod WT and Batf2−/− mice received a daily topical dose of 62.5 mg of 5% imiquimod cream (Beselna Cream 5%, Mochida, Tokyo, Japan) on a shaved patch of back for 6 consecutive days (corresponding to a daily dose of 3.125 mg of the active compound), as previously described (13). Control mice were treated similarly with a control vehicle cream (Vaseline, Unilever, London, UK). Histological analysis Frozen 10-μm tissue sections in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) or cells plated in chamber slides (SCS-008; Matsunami, Osaka, Japan) were fixed in 4% paraformaldehyde and blocked with 3% BSA in 1× PBS. Hematoxylin and eosin (H&E) or immunofluorescent staining was performed. The antibodies and reagents for immunofluorescence analyses were purchased as follows: anti-F4/80–APC (BM8.1; TONBO Biosciences, San Diego, CA, USA), anti-DUSP2 (PA3-115; Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 647–goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific) and Hoechst 33342 solution (Dojindo, Kumamoto, Japan). The dilution ratios for anti-F4/80–APC, anti-DUSP2, anti-rabbit IgG (H+L) and Hoechst 33342 solution were 1:100, 1:500, 1:100 and 1:5000, respectively. Images were obtained using a BZ-9000 (Keyence, Osaka, Japan), FV1200 (Olympus, Tokyo, Japan) or IN Cell Analyzer 6000 (GE Healthcare, Little Chalfont, UK). Quantitative PCR Quantitative PCR was performed as previously described (3). RNA was extracted from cells using a High Pure RNA Isolation kit (Roche) according to the manufacturer’s instructions. Reverse transcription was performed with 4 µl of ReverTra Ace (Toyobo, Osaka, Japan) in a total volume of 20 µl according to the manufacturer’s recommendations. Subsequently, 1 µl of cDNA fragments was amplified using 10 µl of real-time PCR Master Mix (Toyobo) with 1 µl of TaqMan probes (Life Technologies, Carlsbad, CA, USA) in a total volume of 20 µl. Fluorescence from the TaqMan probe for each gene was detected using a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The mRNA expression level of each gene was normalized to the 18S rRNA expression level (18S rRNA, Applied Biosystems). TaqMan probes for quantitative PCR were purchased with the following assay ID numbers: Batf2 (Mm01231591_m1) and Dusp2 (Mm00839675_g1). Dusp2 transfection For Dusp2 transfections, 1 × 106 RAW 264.7 cells were suspended in 100 µl of buffer R (Neon Transfection System, Thermo Fisher Scientific) containing 8 µg of the Dusp2 expression vector or empty vector control. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Twenty-four hours after electroporation, the cells were stimulated with R848 (100 ng ml−1) for 24 h. Following activation, cell extracts were prepared and analyzed by immunoblot analysis. Cell lysates were prepared in lysis buffer as described above, and nuclear extracts were obtained in buffer (50 mM HEPES-KOH [pH 7.8], 420 mM KCl, 0.1 mM EDTA [pH 8.0], 5 mM MgCl2, protease inhibitor cocktail [Roche] and 20% glycerol) as previously described (14, 15). Immunoblot analysis Immunoblot analysis was performed as previously described (3). Antibodies for western blotting were purchased as follows: anti-DUSP2 (PA3-115) (Thermo Fisher Scientific), anti-STAT3 (C-20) (sc-482; Santa Cruz Biotechnology), anti-phospho-STAT3 (phospho Y705) (EP2147Y) (ab76315; Abcam), anti-NF-κB p50 (H-119) (sc-7178; Santa Cruz Biotechnology), anti-NF-κB p65 (C-20) (sc-372; Santa Cruz Biotechnology), anti-NF-κB c-Rel (C) (sc-71; Santa Cruz Biotechnology), anti-IκB-α (C-21) (sc-371; Santa Cruz Biotechnology), HRP–anti-actin (C-11) (sc-1615 HRP; Santa Cruz Biotechnology), anti-c-Myc (A-14) (sc-789; Santa Cruz Biotechnology) and HRP–anti-rabbit IgG (NA934V; GE Healthcare). The dilution ratio was 1:1000 for each antibody. Enzyme-linked immunosorbent assays IL-12 p40 levels were determined by ELISAs performed according to the manufacturer’s instructions (DuoSet ELISA; R&D Systems, Minneapolis, MN, USA). The absorbance of each ELISA plate was measured using a microplate reader (iMark; Bio-Rad, Hercules, CA, USA). Flow cytometry Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Data were collected using a FACSCantoII instrument (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo analysis software (Tree Star, Ashland, OR, USA). After washing with magnetically activated cell sorting (MACS) buffer, the cells were incubated with antibodies for 15 min and washed twice. For T-bet and GATA3 staining, a Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) was used. Antibodies for flow cytometry were purchased from commercial sources as follows: PerCP/Cy5.5–anti-CD4 (RM4-5; BD Pharmingen, San Diego, CA, USA), Pacific Blue–anti-CD3ε (145-2C11; BioLegend, San Diego, CA, USA), FITC–anti-T-bet (4B10; BioLegend) and APC–anti-GATA3 (16E10A23; BioLegend). The dilution ratio was 1:100–200 for each antibody. Luciferase reporter assays Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). For luciferase transfections, 1 × 106 RAW 264.7 cells were suspended in 100 µl of buffer R containing 8 µg of the Batf2 or Dusp2 expression vector or empty vector control, 32 µg of the Dusp2 pro-Luc or Il12b pro-Luc plasmid vector, and 1 µg of the pRL-TK vector. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Each transfection product was plated in a 24-well plate with 1 × 105 cells per well. Forty-eight hours after electroporation, cell extracts were prepared by using Passive Lysis Buffer (Promega). Luciferase activity was determined from a 20-µl cell extract and measured on the microplate reader Centro X3 LB 960 (Berthold Technologies, Bad Wildbad, Germany). For deletion or substitution mutants, the mutant DNA fragments were produced using a PrimeSTAR Mutagenesis Basal Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. All plasmids were verified by restriction mapping and by sequencing. The potential transcription factor-binding regions were estimated using TFBIND software (RIKEN, Yokohama, Japan) (16). Microwell NF-κB DNA-binding assay The NF-κB p65 DNA-binding activity was measured with the TransAM NF-κB Flexi ELISA kit according to the manufacturer’s recommendations (Active Motif) (17, 18). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant NF-κB p65 was purchased from Active Motif (No: 31102). The NF-κB c-Rel DNA-binding activity was measured similarly. DNA affinity precipitation assay DNA affinity precipitation (DNAP) assays were performed as previously described with some modifications (19). Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared. To produce RAW 264.7 cells over-expressing Myc-tagged Batf2, 1 × 107 RAW 264.7 cells were suspended in 1 ml of buffer R containing 40 µg of the Myc-tagged Batf2 expression vector. These cells were then electroporated using the Neon Transfection System at 1680 V for 20 ms. Twelve hours after electroporation, cell extracts were prepared. For DNAP assays, the biotinylated DNA probe (10 µg) was incubated with binding buffer (10 mM HEPES-KOH [pH 7.8], 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol) containing 1 mg of cell lysate and 15 µg of poly(dI-dC) (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 30 min. A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Dynabeads M–280 Streptavidin (50 µl, Thermo Fisher Scientific) were added and mixed by rotation at room temperature for 3 h. The Dynabeads were then collected with a magnet and washed three times with binding buffer. The trapped proteins were separated by standard SDS–PAGE (e-PAGEL; ATTO, Tokyo, Japan) and analyzed by western blotting. For DNAP assays with a p65 depletion, 20 µg of anti-NF-κB p65 (C-20) X (sc-372X; Santa Cruz Biotechnology) was added to 1 mg of cell lysate. The solution was incubated at 4°C for 1 h on a tube rotator. Protein G-Sepharose beads (250 µl of 50% slurry; GE Healthcare) were added, and the mixture was agitated at 4°C for 1 h. After centrifugation, the supernatant was analyzed by DNAP assays as described above. Co-immunoprecipitation For co-immunoprecipitations, 1 × 106 cells were suspended in 100 µl of buffer R containing 8 µg of the Myc-tagged Batf2 expression vector and 8 µg of the Rela plasmid vector. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Transfected cells were plated at 5 × 106 cells per dish. Twelve hours after electroporation, cell extracts were prepared using lysis buffer as described above, and 4 µg of normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) or rabbit anti-NF-κB p65 (C-20) (sc-372, Santa Cruz Biotechnology) were added to the lysates (50 µg) in a total volume of 100 µl. The solution was incubated at 4°C for 1 h on a tube rotator. Protein G-Sepharose beads (50 µl of 50% slurry, GE Healthcare) were added, and the mixture was agitated at 4°C for 1 h. After centrifugation, the pellet was washed four times with TBS (50 mM Tris, 150 mM NaCl, pH 7.5). Samples were analyzed by standard SDS–PAGE immunoblotting using mouse anti-c-Myc antibody (9E10) (sc-40; Santa Cruz Biotechnology). Statistical analysis A two-tailed Student’s t-test or Welch’s t-test was carried out for the comparison of two independent normally distributed groups. A Mann–Whitney’s U-test was used to compare two sets of non-normally distributed data. A P-value of <0.05 was considered statistically significant for all tests. Results DUSP2 levels are significantly lower in Batf2−/− macrophages than in WT macrophages both in vivo and in vitro First, we analyzed the genome-wide status of H3K27Ac in R848-stimulated WT and Batf2−/− BMDMs by using the ChIP-Seq technique. Of the genes most highly induced by R848 in our previously reported microarray results (3), we selected the top eight R848-inducible genes that had a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs on the basis of our ChIP-Seq data. Notably, we found that Dusp2 had a significantly lower acetyl-histone status in Batf2−/− BMDMs compared with WT BMDMs (Fig. 1A and B). We next used ChIP-Seq to analyze the status of BATF2 in R848-simulated WT BMDMs, and we found an increase in the promoter region close to the transcription start site of Dusp2 (Fig. 1B). Since these results suggested that the transcriptional activities on the promoter region of Dusp2 would likely be decreased in Batf2−/− BMDMs, we performed quantitative PCR analyses and western blotting assays to measure the Dusp2 mRNA and protein expression levels in BMDMs from WT or Batf2−/− mice. As expected, the Batf2 mRNA expression levels were confirmed to be highly induced by R848 in WT BMDMs (Fig. 1C). However, although the Dusp2 mRNA expression levels were also highly induced by R848 in WT BMDMs, they were significantly lower in R848-stimulated BMDMs from Batf2−/− mice (Fig. 1C). These findings were further confirmed by the results of western blotting analyses (Fig. 1D). Next, we assessed the in vivo responses to TLR7 ligands by using the topical application of imiquimod in a mouse model. Histological observation revealed that epidermal hyperplasia occurred in the skin of both Batf2−/− and WT mice that had been treated with imiquimod for 6 days, whereas epidermal hyperplasia was not observed in Vaseline-treated controls (Fig. 1E). However, in imiquimod-treated skin, DUSP2 was expressed by the macrophages of WT mice but not by the macrophages of Batf2−/− mice (Fig. 1F). Fig. 1. View largeDownload slide The expression levels of Dusp2 in macrophages from WT or Batf2−/− mice. (A) The top eight R848-inducible genes with a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs were selected from microarray and ChIP-Seq data. (B) ChIP-Seq enrichment profiles for BATF2 and H3K27Ac at the Dusp2 locus were generated using WT or Batf2−/− BMDMs that had been stimulated by R848 for 6 h. (C) Relative expression levels of Batf2 and Dusp2 in BMDMs stimulated by R848 or control for 8 h were quantified using qPCR (n = 6). Bars show means. **P < 0.01. (D) The DUSP2 expression in WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h were analyzed by western blotting. Representative results of two independent experiments are shown. (E) Histology of back skin from WT or Batf2−/− mice that had been treated with imiquimod or control cream (Vaseline) for 6 days was performed via hematoxylin and eosin (H&E) staining. Scale bars represent 100 µm. (F) The F4/80 expression (blue) and DUSP2 expression (green) in back skin of WT or Batf2−/− mice that had been treated with imiquimod for 6 days were detected by immunofluorescence. Scale bars represent 60 µm. Fig. 1. View largeDownload slide The expression levels of Dusp2 in macrophages from WT or Batf2−/− mice. (A) The top eight R848-inducible genes with a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs were selected from microarray and ChIP-Seq data. (B) ChIP-Seq enrichment profiles for BATF2 and H3K27Ac at the Dusp2 locus were generated using WT or Batf2−/− BMDMs that had been stimulated by R848 for 6 h. (C) Relative expression levels of Batf2 and Dusp2 in BMDMs stimulated by R848 or control for 8 h were quantified using qPCR (n = 6). Bars show means. **P < 0.01. (D) The DUSP2 expression in WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h were analyzed by western blotting. Representative results of two independent experiments are shown. (E) Histology of back skin from WT or Batf2−/− mice that had been treated with imiquimod or control cream (Vaseline) for 6 days was performed via hematoxylin and eosin (H&E) staining. Scale bars represent 100 µm. (F) The F4/80 expression (blue) and DUSP2 expression (green) in back skin of WT or Batf2−/− mice that had been treated with imiquimod for 6 days were detected by immunofluorescence. Scale bars represent 60 µm. Phospho-STAT3 is up-regulated and NF-κB p50/p65 are down-regulated in Batf2−/−BMDMs compared with WT controls We confirmed via immunohistochemistry that DUSP2 localizes in the nucleus (Fig. 2A), which is consistent with previously reported data (7). It has also been reported that DUSP2 associates with phospho-STAT3 (p-STAT3) and attenuates its activity through the dephosphorylation of p-STAT3 at Tyr705 (20). In agreement with these reports, we found that the over-expression of Dusp2 down-regulated the expression of p-STAT3 compared with the empty control in nuclear extracts from RAW 264.7 cells (Fig. 2B). Additionally, STAT3 has been reported as a significantly enriched upstream regulator of the genes that are up-regulated in Batf2−/− macrophages (21). On the basis of this, we measured the p-STAT3 expression levels in BMDMs, and the results show that, after stimulation with R848 for 24 h, the p-STAT3 expression is up-regulated in nuclear extracts from Batf2−/− BMDMs compared with WT BMDMs (Fig. 2C). Because p-STAT3 has also been reported to be capable of inhibiting IκB kinase in normal immune cells (22, 23), and thereby reducing NF-κB-associated Th1 immunity (24), we next examined the IκB expression levels. The results show that the expression levels of IκB in R848-stimulated BMDMs were higher in cells from Batf2−/− mice than in cells from WT controls (Fig. 2D). Consistently with these results, down-regulation of the p50, p65 and c-Rel expression levels was observed in nuclear extracts from Batf2−/− BMDMs compared with their levels in nuclear extracts from WT BMDMs, while there were no corresponding significant differences in the cytosolic extracts (Fig. 2E). Fig. 2. View largeDownload slide The expression levels of STAT3 and NF-κB in BMDMs from WT or Batf2−/− mice. (A) The nuclei (Hoechst 33342, blue) and DUSP2 expression (green) in RAW 264.7 cells stimulated with R848 or control for 24 h were detected by immunofluorescence. Scale bars represent 10 µm. (B) RAW 264.7 cells were transfected with the Dusp2 or empty control vector. Twenty-four hours after transfection, the cells were treated with R848 for 24 h. Following activation, the DUSP2, STAT3 and p-STAT3 expression in nuclear extracts was analyzed by western blotting. (C) The STAT3 and p-STAT3 expression in nuclear extracts of WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h was analyzed by western blotting. (D) The IκB-α expression in cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 20, 40 or 60 min was analyzed by western blotting. (E) The p50, p65 and c-Rel expression in nuclear or cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 3 or 6 h was analyzed by western blotting. Data are representative of at least two independent experiments. Fig. 2. View largeDownload slide The expression levels of STAT3 and NF-κB in BMDMs from WT or Batf2−/− mice. (A) The nuclei (Hoechst 33342, blue) and DUSP2 expression (green) in RAW 264.7 cells stimulated with R848 or control for 24 h were detected by immunofluorescence. Scale bars represent 10 µm. (B) RAW 264.7 cells were transfected with the Dusp2 or empty control vector. Twenty-four hours after transfection, the cells were treated with R848 for 24 h. Following activation, the DUSP2, STAT3 and p-STAT3 expression in nuclear extracts was analyzed by western blotting. (C) The STAT3 and p-STAT3 expression in nuclear extracts of WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h was analyzed by western blotting. (D) The IκB-α expression in cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 20, 40 or 60 min was analyzed by western blotting. (E) The p50, p65 and c-Rel expression in nuclear or cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 3 or 6 h was analyzed by western blotting. Data are representative of at least two independent experiments. IL-12 p40 levels are significantly lower in Batf2−/− macrophages than in WT macrophages, and Th1 immunity is impaired in Batf2−/− mice Since the above results suggested the expression of NF-κB-associated cytokines in response to TLR7 stimulation would be impaired in Batf2−/− mice, we performed ELISAs to examine the levels of IL-12 p40 secreted by Batf2−/− and WT BMDMs following their stimulation by R848. The results show that the IL-12 p40 levels were significantly lower in the culture medium of Batf2−/− BMDMs than in that of WT BMDMs (Fig. 3A). To investigate the effect of Dusp2 on IL-12 p40 levels, we performed luciferase reporter assays with a full-length Dusp2 construct and an Il12b promoter linked to a luciferase reporter gene (11). We found that RAW 264.7 cells over-expressing Dusp2 displayed an enhanced level of luciferase reporter activity following stimulation with R848, compared with cells transfected with the empty control (Fig. 3B). Additionally, we observed that the proportion of T-bet+ GATA-3− CD4+ T cells in R848-treated Batf2−/− mice was significantly lower compared with that in R848-treated WT controls (Fig. 3C), indicating that Th1 immunity is impaired in Batf2−/− mice. Fig. 3. View largeDownload slide Analyses of Th1 immunity in WT and Batf2−/− mice. (A) Levels of IL-12 p40 secreted by WT or Batf2−/− BMDMs following stimulation with R848 (100 ng ml−1) or control medium for 24 h were measured by ELISA. Data are expressed as the mean ± SD of two independent experiments (n = 4). *P < 0.05. (B) The mouse Il12b promoter–luciferase reporter construct (Il12b pro-Luc) was transfected into RAW 264.7 cells along with a Dusp2 or empty vector construct. Cells were treated with control medium or R848 at 24 h post-transfection, and the luciferase activity was measured 24 h after stimulation. Data are expressed as the mean ± SD from two independent experiments. *P < 0.05. (C) Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Numbers represent the percentages of T-bet+ GATA3− CD4+ T cells within the CD4+ T-cell population (left). Data are from four independent experiments (right, n = 6–8). Bars show means. *P < 0.05. Fig. 3. View largeDownload slide Analyses of Th1 immunity in WT and Batf2−/− mice. (A) Levels of IL-12 p40 secreted by WT or Batf2−/− BMDMs following stimulation with R848 (100 ng ml−1) or control medium for 24 h were measured by ELISA. Data are expressed as the mean ± SD of two independent experiments (n = 4). *P < 0.05. (B) The mouse Il12b promoter–luciferase reporter construct (Il12b pro-Luc) was transfected into RAW 264.7 cells along with a Dusp2 or empty vector construct. Cells were treated with control medium or R848 at 24 h post-transfection, and the luciferase activity was measured 24 h after stimulation. Data are expressed as the mean ± SD from two independent experiments. *P < 0.05. (C) Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Numbers represent the percentages of T-bet+ GATA3− CD4+ T cells within the CD4+ T-cell population (left). Data are from four independent experiments (right, n = 6–8). Bars show means. *P < 0.05. BATF2 promotes DUSP2 expression From our earlier results, we hypothesized that Batf2 might be a positive regulator of DUSP2 induction. To test this, we next performed luciferase reporter assays. Because the ChIP-Seq enrichment profiles for BATF2 suggested that BATF2 would interact with the Dusp2 promoter position −451 to the transcription start site, we used this promoter region linked to a luciferase reporter gene (Fig. 4A). RAW 264.7 cells over-expressing Batf2 displayed an enhanced level of luciferase reporter activity compared with empty control vector-transfected cells (Fig. 4B), and these luciferase enhancements were observed in a dose-dependent manner (Fig. 4C). Additionally, the level of luciferase enhancement following stimulation with R848 was significantly lower in Batf2−/− BMDMs transfected with the Dusp2 promoter–luciferase reporter construct than that in WT BMDMs (Fig. 4D). Fig. 4. View largeDownload slide Regulation of the Dusp2 proximal promoter region by BATF2 in RAW 264.7 cells. (A) ChIP-Seq enrichment profiles for BATF2 at the mouse Dusp2 promoter locus were generated from WT murine BMDMs stimulated by R848 for 6 h. A schematic of the Dusp2 promoter region used for the luciferase reporter construct is shown. TSS: transcription start site. The number −451 indicates the position from the TSS. (B) The mouse Dusp2 promoter–luciferase reporter construct was transfected into RAW 264.7 cells along with a Batf2 or empty vector construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of duplicates. **P < 0.01. (C) The mouse Dusp2 promoter–luciferase reporter construct was co-transfected with increasing amounts of the Batf2 vector construct. Luciferase activities relative to the empty vector control are shown as a representative mean ± SD. (D) The mouse Dusp2 promoter–luciferase reporter construct was transfected into BMDMs from WT or Batf2−/− mice. Cells were activated with R848 at 24 h post-transfection, and luciferase activity was measured 24 h after stimulation. Luciferase activities are shown as a representative mean ± SD. *P < 0.05. Similar results were obtained in at least two independent experiments. Fig. 4. View largeDownload slide Regulation of the Dusp2 proximal promoter region by BATF2 in RAW 264.7 cells. (A) ChIP-Seq enrichment profiles for BATF2 at the mouse Dusp2 promoter locus were generated from WT murine BMDMs stimulated by R848 for 6 h. A schematic of the Dusp2 promoter region used for the luciferase reporter construct is shown. TSS: transcription start site. The number −451 indicates the position from the TSS. (B) The mouse Dusp2 promoter–luciferase reporter construct was transfected into RAW 264.7 cells along with a Batf2 or empty vector construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of duplicates. **P < 0.01. (C) The mouse Dusp2 promoter–luciferase reporter construct was co-transfected with increasing amounts of the Batf2 vector construct. Luciferase activities relative to the empty vector control are shown as a representative mean ± SD. (D) The mouse Dusp2 promoter–luciferase reporter construct was transfected into BMDMs from WT or Batf2−/− mice. Cells were activated with R848 at 24 h post-transfection, and luciferase activity was measured 24 h after stimulation. Luciferase activities are shown as a representative mean ± SD. *P < 0.05. Similar results were obtained in at least two independent experiments. BATF2 promotes DUSP2 expression through NF-κB-binding sites in the Dusp2 promoter The above results suggest that BATF2 recruitment to the Dusp2 promoter region is important for its transcriptional activation. To identify the Dusp2 promoter elements that are responsible for transcriptional activation by BATF2, we performed luciferase reporter assays using Dusp2 promoter sequences containing mutations. First, we searched for potential transcription factor-binding regions using TFBIND software (16) and found nine potential NF-κB-binding regions on the Dusp2 promoter (Fig. 5A). On the basis of this finding, we performed serial deletion-mutant analyses. Deletions of the region from position −208 to −118 diminished the enhancement of luciferase activity by BATF2 compared with empty vectors (Fig. 5B). Since the region from position −208 to −118 has three predicted NF-κB-binding regions, we performed substitution-mutant analyses focusing on these three binding regions. The Batf2 luciferase enhancement was decreased in the substitution mutant for each of the three binding regions (Fig. 5C), suggesting that all three binding regions are important for Batf2 activation. Fig. 5. View largeDownload slide Analyses of the mechanism responsible for the effect of BATF2 on DUSP2 expression. (A) The sequence of the Dusp2 proximal promoter is shown. Estimated potential transcription factor-binding sequences are boxed and labeled. (B and C) Deletion-mutant (B) and substitution-mutant (C) analyses of the Dusp2 promoter. Mutants were transfected into RAW 264.7 cells with a Batf2 or empty construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of triplicates. Similar results were obtained from two independent experiments. Fig. 5. View largeDownload slide Analyses of the mechanism responsible for the effect of BATF2 on DUSP2 expression. (A) The sequence of the Dusp2 proximal promoter is shown. Estimated potential transcription factor-binding sequences are boxed and labeled. (B and C) Deletion-mutant (B) and substitution-mutant (C) analyses of the Dusp2 promoter. Mutants were transfected into RAW 264.7 cells with a Batf2 or empty construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of triplicates. Similar results were obtained from two independent experiments. BATF2 interacts with p65, and together they bind to the region from position −203 to −121 in the Dusp2 promoter We next performed DNA-binding ELISAs using a biotinylated probe against Dusp2 promoter position −203 to −121 to confirm that NF-κB p65 binds to this region. Compared with the blank control, we observed an increase in the amount of NF-κB p65 bound to the biotinylated probe when recombinant p65 was used directly as a positive control (Fig. 6A), suggesting the direct binding of p65 to this position. The up-regulated activity of p65 was also observed in nuclear extracts from RAW 264.7 cells that were stimulated with R848 compared with non-stimulated cells (Fig. 6A). Similar results were also obtained with analogous experiments using c-Rel (Fig. 6A). We then performed DNAP assays using cell extracts from RAW 264.7 cells over-expressing Myc-tagged Batf2. The results suggest that BATF2, p65 and c-Rel bind to the region at position −203 to −121 in the Dusp2 promoter (Fig. 6B). Next, we repeated the DNAP assays using p65-depleted samples. The results indicate that p65 depletion from the lysates attenuated the recruitment of BATF2 on the Dusp2 promoter (Fig. 6C), suggesting that BATF2 is recruited on the Dusp2 promoter through binding to p65. To confirm this, we performed co-immunoprecipitation experiments using cell extracts from RAW 264.7 cells over-expressing Myc-tagged Batf2 and Rela (p65), and the results verify that BATF2 interacts with p65 (Fig. 6D). Fig. 6. View largeDownload slide Analyses of the DNA-binding activities of NF-κB p65, c-Rel and BATF2. (A) The DNA-binding activities of p65 were measured with a TransAM Flexi NF-κB Transcription Factor Assay Kit (schematic, left). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant p65 (500 ng, middle) or nuclear extracts from cells over-expressing c-Rel (5 µg, right) were used as positive controls (ctrls). Sample nuclear extracts were obtained from RAW 264.7 cells activated with R848 or treated with a medium control for 1 h (middle and right). Error bars indicate ± SD from two independent experiments. *P < 0.05. (B) Results from a DNAP assay (schematic, left) are shown. Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared and incubated with the biotinylated DNA probe. Streptavidin-coupled Dynabeads were added, and the trapped proteins were analyzed by western blotting (WB; right). Data are representative of two independent experiments. (C) For DNAP assays on samples with a depletion of p65, the samples were first subjected to a p65 depletion performed via immunoprecipitation with anti-p65 antibody and Protein G-Sepharose beads prior to their use in a DNAP assay (schematic, left). The resulting supernatant was then analyzed by DNAP assays as described in (B), and the proteins trapped by the streptavidin-coupled Dynabeads were analyzed by western blotting (WB; right). (D) Co-immunoprecipitation experiments assessing BATF2 binding to p65. Cell extracts from RAW 264.7 cells over-expressing both RelA (p65) and Myc-tagged BATF2 were prepared. After immunoprecipitation (IP) with anti-p65 antibody or normal control IgG, samples were analyzed with anti-c-Myc antibody by western blotting (WB). Similar results were obtained in three independent experiments. Fig. 6. View largeDownload slide Analyses of the DNA-binding activities of NF-κB p65, c-Rel and BATF2. (A) The DNA-binding activities of p65 were measured with a TransAM Flexi NF-κB Transcription Factor Assay Kit (schematic, left). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant p65 (500 ng, middle) or nuclear extracts from cells over-expressing c-Rel (5 µg, right) were used as positive controls (ctrls). Sample nuclear extracts were obtained from RAW 264.7 cells activated with R848 or treated with a medium control for 1 h (middle and right). Error bars indicate ± SD from two independent experiments. *P < 0.05. (B) Results from a DNAP assay (schematic, left) are shown. Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared and incubated with the biotinylated DNA probe. Streptavidin-coupled Dynabeads were added, and the trapped proteins were analyzed by western blotting (WB; right). Data are representative of two independent experiments. (C) For DNAP assays on samples with a depletion of p65, the samples were first subjected to a p65 depletion performed via immunoprecipitation with anti-p65 antibody and Protein G-Sepharose beads prior to their use in a DNAP assay (schematic, left). The resulting supernatant was then analyzed by DNAP assays as described in (B), and the proteins trapped by the streptavidin-coupled Dynabeads were analyzed by western blotting (WB; right). (D) Co-immunoprecipitation experiments assessing BATF2 binding to p65. Cell extracts from RAW 264.7 cells over-expressing both RelA (p65) and Myc-tagged BATF2 were prepared. After immunoprecipitation (IP) with anti-p65 antibody or normal control IgG, samples were analyzed with anti-c-Myc antibody by western blotting (WB). Similar results were obtained in three independent experiments. Discussion DUSP2 plays a critical role in inflammatory responses by dephosphorylating p-STAT3 at Tyr705 and attenuating its activity (20). In agreement with previous reports, our results show that the over-expression of Dusp2 down-regulated the expression of p-STAT3 compared with the empty control (Fig. 2B) and that Batf2−/− mice, which have a significantly lower level of DUSP2 than WT controls, have up-regulated p-STAT3 expression compared with WT mice (Fig. 2C). Although IL-6 is a key activator of STAT3 via phosphorylation (24), the expression levels of IL-6 are similar between WT and Batf2−/− mice (3). Therefore, phosphatases, including DUSP2, are likely important in the up-regulation of p-STAT3 in Batf2−/− BMDMs. Notably, it was also reported that p-STAT3 can inhibit IκB kinase and thereby reduce NF-κB-associated Th1 immunity (22–24), although the precise mechanisms are still unknown. In addition, myeloid-specific Stat3 gene ablation induced the release of IL-12 by dendritic cells (25, 26). Our results show that IκB was up-regulated in the cytosolic extracts from Batf2−/− BMDMs compared with WT controls (Fig. 2D). Additionally, NF-κB p50, p65 and c-Rel were all down-regulated in the nuclear extracts from Batf2−/− BMDMs compared with WT controls (Fig. 2E). These data are also consistent with our finding that the IL-12 p40 levels were significantly lower in the culture medium of Batf2−/− BMDMs than in that of WT BMDMs (Fig. 3A). Moreover, we additionally observed a significantly lower proportion of T-bet+ GATA-3− CD4+ T cells in R848-treated Batf2−/− mice than in R848-treated WT controls (Fig. 3C), indicating a reduction of Th1 immunity in Batf2−/− mice. Given that p-STAT3 can attenuate NF-κB-associated Th1 immunity through its inhibition of IκB kinase, the reduction of DUSP2 and the up-regulation of p-STAT3 via its attenuated dephosphorylation in Batf2−/− mice indicate their involvements in the impairment of NF-κB-associated Th1 immunity in Batf2−/− mice (Fig. 7). Fig. 7. View largeDownload slide A schematic model of how BATF2 may contribute to up-regulation of the phosphatase DUSP2 and enhancement of Th1 immunity after TLR7 stimulation. In this proposed model, BATF2 up-regulates the expression of DUSP2, which may be involved in the promotion of the downstream expression of IL-12 p40 through the down-regulation of p-STAT3 and the up-regulation of NF-κB. This increase in IL-12 p40 leads to an enhancement of NF-κB-associated Th1 immunity. Fig. 7. View largeDownload slide A schematic model of how BATF2 may contribute to up-regulation of the phosphatase DUSP2 and enhancement of Th1 immunity after TLR7 stimulation. In this proposed model, BATF2 up-regulates the expression of DUSP2, which may be involved in the promotion of the downstream expression of IL-12 p40 through the down-regulation of p-STAT3 and the up-regulation of NF-κB. This increase in IL-12 p40 leads to an enhancement of NF-κB-associated Th1 immunity. These results suggest that BATF2 activates transcription of Dusp2 through its recruitment to the Dusp2 promoter region. Although it was previously thought that BATF family members function only as inhibitors of AP-1 (27, 28), our findings add to the growing evidence indicating that an interaction with a non-AP-1 factor is likely involved in BATF-specific positive transcriptional activities (2). While BATF family proteins lack a transcriptional activation domain, they are still able to support other transcription factors and exert unique and positive transcriptional activities. For example, BATF2 interacts with IRF1 to induce inflammatory responses during Mycobacterium tuberculosis infection (9, 29). In addition, BATF/IRF4 and BATF/IRF8 interactions are important for compensatory dendritic cell development (4). An interaction between BATF2 and NF-κB p65 was reported previously by using yeast two-hybrid technology (30). Here, we demonstrate that BATF2 interacts with NF-κB p65 and up-regulates the expression of DUSP2 via binding to the region at position −203 to −121 in the Dusp2 promoter (Figs 5 and 6). Although the reason for this specificity is still unclear, it may be connected to the ability of BATF2 to function as either an inhibitor or an up-regulator depending on how it interacts with other transcription factors. For example, although our results show that BATF2 is an up-regulator of NF-κB, BATF family members, including BATF2, also function as AP-1 inhibitors (1). Furthermore, BATF was recently proposed to be a pioneer factor (31), which can bind to a target site in condensed chromatin and allow the rapid recruitment of other transcription factors. It is possible that BATF2 may also have some similar epigenetic functions in the Dusp2 promoter. Although our data suggest the possible involvement of DUSP2 in the promotion of Th1 immunity through up-regulation of Il12b expression (Figs 3B and 7), additional work is still needed to confirm this model because the data from this study do not directly show the impairment of Th1 immunity in Dusp2-deficient macrophages. Specifically, our results do not demonstrate an impairment of the Th1 response in Dusp2-deficient mice following their stimulation with TLR7 ligands. Therefore, further studies are needed to fully elucidate the relationship between DUSP2 and the BATF2-mediated Th1 response. In this study, we focused on the involvement of BATF2 in the up-regulation of the phosphatase DUSP2. Although BATF2 was previously known only for its inhibitory functions, this study has revealed that BATF2 has other unique and positive transcriptional activities. Further studies are needed to elucidate the full role of BATF2. Funding This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Japan Society for the Promotion of Science (JSPS) through funding for Specially Promoted Research (Grant Number 15H05704) and by a grant from Project MEET; Osaka University Graduate School of Medicine, Mitsubishi Tanabe Pharma Corporation. This study was supported in part by Yuri Terao and Center for Medical Research and Education, Graduate School of Medicine, Osaka University. Acknowledgements We thank T. Machida, T. Kawasaki, T. Matsuki, K. Fukushima, K. J. Yoshida, H. Nabeshima, I. Ebina, K. Kuniyoshi, X. Sun, M. Shimoda, S. K. Singh, Y. Nagahama, Y. Kozakai, K. Maruyama, K. Iwamoto, M. Okada, Y. Kishi, H. Nakamura, T. Suzuki and T. Sugita for discussions; A. Otsuka, A. Yamazaki, M. Tasai, K. Asakawa, C. Funamoto, A. Wataki and R. Kawaguchi for technical assistance; and E. Kamada for secretarial assistance. We also thank K. Oakley from Edanz Group for editing a draft of this manuscript. Conflicts of interest statement: the authors declared no conflicts of interest. References 1 Su , Z. Z. , Lee , S. G. , Emdad , L. et al. 2008 . Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN) . Proc. Natl Acad. Sci. USA 105 : 20906 . Google Scholar CrossRef Search ADS 2 Murphy , T. L. , Tussiwand , R. and Murphy , K. M . 2013 . Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks . Nat. Rev. Immunol . 13 : 499 . Google Scholar CrossRef Search ADS PubMed 3 Kanemaru , H. , Yamane , F. , Fukushima , K. et al. 2017 . Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages . Proc. Natl Acad. Sci. USA 114 : E7331 . Google Scholar CrossRef Search ADS 4 Tussiwand , R. , Lee , W. L. , Murphy , T. L. et al. 2012 . Compensatory dendritic cell development mediated by BATF-IRF interactions . Nature 490 : 502 . Google Scholar CrossRef Search ADS PubMed 5 Jeffrey , K. L. , Brummer , T. , Rolph , M. S. et al. 2006 . Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1 . Nat. Immunol . 7 : 274 . Google Scholar CrossRef Search ADS PubMed 6 Ward , Y. , Gupta , S. , Jensen , P. , Wartmann , M. , Davis , R. J. and Kelly , K . 1994 . Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1 . Nature 367 : 651 . Google Scholar CrossRef Search ADS PubMed 7 Rohan , P. J. , Davis , P. , Moskaluk , C. A. et al. 1993 . PAC-1: a mitogen-induced nuclear protein tyrosine phosphatase . Science 259 : 1763 . Google Scholar CrossRef Search ADS PubMed 8 Yamamoto , M. , Uematsu , S. , Okamoto , T. et al. 2007 . Enhanced TLR-mediated NF-IL6 dependent gene expression by Trib1 deficiency . J. Exp. Med . 204 : 2233 . Google Scholar CrossRef Search ADS PubMed 9 Roy , S. , Guler , R. , Parihar , S. P. et al. 2015 . Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection . J. Immunol . 194 : 6035 . Google Scholar CrossRef Search ADS PubMed 10 Murphy , T. L. , Cleveland , M. G. , Kulesza , P. , Magram , J. and Murphy , K. M . 1995 . Regulation of interleukin 12 p40 expression through an NF-κB half-site . Mol. Cell. Biol . 15 : 5258 . Google Scholar CrossRef Search ADS PubMed 11 Plevy , S. E. , Gemberling , J. H. , Hsu , S. , Dorner , A. J. and Smale , S. T . 1997 . Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins . Mol. Cell. Biol . 17 : 4572 . Google Scholar CrossRef Search ADS PubMed 12 Sanjabi , S. , Williams , K. J. , Saccani , S. et al. 2005 . A c-Rel subdomain responsible for enhanced DNA-binding affinity and selective gene activation . Genes Dev . 19 : 2138 . Google Scholar CrossRef Search ADS PubMed 13 van der Fits , L. , Mourits , S. , Voerman , J. S. et al. 2009 . Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis . J. Immunol . 182 : 5836 . Google Scholar CrossRef Search ADS PubMed 14 Dignam , J. D. , Lebovitz , R. M. and Roeder , R. G . 1983 . Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei . Nucleic Acids Res . 11 : 1475 . Google Scholar CrossRef Search ADS PubMed 15 Lo , K. , Landau , N. R. and Smale , S. T . 1991 . LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes . Mol. Cell. Biol . 11 : 5229 . Google Scholar CrossRef Search ADS PubMed 16 Tsunoda , T. and Takagi , T . 1999 . Estimating transcription factor bindability on DNA . Bioinformatics 15 : 622 . Google Scholar CrossRef Search ADS PubMed 17 Renard , P. , Ernest , I. , Houbion , A. et al. 2001 . Development of a sensitive multi-well colorimetric assay for active NF-κB . Nucleic Acids Res . 29 : E21 . Google Scholar CrossRef Search ADS PubMed 18 Maruyama , K. , Fukasaka , M. , Vandenbon , A. et al. 2012 . The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation . Immunity 37 : 1024 . Google Scholar CrossRef Search ADS PubMed 19 Suzuki , T. , Fujisawa , J. I. , Toita , M. and Yoshida , M . 1993 . The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1 . Proc. Natl Acad. Sci. USA 90 : 610 . Google Scholar CrossRef Search ADS 20 Lu , D. , Liu , L. , Ji , X. et al. 2015 . The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation . Nat. Immunol . 16 : 1263 . Google Scholar CrossRef Search ADS PubMed 21 Kitada , S. , Kayama , H. , Okuzaki , D. et al. 2017 . BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Trypanosoma cruzi infection . J. Exp. Med . 214 : 1313 . Google Scholar CrossRef Search ADS PubMed 22 Welte , T. , Zhang , S. S. , Wang , T. et al. 2003 . STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity . Proc. Natl Acad. Sci. USA 100 : 1879 . Google Scholar CrossRef Search ADS 23 Lee , H. , Herrmann , A. , Deng , J. H. et al. 2009 . Persistently activated Stat3 maintains constitutive NF-κB activity in tumors . Cancer Cell 15 : 283 . Google Scholar CrossRef Search ADS PubMed 24 Yu , H. , Pardoll , D. and Jove , R . 2009 . STATs in cancer inflammation and immunity: a leading role for STAT3 . Nat. Rev. Cancer 9 : 798 . Google Scholar CrossRef Search ADS PubMed 25 Kortylewski , M. , Xin , H. , Kujawski , M. et al. 2009 . Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment . Cancer Cell 15 : 114 . Google Scholar CrossRef Search ADS PubMed 26 Kortylewski , M. , Kujawski , M. , Wang , T. et al. 2005 . Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity . Nat. Med . 11 : 1314 . Google Scholar CrossRef Search ADS PubMed 27 Dorsey , M. J. , Tae , H. J. , Sollenberger , K. G. , Mascarenhas , N. T. , Johansen , L. M. and Taparowsky , E. J . 1995 . B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family . Oncogene 11 : 2255 . Google Scholar PubMed 28 Echlin , D. R. , Tae , H. J. , Mitin , N. and Taparowsky , E. J . 2000 . B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos . Oncogene 19 : 1752 . Google Scholar CrossRef Search ADS PubMed 29 Guler , R. , Roy , S. , Suzuki , H. and Brombacher , F . 2015 . Targeting Batf2 for infectious diseases and cancer . Oncotarget 6 : 26575 . Google Scholar CrossRef Search ADS PubMed 30 Wang , J. , Huo , K. , Ma , L. et al. 2011 . Toward an understanding of the protein interaction network of the human liver . Mol. Syst. Biol . 7 : 536 . Google Scholar CrossRef Search ADS PubMed 31 Ciofani , M. , Madar , A. , Galan , C. et al. 2012 . A validated regulatory network for Th17 cell specification . Cell 151 : 289 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Immunology Oxford University Press

BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3 dephosphorylation

Loading next page...
 
/lp/ou_press/batf2-activates-dusp2-gene-expression-and-up-regulates-nf-b-activity-2t7OGYvJ5c
Publisher
Oxford University Press
Copyright
© The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
0953-8178
eISSN
1460-2377
D.O.I.
10.1093/intimm/dxy023
Publisher site
See Article on Publisher Site

Abstract

Abstract Growing evidence has revealed that the transcription factor basic leucine zipper transcription factor ATF-like 2 (BATF2) has unique transcriptional activities, including regulating cytokines via TLR signals in macrophages, which affect mortality due to infection and cancer. On the basis of genome-wide analyses using the chromatin immunoprecipitation-sequencing technique, we found that dual-specificity phosphatase 2 (Dusp2) had a significantly lower acetyl-histone status in Batf2−/− bone marrow-derived macrophages (BMDMs) compared with wild-type (WT) BMDMs. The phosphatase DUSP2 has been reported to play a critical role in inflammatory responses. Therefore, we evaluated the BATF2 transcriptional activities on the Dusp2 promoter. We found that the DUSP2 and IL-12 p40 expression levels were significantly lower in Batf2−/− BMDMs than in WT controls following their stimulation with TLR7 ligands. Further in vitro studies revealed that phospho-STAT3 was up-regulated and NF-κB p50/p65 were down-regulated in Batf2−/− BMDMs compared with their levels in WT controls. Additionally, Th1 immunity was impaired in Batf2−/− mice following their stimulation with TLR7 ligands. We also found that BATF2 interacts with NF-κB p65 and promotes DUSP2 expression through the NF-κB-binding site in the Dusp2 promoter at −203 to −121. Collectively, our findings suggest that BATF2 activates DUSP2 gene expression and up-regulates NF-κB activity via phospho-STAT3 dephosphorylation. IL-12, inflammation, macrophage, Th1, TLR7 Introduction The basic leucine zipper transcription factor ATF-like 2 (BATF2) is a member of the BATF family. Along with BATF2, the BATF family includes BATF (SFA2) and BATF3 (JDP1; p21SNFT), and its members also belong to the AP-1 basic leucine zipper transcription factor family. Although BATF family members were initially thought to function only as inhibitors of AP-1 (1), recent studies have suggested that these factors additionally have unique and positive transcriptional activities (2). It was recently reported that the IL-12 p40 expression was significantly lower in Batf2−/− macrophages following their stimulation by TLR7 ligands, leading to the impairment of Th1 immunity and the attenuation of the anti-tumor effect in Batf2−/− mice (3). Additionally, Batf2-deficient mice have a higher mortality from Toxoplasma gondii infection compared with wild-type (WT) mice (4). On the basis of these findings, we analyzed the genome-wide status of histone acetylation (H3K27Ac) in WT and Batf2−/− bone marrow-derived macrophages (BMDMs) that had been stimulated with R848, a TLR7 ligand, by using the chromatin immunoprecipitation-sequencing (ChIP-Seq) technique. The results indicate that dual-specificity phosphatase 2 (Dusp2) had a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs. DUSP2, also called phosphatase of activated cells 1 (PAC-1), is a member of the DUSP family. Unlike other DUSP family members, the expression of DUSP2 is restricted to immune cells (5) and has been mostly associated with immune tissues, such as the spleen and thymus (6, 7). As with Batf2−/− macrophages, Dusp2−/− macrophages have impaired effector responses, including inflammatory mediator production (5). Additionally, the IL-12 p40 expression level is lower in Dusp2-deficient macrophages following their stimulation by LPS (5). Therefore, in this study, we further assessed the role of BATF2, particularly its positive transcriptional activities via TLR signals, and evaluated its transcriptional activities on the Dusp2 promoter. Methods Mice Batf2−/− mice were generated as previously described (3). Mice were maintained in our specific pathogen-free animal facility according to the institutional guidelines. Experiments were generally performed at 6–12 weeks of age. All animal experiments were performed with approval from the Animal Research Committee of the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). Cells RAW 264.7 cells, which were described previously (8), were grown in RPMI-1640 containing 10% FCS. BMDMs were generated as described previously (9). Briefly, mouse bone marrow cells were obtained from femurs and differentiated into macrophages in RPMI-1640 with 10% FCS containing 3 ng ml−1 macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA). Medium was changed on day 3. Macrophages were harvested and stimulated with R848 (100 ng ml−1; InvivoGen, San Diego, CA, USA) on day 5. Plasmids Batf2 cDNA was obtained via PCR from a mouse cDNA library as described previously (3). Dusp2 and Myc-tagged Batf2 were purchased from OriGene Technologies (MC204715 and MR203728, respectively; Rockville, MD, USA). Dusp2 pro-Luc plasmid was produced by cloning. The mouse Dusp2 promoter was obtained via PCR from normal genomic DNA and subsequently cloned into the Kpn I–Bgl II sites of the restricted Il12b pro-Luc construct as previously described (10). The Il12b 400-bp pro-Luc, RelA cFlag pcDNA3 and c-Rel cFlag pcDNA3 were gifts from Stephen Smale; Howard Hughes Medical Institute, Chevy Chase, MD, USA; and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA, USA, respectively (Addgene plasmid 20020, 20012 and 20013, respectively; Cambridge, MA, USA) (11, 12). ChIP-Seq analysis ChIP analysis was performed using ChIP-IT Express Enzymatic (Active Motif, Carlsbad, CA, USA). First, 2 × 107 BMDMs were stimulated with R848 (100 ng ml−1). Six hours later, the cells were fixed with 20 ml of 1% formaldehyde for 10 min, followed by a wash with 10 ml of PBS, and the fixation reaction was stopped by adding 10 ml of glycine stop fix solution for 5 min. Cell extracts were prepared in lysis buffer (10 mM HEPES-KOH [pH 7.8], 10 mM KCl, 0.1 mM EDTA [pH 8.0], protease inhibitor cocktail [Roche, Basel, Switzerland], 0.1% Nonidet P-40) and were homogenized using a Dounce homogenizer (BioMasherII; Nippi, Tokyo, Japan) on ice. Enzymatic shearing to produce DNA fragments was performed for 30 min. Immunoprecipitation then was performed on these fragments using 8 µg of one of the following antibodies: anti-BATF2 (L-24) (sc-130972; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-histone H3 (acetyl K27) (ab4729; Abcam, Cambridge, UK). After the samples had been incubated overnight with 50 µl of protein G magnetic beads, the beads were washed. DNA was eluted in 40 µl of elution buffer AM2, and 40 µl of reverse cross-linking buffer was then added. After centrifugation, the resulting supernatants were collected and incubated at 95°C for 15 min. After samples were returned to room temperature, 2 µg of proteinase K was added, and the samples were incubated for 1 h at 37°C. DNA was then purified via ethanol precipitation. The ChIP-Seq was performed by Filgen (Aichi, Japan), and the resulting data were analyzed by CLC Genomics Workbench 10 (Qiagen, Hilden, Germany). Topical application of imiquimod WT and Batf2−/− mice received a daily topical dose of 62.5 mg of 5% imiquimod cream (Beselna Cream 5%, Mochida, Tokyo, Japan) on a shaved patch of back for 6 consecutive days (corresponding to a daily dose of 3.125 mg of the active compound), as previously described (13). Control mice were treated similarly with a control vehicle cream (Vaseline, Unilever, London, UK). Histological analysis Frozen 10-μm tissue sections in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) or cells plated in chamber slides (SCS-008; Matsunami, Osaka, Japan) were fixed in 4% paraformaldehyde and blocked with 3% BSA in 1× PBS. Hematoxylin and eosin (H&E) or immunofluorescent staining was performed. The antibodies and reagents for immunofluorescence analyses were purchased as follows: anti-F4/80–APC (BM8.1; TONBO Biosciences, San Diego, CA, USA), anti-DUSP2 (PA3-115; Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 647–goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific) and Hoechst 33342 solution (Dojindo, Kumamoto, Japan). The dilution ratios for anti-F4/80–APC, anti-DUSP2, anti-rabbit IgG (H+L) and Hoechst 33342 solution were 1:100, 1:500, 1:100 and 1:5000, respectively. Images were obtained using a BZ-9000 (Keyence, Osaka, Japan), FV1200 (Olympus, Tokyo, Japan) or IN Cell Analyzer 6000 (GE Healthcare, Little Chalfont, UK). Quantitative PCR Quantitative PCR was performed as previously described (3). RNA was extracted from cells using a High Pure RNA Isolation kit (Roche) according to the manufacturer’s instructions. Reverse transcription was performed with 4 µl of ReverTra Ace (Toyobo, Osaka, Japan) in a total volume of 20 µl according to the manufacturer’s recommendations. Subsequently, 1 µl of cDNA fragments was amplified using 10 µl of real-time PCR Master Mix (Toyobo) with 1 µl of TaqMan probes (Life Technologies, Carlsbad, CA, USA) in a total volume of 20 µl. Fluorescence from the TaqMan probe for each gene was detected using a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The mRNA expression level of each gene was normalized to the 18S rRNA expression level (18S rRNA, Applied Biosystems). TaqMan probes for quantitative PCR were purchased with the following assay ID numbers: Batf2 (Mm01231591_m1) and Dusp2 (Mm00839675_g1). Dusp2 transfection For Dusp2 transfections, 1 × 106 RAW 264.7 cells were suspended in 100 µl of buffer R (Neon Transfection System, Thermo Fisher Scientific) containing 8 µg of the Dusp2 expression vector or empty vector control. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Twenty-four hours after electroporation, the cells were stimulated with R848 (100 ng ml−1) for 24 h. Following activation, cell extracts were prepared and analyzed by immunoblot analysis. Cell lysates were prepared in lysis buffer as described above, and nuclear extracts were obtained in buffer (50 mM HEPES-KOH [pH 7.8], 420 mM KCl, 0.1 mM EDTA [pH 8.0], 5 mM MgCl2, protease inhibitor cocktail [Roche] and 20% glycerol) as previously described (14, 15). Immunoblot analysis Immunoblot analysis was performed as previously described (3). Antibodies for western blotting were purchased as follows: anti-DUSP2 (PA3-115) (Thermo Fisher Scientific), anti-STAT3 (C-20) (sc-482; Santa Cruz Biotechnology), anti-phospho-STAT3 (phospho Y705) (EP2147Y) (ab76315; Abcam), anti-NF-κB p50 (H-119) (sc-7178; Santa Cruz Biotechnology), anti-NF-κB p65 (C-20) (sc-372; Santa Cruz Biotechnology), anti-NF-κB c-Rel (C) (sc-71; Santa Cruz Biotechnology), anti-IκB-α (C-21) (sc-371; Santa Cruz Biotechnology), HRP–anti-actin (C-11) (sc-1615 HRP; Santa Cruz Biotechnology), anti-c-Myc (A-14) (sc-789; Santa Cruz Biotechnology) and HRP–anti-rabbit IgG (NA934V; GE Healthcare). The dilution ratio was 1:1000 for each antibody. Enzyme-linked immunosorbent assays IL-12 p40 levels were determined by ELISAs performed according to the manufacturer’s instructions (DuoSet ELISA; R&D Systems, Minneapolis, MN, USA). The absorbance of each ELISA plate was measured using a microplate reader (iMark; Bio-Rad, Hercules, CA, USA). Flow cytometry Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Data were collected using a FACSCantoII instrument (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo analysis software (Tree Star, Ashland, OR, USA). After washing with magnetically activated cell sorting (MACS) buffer, the cells were incubated with antibodies for 15 min and washed twice. For T-bet and GATA3 staining, a Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) was used. Antibodies for flow cytometry were purchased from commercial sources as follows: PerCP/Cy5.5–anti-CD4 (RM4-5; BD Pharmingen, San Diego, CA, USA), Pacific Blue–anti-CD3ε (145-2C11; BioLegend, San Diego, CA, USA), FITC–anti-T-bet (4B10; BioLegend) and APC–anti-GATA3 (16E10A23; BioLegend). The dilution ratio was 1:100–200 for each antibody. Luciferase reporter assays Luciferase reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). For luciferase transfections, 1 × 106 RAW 264.7 cells were suspended in 100 µl of buffer R containing 8 µg of the Batf2 or Dusp2 expression vector or empty vector control, 32 µg of the Dusp2 pro-Luc or Il12b pro-Luc plasmid vector, and 1 µg of the pRL-TK vector. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Each transfection product was plated in a 24-well plate with 1 × 105 cells per well. Forty-eight hours after electroporation, cell extracts were prepared by using Passive Lysis Buffer (Promega). Luciferase activity was determined from a 20-µl cell extract and measured on the microplate reader Centro X3 LB 960 (Berthold Technologies, Bad Wildbad, Germany). For deletion or substitution mutants, the mutant DNA fragments were produced using a PrimeSTAR Mutagenesis Basal Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. All plasmids were verified by restriction mapping and by sequencing. The potential transcription factor-binding regions were estimated using TFBIND software (RIKEN, Yokohama, Japan) (16). Microwell NF-κB DNA-binding assay The NF-κB p65 DNA-binding activity was measured with the TransAM NF-κB Flexi ELISA kit according to the manufacturer’s recommendations (Active Motif) (17, 18). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant NF-κB p65 was purchased from Active Motif (No: 31102). The NF-κB c-Rel DNA-binding activity was measured similarly. DNA affinity precipitation assay DNA affinity precipitation (DNAP) assays were performed as previously described with some modifications (19). Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared. To produce RAW 264.7 cells over-expressing Myc-tagged Batf2, 1 × 107 RAW 264.7 cells were suspended in 1 ml of buffer R containing 40 µg of the Myc-tagged Batf2 expression vector. These cells were then electroporated using the Neon Transfection System at 1680 V for 20 ms. Twelve hours after electroporation, cell extracts were prepared. For DNAP assays, the biotinylated DNA probe (10 µg) was incubated with binding buffer (10 mM HEPES-KOH [pH 7.8], 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol) containing 1 mg of cell lysate and 15 µg of poly(dI-dC) (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 30 min. A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Dynabeads M–280 Streptavidin (50 µl, Thermo Fisher Scientific) were added and mixed by rotation at room temperature for 3 h. The Dynabeads were then collected with a magnet and washed three times with binding buffer. The trapped proteins were separated by standard SDS–PAGE (e-PAGEL; ATTO, Tokyo, Japan) and analyzed by western blotting. For DNAP assays with a p65 depletion, 20 µg of anti-NF-κB p65 (C-20) X (sc-372X; Santa Cruz Biotechnology) was added to 1 mg of cell lysate. The solution was incubated at 4°C for 1 h on a tube rotator. Protein G-Sepharose beads (250 µl of 50% slurry; GE Healthcare) were added, and the mixture was agitated at 4°C for 1 h. After centrifugation, the supernatant was analyzed by DNAP assays as described above. Co-immunoprecipitation For co-immunoprecipitations, 1 × 106 cells were suspended in 100 µl of buffer R containing 8 µg of the Myc-tagged Batf2 expression vector and 8 µg of the Rela plasmid vector. Cells were electroporated using the Neon Transfection System at 1680 V for 20 ms. Transfected cells were plated at 5 × 106 cells per dish. Twelve hours after electroporation, cell extracts were prepared using lysis buffer as described above, and 4 µg of normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) or rabbit anti-NF-κB p65 (C-20) (sc-372, Santa Cruz Biotechnology) were added to the lysates (50 µg) in a total volume of 100 µl. The solution was incubated at 4°C for 1 h on a tube rotator. Protein G-Sepharose beads (50 µl of 50% slurry, GE Healthcare) were added, and the mixture was agitated at 4°C for 1 h. After centrifugation, the pellet was washed four times with TBS (50 mM Tris, 150 mM NaCl, pH 7.5). Samples were analyzed by standard SDS–PAGE immunoblotting using mouse anti-c-Myc antibody (9E10) (sc-40; Santa Cruz Biotechnology). Statistical analysis A two-tailed Student’s t-test or Welch’s t-test was carried out for the comparison of two independent normally distributed groups. A Mann–Whitney’s U-test was used to compare two sets of non-normally distributed data. A P-value of <0.05 was considered statistically significant for all tests. Results DUSP2 levels are significantly lower in Batf2−/− macrophages than in WT macrophages both in vivo and in vitro First, we analyzed the genome-wide status of H3K27Ac in R848-stimulated WT and Batf2−/− BMDMs by using the ChIP-Seq technique. Of the genes most highly induced by R848 in our previously reported microarray results (3), we selected the top eight R848-inducible genes that had a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs on the basis of our ChIP-Seq data. Notably, we found that Dusp2 had a significantly lower acetyl-histone status in Batf2−/− BMDMs compared with WT BMDMs (Fig. 1A and B). We next used ChIP-Seq to analyze the status of BATF2 in R848-simulated WT BMDMs, and we found an increase in the promoter region close to the transcription start site of Dusp2 (Fig. 1B). Since these results suggested that the transcriptional activities on the promoter region of Dusp2 would likely be decreased in Batf2−/− BMDMs, we performed quantitative PCR analyses and western blotting assays to measure the Dusp2 mRNA and protein expression levels in BMDMs from WT or Batf2−/− mice. As expected, the Batf2 mRNA expression levels were confirmed to be highly induced by R848 in WT BMDMs (Fig. 1C). However, although the Dusp2 mRNA expression levels were also highly induced by R848 in WT BMDMs, they were significantly lower in R848-stimulated BMDMs from Batf2−/− mice (Fig. 1C). These findings were further confirmed by the results of western blotting analyses (Fig. 1D). Next, we assessed the in vivo responses to TLR7 ligands by using the topical application of imiquimod in a mouse model. Histological observation revealed that epidermal hyperplasia occurred in the skin of both Batf2−/− and WT mice that had been treated with imiquimod for 6 days, whereas epidermal hyperplasia was not observed in Vaseline-treated controls (Fig. 1E). However, in imiquimod-treated skin, DUSP2 was expressed by the macrophages of WT mice but not by the macrophages of Batf2−/− mice (Fig. 1F). Fig. 1. View largeDownload slide The expression levels of Dusp2 in macrophages from WT or Batf2−/− mice. (A) The top eight R848-inducible genes with a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs were selected from microarray and ChIP-Seq data. (B) ChIP-Seq enrichment profiles for BATF2 and H3K27Ac at the Dusp2 locus were generated using WT or Batf2−/− BMDMs that had been stimulated by R848 for 6 h. (C) Relative expression levels of Batf2 and Dusp2 in BMDMs stimulated by R848 or control for 8 h were quantified using qPCR (n = 6). Bars show means. **P < 0.01. (D) The DUSP2 expression in WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h were analyzed by western blotting. Representative results of two independent experiments are shown. (E) Histology of back skin from WT or Batf2−/− mice that had been treated with imiquimod or control cream (Vaseline) for 6 days was performed via hematoxylin and eosin (H&E) staining. Scale bars represent 100 µm. (F) The F4/80 expression (blue) and DUSP2 expression (green) in back skin of WT or Batf2−/− mice that had been treated with imiquimod for 6 days were detected by immunofluorescence. Scale bars represent 60 µm. Fig. 1. View largeDownload slide The expression levels of Dusp2 in macrophages from WT or Batf2−/− mice. (A) The top eight R848-inducible genes with a significantly lower H3K27Ac status in Batf2−/− BMDMs compared with WT BMDMs were selected from microarray and ChIP-Seq data. (B) ChIP-Seq enrichment profiles for BATF2 and H3K27Ac at the Dusp2 locus were generated using WT or Batf2−/− BMDMs that had been stimulated by R848 for 6 h. (C) Relative expression levels of Batf2 and Dusp2 in BMDMs stimulated by R848 or control for 8 h were quantified using qPCR (n = 6). Bars show means. **P < 0.01. (D) The DUSP2 expression in WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h were analyzed by western blotting. Representative results of two independent experiments are shown. (E) Histology of back skin from WT or Batf2−/− mice that had been treated with imiquimod or control cream (Vaseline) for 6 days was performed via hematoxylin and eosin (H&E) staining. Scale bars represent 100 µm. (F) The F4/80 expression (blue) and DUSP2 expression (green) in back skin of WT or Batf2−/− mice that had been treated with imiquimod for 6 days were detected by immunofluorescence. Scale bars represent 60 µm. Phospho-STAT3 is up-regulated and NF-κB p50/p65 are down-regulated in Batf2−/−BMDMs compared with WT controls We confirmed via immunohistochemistry that DUSP2 localizes in the nucleus (Fig. 2A), which is consistent with previously reported data (7). It has also been reported that DUSP2 associates with phospho-STAT3 (p-STAT3) and attenuates its activity through the dephosphorylation of p-STAT3 at Tyr705 (20). In agreement with these reports, we found that the over-expression of Dusp2 down-regulated the expression of p-STAT3 compared with the empty control in nuclear extracts from RAW 264.7 cells (Fig. 2B). Additionally, STAT3 has been reported as a significantly enriched upstream regulator of the genes that are up-regulated in Batf2−/− macrophages (21). On the basis of this, we measured the p-STAT3 expression levels in BMDMs, and the results show that, after stimulation with R848 for 24 h, the p-STAT3 expression is up-regulated in nuclear extracts from Batf2−/− BMDMs compared with WT BMDMs (Fig. 2C). Because p-STAT3 has also been reported to be capable of inhibiting IκB kinase in normal immune cells (22, 23), and thereby reducing NF-κB-associated Th1 immunity (24), we next examined the IκB expression levels. The results show that the expression levels of IκB in R848-stimulated BMDMs were higher in cells from Batf2−/− mice than in cells from WT controls (Fig. 2D). Consistently with these results, down-regulation of the p50, p65 and c-Rel expression levels was observed in nuclear extracts from Batf2−/− BMDMs compared with their levels in nuclear extracts from WT BMDMs, while there were no corresponding significant differences in the cytosolic extracts (Fig. 2E). Fig. 2. View largeDownload slide The expression levels of STAT3 and NF-κB in BMDMs from WT or Batf2−/− mice. (A) The nuclei (Hoechst 33342, blue) and DUSP2 expression (green) in RAW 264.7 cells stimulated with R848 or control for 24 h were detected by immunofluorescence. Scale bars represent 10 µm. (B) RAW 264.7 cells were transfected with the Dusp2 or empty control vector. Twenty-four hours after transfection, the cells were treated with R848 for 24 h. Following activation, the DUSP2, STAT3 and p-STAT3 expression in nuclear extracts was analyzed by western blotting. (C) The STAT3 and p-STAT3 expression in nuclear extracts of WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h was analyzed by western blotting. (D) The IκB-α expression in cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 20, 40 or 60 min was analyzed by western blotting. (E) The p50, p65 and c-Rel expression in nuclear or cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 3 or 6 h was analyzed by western blotting. Data are representative of at least two independent experiments. Fig. 2. View largeDownload slide The expression levels of STAT3 and NF-κB in BMDMs from WT or Batf2−/− mice. (A) The nuclei (Hoechst 33342, blue) and DUSP2 expression (green) in RAW 264.7 cells stimulated with R848 or control for 24 h were detected by immunofluorescence. Scale bars represent 10 µm. (B) RAW 264.7 cells were transfected with the Dusp2 or empty control vector. Twenty-four hours after transfection, the cells were treated with R848 for 24 h. Following activation, the DUSP2, STAT3 and p-STAT3 expression in nuclear extracts was analyzed by western blotting. (C) The STAT3 and p-STAT3 expression in nuclear extracts of WT or Batf2−/− BMDMs stimulated with R848 for 0, 8 or 24 h was analyzed by western blotting. (D) The IκB-α expression in cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 20, 40 or 60 min was analyzed by western blotting. (E) The p50, p65 and c-Rel expression in nuclear or cytosolic extracts of WT or Batf2−/− BMDMs activated with R848 for 0, 3 or 6 h was analyzed by western blotting. Data are representative of at least two independent experiments. IL-12 p40 levels are significantly lower in Batf2−/− macrophages than in WT macrophages, and Th1 immunity is impaired in Batf2−/− mice Since the above results suggested the expression of NF-κB-associated cytokines in response to TLR7 stimulation would be impaired in Batf2−/− mice, we performed ELISAs to examine the levels of IL-12 p40 secreted by Batf2−/− and WT BMDMs following their stimulation by R848. The results show that the IL-12 p40 levels were significantly lower in the culture medium of Batf2−/− BMDMs than in that of WT BMDMs (Fig. 3A). To investigate the effect of Dusp2 on IL-12 p40 levels, we performed luciferase reporter assays with a full-length Dusp2 construct and an Il12b promoter linked to a luciferase reporter gene (11). We found that RAW 264.7 cells over-expressing Dusp2 displayed an enhanced level of luciferase reporter activity following stimulation with R848, compared with cells transfected with the empty control (Fig. 3B). Additionally, we observed that the proportion of T-bet+ GATA-3− CD4+ T cells in R848-treated Batf2−/− mice was significantly lower compared with that in R848-treated WT controls (Fig. 3C), indicating that Th1 immunity is impaired in Batf2−/− mice. Fig. 3. View largeDownload slide Analyses of Th1 immunity in WT and Batf2−/− mice. (A) Levels of IL-12 p40 secreted by WT or Batf2−/− BMDMs following stimulation with R848 (100 ng ml−1) or control medium for 24 h were measured by ELISA. Data are expressed as the mean ± SD of two independent experiments (n = 4). *P < 0.05. (B) The mouse Il12b promoter–luciferase reporter construct (Il12b pro-Luc) was transfected into RAW 264.7 cells along with a Dusp2 or empty vector construct. Cells were treated with control medium or R848 at 24 h post-transfection, and the luciferase activity was measured 24 h after stimulation. Data are expressed as the mean ± SD from two independent experiments. *P < 0.05. (C) Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Numbers represent the percentages of T-bet+ GATA3− CD4+ T cells within the CD4+ T-cell population (left). Data are from four independent experiments (right, n = 6–8). Bars show means. *P < 0.05. Fig. 3. View largeDownload slide Analyses of Th1 immunity in WT and Batf2−/− mice. (A) Levels of IL-12 p40 secreted by WT or Batf2−/− BMDMs following stimulation with R848 (100 ng ml−1) or control medium for 24 h were measured by ELISA. Data are expressed as the mean ± SD of two independent experiments (n = 4). *P < 0.05. (B) The mouse Il12b promoter–luciferase reporter construct (Il12b pro-Luc) was transfected into RAW 264.7 cells along with a Dusp2 or empty vector construct. Cells were treated with control medium or R848 at 24 h post-transfection, and the luciferase activity was measured 24 h after stimulation. Data are expressed as the mean ± SD from two independent experiments. *P < 0.05. (C) Twenty-four hours after an intra-peritoneal injection of R848 (10 µg) in 100 µl of PBS, the T-bet and GATA3 expression levels in splenic CD4+ T cells from Batf2−/− and WT mice were analyzed by flow cytometry. Numbers represent the percentages of T-bet+ GATA3− CD4+ T cells within the CD4+ T-cell population (left). Data are from four independent experiments (right, n = 6–8). Bars show means. *P < 0.05. BATF2 promotes DUSP2 expression From our earlier results, we hypothesized that Batf2 might be a positive regulator of DUSP2 induction. To test this, we next performed luciferase reporter assays. Because the ChIP-Seq enrichment profiles for BATF2 suggested that BATF2 would interact with the Dusp2 promoter position −451 to the transcription start site, we used this promoter region linked to a luciferase reporter gene (Fig. 4A). RAW 264.7 cells over-expressing Batf2 displayed an enhanced level of luciferase reporter activity compared with empty control vector-transfected cells (Fig. 4B), and these luciferase enhancements were observed in a dose-dependent manner (Fig. 4C). Additionally, the level of luciferase enhancement following stimulation with R848 was significantly lower in Batf2−/− BMDMs transfected with the Dusp2 promoter–luciferase reporter construct than that in WT BMDMs (Fig. 4D). Fig. 4. View largeDownload slide Regulation of the Dusp2 proximal promoter region by BATF2 in RAW 264.7 cells. (A) ChIP-Seq enrichment profiles for BATF2 at the mouse Dusp2 promoter locus were generated from WT murine BMDMs stimulated by R848 for 6 h. A schematic of the Dusp2 promoter region used for the luciferase reporter construct is shown. TSS: transcription start site. The number −451 indicates the position from the TSS. (B) The mouse Dusp2 promoter–luciferase reporter construct was transfected into RAW 264.7 cells along with a Batf2 or empty vector construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of duplicates. **P < 0.01. (C) The mouse Dusp2 promoter–luciferase reporter construct was co-transfected with increasing amounts of the Batf2 vector construct. Luciferase activities relative to the empty vector control are shown as a representative mean ± SD. (D) The mouse Dusp2 promoter–luciferase reporter construct was transfected into BMDMs from WT or Batf2−/− mice. Cells were activated with R848 at 24 h post-transfection, and luciferase activity was measured 24 h after stimulation. Luciferase activities are shown as a representative mean ± SD. *P < 0.05. Similar results were obtained in at least two independent experiments. Fig. 4. View largeDownload slide Regulation of the Dusp2 proximal promoter region by BATF2 in RAW 264.7 cells. (A) ChIP-Seq enrichment profiles for BATF2 at the mouse Dusp2 promoter locus were generated from WT murine BMDMs stimulated by R848 for 6 h. A schematic of the Dusp2 promoter region used for the luciferase reporter construct is shown. TSS: transcription start site. The number −451 indicates the position from the TSS. (B) The mouse Dusp2 promoter–luciferase reporter construct was transfected into RAW 264.7 cells along with a Batf2 or empty vector construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of duplicates. **P < 0.01. (C) The mouse Dusp2 promoter–luciferase reporter construct was co-transfected with increasing amounts of the Batf2 vector construct. Luciferase activities relative to the empty vector control are shown as a representative mean ± SD. (D) The mouse Dusp2 promoter–luciferase reporter construct was transfected into BMDMs from WT or Batf2−/− mice. Cells were activated with R848 at 24 h post-transfection, and luciferase activity was measured 24 h after stimulation. Luciferase activities are shown as a representative mean ± SD. *P < 0.05. Similar results were obtained in at least two independent experiments. BATF2 promotes DUSP2 expression through NF-κB-binding sites in the Dusp2 promoter The above results suggest that BATF2 recruitment to the Dusp2 promoter region is important for its transcriptional activation. To identify the Dusp2 promoter elements that are responsible for transcriptional activation by BATF2, we performed luciferase reporter assays using Dusp2 promoter sequences containing mutations. First, we searched for potential transcription factor-binding regions using TFBIND software (16) and found nine potential NF-κB-binding regions on the Dusp2 promoter (Fig. 5A). On the basis of this finding, we performed serial deletion-mutant analyses. Deletions of the region from position −208 to −118 diminished the enhancement of luciferase activity by BATF2 compared with empty vectors (Fig. 5B). Since the region from position −208 to −118 has three predicted NF-κB-binding regions, we performed substitution-mutant analyses focusing on these three binding regions. The Batf2 luciferase enhancement was decreased in the substitution mutant for each of the three binding regions (Fig. 5C), suggesting that all three binding regions are important for Batf2 activation. Fig. 5. View largeDownload slide Analyses of the mechanism responsible for the effect of BATF2 on DUSP2 expression. (A) The sequence of the Dusp2 proximal promoter is shown. Estimated potential transcription factor-binding sequences are boxed and labeled. (B and C) Deletion-mutant (B) and substitution-mutant (C) analyses of the Dusp2 promoter. Mutants were transfected into RAW 264.7 cells with a Batf2 or empty construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of triplicates. Similar results were obtained from two independent experiments. Fig. 5. View largeDownload slide Analyses of the mechanism responsible for the effect of BATF2 on DUSP2 expression. (A) The sequence of the Dusp2 proximal promoter is shown. Estimated potential transcription factor-binding sequences are boxed and labeled. (B and C) Deletion-mutant (B) and substitution-mutant (C) analyses of the Dusp2 promoter. Mutants were transfected into RAW 264.7 cells with a Batf2 or empty construct. Luciferase activity was measured 48 h post-transfection. Data are expressed as the mean ± SD of triplicates. Similar results were obtained from two independent experiments. BATF2 interacts with p65, and together they bind to the region from position −203 to −121 in the Dusp2 promoter We next performed DNA-binding ELISAs using a biotinylated probe against Dusp2 promoter position −203 to −121 to confirm that NF-κB p65 binds to this region. Compared with the blank control, we observed an increase in the amount of NF-κB p65 bound to the biotinylated probe when recombinant p65 was used directly as a positive control (Fig. 6A), suggesting the direct binding of p65 to this position. The up-regulated activity of p65 was also observed in nuclear extracts from RAW 264.7 cells that were stimulated with R848 compared with non-stimulated cells (Fig. 6A). Similar results were also obtained with analogous experiments using c-Rel (Fig. 6A). We then performed DNAP assays using cell extracts from RAW 264.7 cells over-expressing Myc-tagged Batf2. The results suggest that BATF2, p65 and c-Rel bind to the region at position −203 to −121 in the Dusp2 promoter (Fig. 6B). Next, we repeated the DNAP assays using p65-depleted samples. The results indicate that p65 depletion from the lysates attenuated the recruitment of BATF2 on the Dusp2 promoter (Fig. 6C), suggesting that BATF2 is recruited on the Dusp2 promoter through binding to p65. To confirm this, we performed co-immunoprecipitation experiments using cell extracts from RAW 264.7 cells over-expressing Myc-tagged Batf2 and Rela (p65), and the results verify that BATF2 interacts with p65 (Fig. 6D). Fig. 6. View largeDownload slide Analyses of the DNA-binding activities of NF-κB p65, c-Rel and BATF2. (A) The DNA-binding activities of p65 were measured with a TransAM Flexi NF-κB Transcription Factor Assay Kit (schematic, left). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant p65 (500 ng, middle) or nuclear extracts from cells over-expressing c-Rel (5 µg, right) were used as positive controls (ctrls). Sample nuclear extracts were obtained from RAW 264.7 cells activated with R848 or treated with a medium control for 1 h (middle and right). Error bars indicate ± SD from two independent experiments. *P < 0.05. (B) Results from a DNAP assay (schematic, left) are shown. Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared and incubated with the biotinylated DNA probe. Streptavidin-coupled Dynabeads were added, and the trapped proteins were analyzed by western blotting (WB; right). Data are representative of two independent experiments. (C) For DNAP assays on samples with a depletion of p65, the samples were first subjected to a p65 depletion performed via immunoprecipitation with anti-p65 antibody and Protein G-Sepharose beads prior to their use in a DNAP assay (schematic, left). The resulting supernatant was then analyzed by DNAP assays as described in (B), and the proteins trapped by the streptavidin-coupled Dynabeads were analyzed by western blotting (WB; right). (D) Co-immunoprecipitation experiments assessing BATF2 binding to p65. Cell extracts from RAW 264.7 cells over-expressing both RelA (p65) and Myc-tagged BATF2 were prepared. After immunoprecipitation (IP) with anti-p65 antibody or normal control IgG, samples were analyzed with anti-c-Myc antibody by western blotting (WB). Similar results were obtained in three independent experiments. Fig. 6. View largeDownload slide Analyses of the DNA-binding activities of NF-κB p65, c-Rel and BATF2. (A) The DNA-binding activities of p65 were measured with a TransAM Flexi NF-κB Transcription Factor Assay Kit (schematic, left). A biotinylated probe containing positions −203 to −121 within the Dusp2 promoter was used as the DNA probe. Recombinant p65 (500 ng, middle) or nuclear extracts from cells over-expressing c-Rel (5 µg, right) were used as positive controls (ctrls). Sample nuclear extracts were obtained from RAW 264.7 cells activated with R848 or treated with a medium control for 1 h (middle and right). Error bars indicate ± SD from two independent experiments. *P < 0.05. (B) Results from a DNAP assay (schematic, left) are shown. Cell lysates from RAW 264.7 cells over-expressing Myc-tagged Batf2 were prepared and incubated with the biotinylated DNA probe. Streptavidin-coupled Dynabeads were added, and the trapped proteins were analyzed by western blotting (WB; right). Data are representative of two independent experiments. (C) For DNAP assays on samples with a depletion of p65, the samples were first subjected to a p65 depletion performed via immunoprecipitation with anti-p65 antibody and Protein G-Sepharose beads prior to their use in a DNAP assay (schematic, left). The resulting supernatant was then analyzed by DNAP assays as described in (B), and the proteins trapped by the streptavidin-coupled Dynabeads were analyzed by western blotting (WB; right). (D) Co-immunoprecipitation experiments assessing BATF2 binding to p65. Cell extracts from RAW 264.7 cells over-expressing both RelA (p65) and Myc-tagged BATF2 were prepared. After immunoprecipitation (IP) with anti-p65 antibody or normal control IgG, samples were analyzed with anti-c-Myc antibody by western blotting (WB). Similar results were obtained in three independent experiments. Discussion DUSP2 plays a critical role in inflammatory responses by dephosphorylating p-STAT3 at Tyr705 and attenuating its activity (20). In agreement with previous reports, our results show that the over-expression of Dusp2 down-regulated the expression of p-STAT3 compared with the empty control (Fig. 2B) and that Batf2−/− mice, which have a significantly lower level of DUSP2 than WT controls, have up-regulated p-STAT3 expression compared with WT mice (Fig. 2C). Although IL-6 is a key activator of STAT3 via phosphorylation (24), the expression levels of IL-6 are similar between WT and Batf2−/− mice (3). Therefore, phosphatases, including DUSP2, are likely important in the up-regulation of p-STAT3 in Batf2−/− BMDMs. Notably, it was also reported that p-STAT3 can inhibit IκB kinase and thereby reduce NF-κB-associated Th1 immunity (22–24), although the precise mechanisms are still unknown. In addition, myeloid-specific Stat3 gene ablation induced the release of IL-12 by dendritic cells (25, 26). Our results show that IκB was up-regulated in the cytosolic extracts from Batf2−/− BMDMs compared with WT controls (Fig. 2D). Additionally, NF-κB p50, p65 and c-Rel were all down-regulated in the nuclear extracts from Batf2−/− BMDMs compared with WT controls (Fig. 2E). These data are also consistent with our finding that the IL-12 p40 levels were significantly lower in the culture medium of Batf2−/− BMDMs than in that of WT BMDMs (Fig. 3A). Moreover, we additionally observed a significantly lower proportion of T-bet+ GATA-3− CD4+ T cells in R848-treated Batf2−/− mice than in R848-treated WT controls (Fig. 3C), indicating a reduction of Th1 immunity in Batf2−/− mice. Given that p-STAT3 can attenuate NF-κB-associated Th1 immunity through its inhibition of IκB kinase, the reduction of DUSP2 and the up-regulation of p-STAT3 via its attenuated dephosphorylation in Batf2−/− mice indicate their involvements in the impairment of NF-κB-associated Th1 immunity in Batf2−/− mice (Fig. 7). Fig. 7. View largeDownload slide A schematic model of how BATF2 may contribute to up-regulation of the phosphatase DUSP2 and enhancement of Th1 immunity after TLR7 stimulation. In this proposed model, BATF2 up-regulates the expression of DUSP2, which may be involved in the promotion of the downstream expression of IL-12 p40 through the down-regulation of p-STAT3 and the up-regulation of NF-κB. This increase in IL-12 p40 leads to an enhancement of NF-κB-associated Th1 immunity. Fig. 7. View largeDownload slide A schematic model of how BATF2 may contribute to up-regulation of the phosphatase DUSP2 and enhancement of Th1 immunity after TLR7 stimulation. In this proposed model, BATF2 up-regulates the expression of DUSP2, which may be involved in the promotion of the downstream expression of IL-12 p40 through the down-regulation of p-STAT3 and the up-regulation of NF-κB. This increase in IL-12 p40 leads to an enhancement of NF-κB-associated Th1 immunity. These results suggest that BATF2 activates transcription of Dusp2 through its recruitment to the Dusp2 promoter region. Although it was previously thought that BATF family members function only as inhibitors of AP-1 (27, 28), our findings add to the growing evidence indicating that an interaction with a non-AP-1 factor is likely involved in BATF-specific positive transcriptional activities (2). While BATF family proteins lack a transcriptional activation domain, they are still able to support other transcription factors and exert unique and positive transcriptional activities. For example, BATF2 interacts with IRF1 to induce inflammatory responses during Mycobacterium tuberculosis infection (9, 29). In addition, BATF/IRF4 and BATF/IRF8 interactions are important for compensatory dendritic cell development (4). An interaction between BATF2 and NF-κB p65 was reported previously by using yeast two-hybrid technology (30). Here, we demonstrate that BATF2 interacts with NF-κB p65 and up-regulates the expression of DUSP2 via binding to the region at position −203 to −121 in the Dusp2 promoter (Figs 5 and 6). Although the reason for this specificity is still unclear, it may be connected to the ability of BATF2 to function as either an inhibitor or an up-regulator depending on how it interacts with other transcription factors. For example, although our results show that BATF2 is an up-regulator of NF-κB, BATF family members, including BATF2, also function as AP-1 inhibitors (1). Furthermore, BATF was recently proposed to be a pioneer factor (31), which can bind to a target site in condensed chromatin and allow the rapid recruitment of other transcription factors. It is possible that BATF2 may also have some similar epigenetic functions in the Dusp2 promoter. Although our data suggest the possible involvement of DUSP2 in the promotion of Th1 immunity through up-regulation of Il12b expression (Figs 3B and 7), additional work is still needed to confirm this model because the data from this study do not directly show the impairment of Th1 immunity in Dusp2-deficient macrophages. Specifically, our results do not demonstrate an impairment of the Th1 response in Dusp2-deficient mice following their stimulation with TLR7 ligands. Therefore, further studies are needed to fully elucidate the relationship between DUSP2 and the BATF2-mediated Th1 response. In this study, we focused on the involvement of BATF2 in the up-regulation of the phosphatase DUSP2. Although BATF2 was previously known only for its inhibitory functions, this study has revealed that BATF2 has other unique and positive transcriptional activities. Further studies are needed to elucidate the full role of BATF2. Funding This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Japan Society for the Promotion of Science (JSPS) through funding for Specially Promoted Research (Grant Number 15H05704) and by a grant from Project MEET; Osaka University Graduate School of Medicine, Mitsubishi Tanabe Pharma Corporation. This study was supported in part by Yuri Terao and Center for Medical Research and Education, Graduate School of Medicine, Osaka University. Acknowledgements We thank T. Machida, T. Kawasaki, T. Matsuki, K. Fukushima, K. J. Yoshida, H. Nabeshima, I. Ebina, K. Kuniyoshi, X. Sun, M. Shimoda, S. K. Singh, Y. Nagahama, Y. Kozakai, K. Maruyama, K. Iwamoto, M. Okada, Y. Kishi, H. Nakamura, T. Suzuki and T. Sugita for discussions; A. Otsuka, A. Yamazaki, M. Tasai, K. Asakawa, C. Funamoto, A. Wataki and R. Kawaguchi for technical assistance; and E. Kamada for secretarial assistance. We also thank K. Oakley from Edanz Group for editing a draft of this manuscript. Conflicts of interest statement: the authors declared no conflicts of interest. References 1 Su , Z. Z. , Lee , S. G. , Emdad , L. et al. 2008 . Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN) . Proc. Natl Acad. Sci. USA 105 : 20906 . Google Scholar CrossRef Search ADS 2 Murphy , T. L. , Tussiwand , R. and Murphy , K. M . 2013 . Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks . Nat. Rev. Immunol . 13 : 499 . Google Scholar CrossRef Search ADS PubMed 3 Kanemaru , H. , Yamane , F. , Fukushima , K. et al. 2017 . Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages . Proc. Natl Acad. Sci. USA 114 : E7331 . Google Scholar CrossRef Search ADS 4 Tussiwand , R. , Lee , W. L. , Murphy , T. L. et al. 2012 . Compensatory dendritic cell development mediated by BATF-IRF interactions . Nature 490 : 502 . Google Scholar CrossRef Search ADS PubMed 5 Jeffrey , K. L. , Brummer , T. , Rolph , M. S. et al. 2006 . Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1 . Nat. Immunol . 7 : 274 . Google Scholar CrossRef Search ADS PubMed 6 Ward , Y. , Gupta , S. , Jensen , P. , Wartmann , M. , Davis , R. J. and Kelly , K . 1994 . Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1 . Nature 367 : 651 . Google Scholar CrossRef Search ADS PubMed 7 Rohan , P. J. , Davis , P. , Moskaluk , C. A. et al. 1993 . PAC-1: a mitogen-induced nuclear protein tyrosine phosphatase . Science 259 : 1763 . Google Scholar CrossRef Search ADS PubMed 8 Yamamoto , M. , Uematsu , S. , Okamoto , T. et al. 2007 . Enhanced TLR-mediated NF-IL6 dependent gene expression by Trib1 deficiency . J. Exp. Med . 204 : 2233 . Google Scholar CrossRef Search ADS PubMed 9 Roy , S. , Guler , R. , Parihar , S. P. et al. 2015 . Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection . J. Immunol . 194 : 6035 . Google Scholar CrossRef Search ADS PubMed 10 Murphy , T. L. , Cleveland , M. G. , Kulesza , P. , Magram , J. and Murphy , K. M . 1995 . Regulation of interleukin 12 p40 expression through an NF-κB half-site . Mol. Cell. Biol . 15 : 5258 . Google Scholar CrossRef Search ADS PubMed 11 Plevy , S. E. , Gemberling , J. H. , Hsu , S. , Dorner , A. J. and Smale , S. T . 1997 . Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins . Mol. Cell. Biol . 17 : 4572 . Google Scholar CrossRef Search ADS PubMed 12 Sanjabi , S. , Williams , K. J. , Saccani , S. et al. 2005 . A c-Rel subdomain responsible for enhanced DNA-binding affinity and selective gene activation . Genes Dev . 19 : 2138 . Google Scholar CrossRef Search ADS PubMed 13 van der Fits , L. , Mourits , S. , Voerman , J. S. et al. 2009 . Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis . J. Immunol . 182 : 5836 . Google Scholar CrossRef Search ADS PubMed 14 Dignam , J. D. , Lebovitz , R. M. and Roeder , R. G . 1983 . Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei . Nucleic Acids Res . 11 : 1475 . Google Scholar CrossRef Search ADS PubMed 15 Lo , K. , Landau , N. R. and Smale , S. T . 1991 . LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes . Mol. Cell. Biol . 11 : 5229 . Google Scholar CrossRef Search ADS PubMed 16 Tsunoda , T. and Takagi , T . 1999 . Estimating transcription factor bindability on DNA . Bioinformatics 15 : 622 . Google Scholar CrossRef Search ADS PubMed 17 Renard , P. , Ernest , I. , Houbion , A. et al. 2001 . Development of a sensitive multi-well colorimetric assay for active NF-κB . Nucleic Acids Res . 29 : E21 . Google Scholar CrossRef Search ADS PubMed 18 Maruyama , K. , Fukasaka , M. , Vandenbon , A. et al. 2012 . The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation . Immunity 37 : 1024 . Google Scholar CrossRef Search ADS PubMed 19 Suzuki , T. , Fujisawa , J. I. , Toita , M. and Yoshida , M . 1993 . The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1 . Proc. Natl Acad. Sci. USA 90 : 610 . Google Scholar CrossRef Search ADS 20 Lu , D. , Liu , L. , Ji , X. et al. 2015 . The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation . Nat. Immunol . 16 : 1263 . Google Scholar CrossRef Search ADS PubMed 21 Kitada , S. , Kayama , H. , Okuzaki , D. et al. 2017 . BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Trypanosoma cruzi infection . J. Exp. Med . 214 : 1313 . Google Scholar CrossRef Search ADS PubMed 22 Welte , T. , Zhang , S. S. , Wang , T. et al. 2003 . STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity . Proc. Natl Acad. Sci. USA 100 : 1879 . Google Scholar CrossRef Search ADS 23 Lee , H. , Herrmann , A. , Deng , J. H. et al. 2009 . Persistently activated Stat3 maintains constitutive NF-κB activity in tumors . Cancer Cell 15 : 283 . Google Scholar CrossRef Search ADS PubMed 24 Yu , H. , Pardoll , D. and Jove , R . 2009 . STATs in cancer inflammation and immunity: a leading role for STAT3 . Nat. Rev. Cancer 9 : 798 . Google Scholar CrossRef Search ADS PubMed 25 Kortylewski , M. , Xin , H. , Kujawski , M. et al. 2009 . Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment . Cancer Cell 15 : 114 . Google Scholar CrossRef Search ADS PubMed 26 Kortylewski , M. , Kujawski , M. , Wang , T. et al. 2005 . Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity . Nat. Med . 11 : 1314 . Google Scholar CrossRef Search ADS PubMed 27 Dorsey , M. J. , Tae , H. J. , Sollenberger , K. G. , Mascarenhas , N. T. , Johansen , L. M. and Taparowsky , E. J . 1995 . B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family . Oncogene 11 : 2255 . Google Scholar PubMed 28 Echlin , D. R. , Tae , H. J. , Mitin , N. and Taparowsky , E. J . 2000 . B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos . Oncogene 19 : 1752 . Google Scholar CrossRef Search ADS PubMed 29 Guler , R. , Roy , S. , Suzuki , H. and Brombacher , F . 2015 . Targeting Batf2 for infectious diseases and cancer . Oncotarget 6 : 26575 . Google Scholar CrossRef Search ADS PubMed 30 Wang , J. , Huo , K. , Ma , L. et al. 2011 . Toward an understanding of the protein interaction network of the human liver . Mol. Syst. Biol . 7 : 536 . Google Scholar CrossRef Search ADS PubMed 31 Ciofani , M. , Madar , A. , Galan , C. et al. 2012 . A validated regulatory network for Th17 cell specification . Cell 151 : 289 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

International ImmunologyOxford University Press

Published: Mar 9, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off