Type I IFNs Downregulate Myeloid Cell IFN- γ by Inducing Recruitment of an Early Growth Response 3/NGFI-A Binding 1 Complex That Silences ifngr1 This information is current as of September 28, 2021. Staci J. Kearney, Christine Delgado, Emily M. Eshleman, Krista K. Hill, Brian P. O'Connor and Laurel L. Lenz J Immunol 2013; 191:3384-3392; Prepublished online 9 August 2013; Downloaded from doi: 10.4049/jimmunol.1203510 http://www.jimmunol.org/content/191/6/3384 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/08/12/jimmunol.120351 Material 0.DC1 References This article cites 69 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/191/6/3384.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Type I IFNs Downregulate Myeloid Cell IFN-g Receptor by Inducing Recruitment of an Early Growth Response 3/NGFI-A Binding Protein 1 Complex That Silences ifngr1 Transcription

Staci J. Kearney,*,† Christine Delgado,† Emily M. Eshleman,† Krista K. Hill,* Brian P. O’Connor,*,†,‡,1 and Laurel L. Lenz*,†,1

The ability of type I IFNs to increase susceptibility to certain bacterial infections correlates with downregulation of myeloid cell surface IFNGR, the receptor for the type II IFN (IFN-g), and reduced myeloid cell responsiveness to IFN-g. In this study, we show

that the rapid reductions in mouse and human myeloid cell surface IFNGR1 expression that occur in response to type I IFN Downloaded from treatment reflect a rapid silencing of new ifngr1 transcription by repressive transcriptional regulators. Treatment of macrophages with IFN-b reduced cellular abundance of ifngr1 transcripts as rapidly and effectively as actinomycin D treatment. IFN-b treatment also significantly reduced the amounts of activated RNA polymerase II (pol II) and acetylated histones H3 and H4 at the ifngr1 promoter and the activity of an IFNGR1-luc reporter construct in macrophages. The suppression of IFNGR1-luc activity required an intact early growth response factor (Egr) binding site in the proximal ifngr1 promoter. Three Egr

and two Egr/NGFI-A binding (Nab) proteins were found to be expressed in bone macrophages, but only Egr3 and Nab1 were http://www.jimmunol.org/ recruited to the ifngr1 promoter upon IFN-b stimulation. Knockdown of Nab1 in a macrophage cell line prevented downregu- lation of IFNGR1 and prevented the loss of acetylated histones from the ifngr1 promoter. These data suggest that type I IFN stimulation induces a rapid recruitment of a repressive Egr3/Nab1 complex that silences transcription from the ifngr1 promoter. This mechanism of silencing may contribute to the anti-inflammatory effects of type I IFNs. The Journal of Immunology, 2013, 191: 3384–3392.

ype I IFNs (e.g., IFNs a and b) were originally recognized IFNs reduces host resistance to a number of bacterial infections for their ability to induce an antiviral state following (5–7). The mechanisms responsible for these negative effects of T autocrine or paracrine signaling in fibroblasts and other type I IFNs have been unclear. by guest on September 28, 2021 cell types (1, 2). Consequently, responses to type I IFNs are re- The modulation of host cell by type I IFNs quired for optimal host resistance in diverse viral infection mod- requires binding of these cytokines to a ubiquitously expressed cell els, and considerable effort has focused on understanding how surface receptor, IFNAR (1, 2). Such binding elicits signals that these cytokines stimulate protective immune responses (1–4). How- alter expression of hundreds of target (1–3, 8, 9). There is ever, in apparent contrast to the protective effects of type I IFNs considerable information on how type I IFNs positively regulate during viral infections, these cytokines have also been found to gene expression, but the mechanisms they use to negatively reg- suppress or negatively regulate inflammatory and immune respon- ulate gene expression have been less clear. Recently, it was shown ses in other disease settings. For example, responsiveness to type I that the ability of type I IFNs to negatively regulate expression of certain induced genes involves recruitment of repressive epige- netic factors, such as methyltransferases (10) or histone deacety- *Integrated Department of Immunology, National Jewish Health, Denver, CO 80206; †Integrated Department of Immunology, University of Colorado, Denver, CO 80045; lases (HDACs) (11, 12). However, it is not known whether similar and ‡Integrated Center for Genes, Environment and Health, National Jewish Health, mechanisms contribute to the ability of type I IFNs to silence ex- Denver, CO 80206 pression of basally transcribed genes. 1 B.P.O. and L.L.L. contributed equally to this manuscript. Prior studies by our laboratory and others established that type I Received for publication December 28, 2012. Accepted for publication July 8, 2013. IFNs suppress myeloid cell activation during infections by certain This work was supported by the National Institutes of Health Research Grants intracellular bacteria, including Mycobacterium tuberculosis and R56AI65638, RO1AI065638, R21AI102264, and R21AI103782 (to L.L.L.). B.P.O. Listeria monocytogenes (13, 14). The induction of macrophage received support from the Walter Scott Foundation. S.J.K. received support from a Cancer Research Institute training grant. Core facilities used in these studies re- activation in these and other infections involves type II IFN, IFN-g, ceived support from National Institutes of Health Research Grants P01AI22296 and and plays an essential role in host resistance (6, 7, 15–17). Cellular P30CA046934. responsiveness to IFN-g requires cell surface expression of a het- Address correspondence and reprint requests to Dr. Laurel L. Lenz, Integrated De- erodimeric receptor, IFNGR, which is composed of the IFNGR1 partment of Immunology, National Jewish Health, 1400 Jackson Street, Suite K510, Denver, CO 80206. E-mail address: [email protected] and IFNGR2 subunits. Our prior findings using L. monocytogenes The online version of this article contains supplemental material. infection showed that the reduced activation of myeloid cells Abbreviations used in this article: BMDM, bone marrow–derived macrophage; ChIP, corresponded with type I IFN–dependent reductions in myeloid chromatin immunoprecipitation; Egr, early growth response; HDAC, histone deace- cell surface IFNGR1 and IFNGR2, which correlated with reduced tylase; hPBMC, human PBMC; hps, hour poststimulation; MFI, mean fluorescence abundance of ifngr1 (but not ifngr2) transcripts (14). These find- intensity; Nab, NGFI-A binding protein; qPCR, quantitative RT-PCR; wt, wild-type. ings suggest that the abundance of ifngr1 transcripts regulates cell Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 surface IFNGR levels in mouse myeloid cells. In this study, we www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203510 The Journal of Immunology 3385 sought to investigate how type I IFNs negatively regulate myeloid EL4 murine T cells were cultured in DM10 medium (DMEM supple- cell ifngr1 expression. mented with 10% FBS, 1% sodium pyruvate, 1% L-, and 1% The silencing of basally transcribed genes often involves re- penicillin/streptomycin). THP-1 cells were cultured in suspension with RP10 medium (RPMI 1640 supplemented with 10% FBS, 1% sodium cruitment of repressive transcription factors to the target gene pyruvate, 1% L-glutamine, and 1% penicillin/streptomycin). Twenty-four promoter. The early growth response (Egr) family of transcription hours prior to experimentation, THP-1 cells were stimulated with 0.1 mg/ml factors comprises four members (Egr1, Egr2, Egr3, and Egr4). PMA (P-8139; Sigma-Aldrich, St. Louis, MO) to obtain adherent cells. DNA-binding domains in these Egr proteins are formed by three To obtain human PBMCs (hPBMCs), de-identified blood from donors was collected in heparin-containing vacuum tubes, and WBCs were zinc-finger motifs that bind to the consensus sequence CGCCC- separated from whole blood by Ficoll–Paque gradient (Histopaque-1077; CCGC (18). Egr proteins were originally recognized for their role Sigma-Aldrich). Isolated cells were incubated overnight in 6-well culture in the genetic regulation of cell growth and differentiation in re- plates in DMEM supplemented with human serum. BMDMs, RAW264.7, sponse to extracellular stimuli, particularly in the context of the and EL4 cells were treated at various time points with 100 U/ml murine nervous system (19). They are now also known to promote ex- IFN-b (PBL IFN Source, Piscataway, NJ). THP-1 cells and hPBMCs were treated at various time points with 100 U/ml human IFN-b (PBL IFN pression of a diverse group of genes, including several with im- Source). portant immunological functions (20–24). Egr family members can also repress the transcription of certain target genes, particu- Flow cytometry larly in response to external stimuli such as cytokines (25, 26). BMDMs and adherent cell lines were lifted from culture dishes with cold The mechanisms for gene repression include interference with PBS. Adherent hPBMCs were lifted from culture dishes with cold PBS and transcriptional activators such as Sp1 (25‑29) and TATA binding added to nonadherent cells. Murine FcRs were blocked before staining using supernatant from hybridoma 2.4G2 (rat anti-CD16/32), and human protein (30, 31) and recruitment of a family of Egr corepressors FcRs were blocked using pooled human serum in PBS. To detect murine Downloaded from known as NGFI-A binding proteins (Nab) (32, 33). Egr1, Egr2, IFNGR1, cells were stained with biotinylated Abs to IFNGR1/CD119 (BD and Egr3 proteins (but not Egr4) contain a repression domain (R1) Biosciences, San Jose, CA), followed by streptavidin–APC secondary Ab that binds to the highly conserved NCD1 domain present in both (eBioscience, San Diego, CA). To detect human IFNGR1, cells were Nab family members, Nab1 and Nab2 (34‑38). Nab proteins are stained with biotinylated Abs to IFNGR1/CD119 (Caltag Laboratories, Life Technologies, Carlsbad, CA), followed by streptavidin–APC second- unable to bind to DNA alone (38) and thus suppress transcription ary. Primary human T cells and monocytes were detected using CD3-FITC upon recruitment to a DNA-bound Egr family member (32, 33, and CD14-PE Abs (eBioscience), respectively. To detect MHC class I, http://www.jimmunol.org/ 38). The repressive Egr–Nab complexes often silence or maintain EL4 cells were stained with anti–H2Db-FITC (eBioscience). All Abs were repression of gene expression by recruiting factors that can induce diluted in surface staining buffer (PBS/1%BSA/0.01% NaN3). The mean fluorescence intensities (MFIs) for each of three treated samples per time epigenetic gene silencing, such as HDACs (33, 39). point were normalized to mean MFI for three untreated samples using the In this study, we showed that type I IFN treatment rapidly following formula: relative surface staining = (MFI treated)/(MFI un- silences ifngr1 transcription in mouse and human macrophages, treated). For statistical analyses, we pooled the relative MFI values from but not T cells, and describe a mechanism contributing to this three separate experiments each using at least three control and three treated silencing. We identified putative Egr binding sites in the mouse samples. and human ifngr1 promoters and showed that a proximal Egr site Real-time quantitative PCR is required for silencing of ifngr1 transcription in mouse myeloid by guest on September 28, 2021 Preparation and analysis of samples for quantitative RT-PCR (qPCR) was cells treated with IFN-b. Chromatin immunoprecipitation analysis described previously (14). Briefly, 9 3 106 BMM or RAW264.7 cells were further indicated that type I IFNs induce rapid recruitment of Egr3 distributed into 3 wells of a 6-well plate (Cell Star; Sigma-Aldrich) for each to a region of the ifngr1 promoter containing this proximal Egr treatment time point. Cells were pooled from 3 wells, and the RNA was binding site in myeloid but not T cells. Recruitment of Egr3 isolated using the RNeasy kit (Qiagen, Valencia, CA). cDNA synthesis was conducted with 1 mg total RNA using oligo(dT) primers (Promega, Madison, correlated with reductions in activated RNA polymerase II (pol II) WI). Commercial oligonucleotide primer sets from Applied Biosystems (Life and preceded recruitment of Nab1. Nab1 recruitment coincided Technologies) were used to quantify mouse ifngr1 and human ifngr1 tran- with and was required for deacetylation of the ifngr1 promoter and scripts and the primer set, sense, 59-GCACTGGGTGGAATGAGACTATTG- downregulation of cell surface IFNGR. These data demonstrate 39, and antisense, 59-GACCTGTCAGTTGATGCCTCAGAA-39,wasusedto b involvement of a Egr3/Nab1 complex in the silencing of ifngr1 quantify mouse IFN- transcripts. Real-time quantitative PCR was performed using Sybr Green PCR MasterMix (Applied Biosystems) and an ABI Prism transcription and downregulation of IFNGR by type I IFNs. Pu- 7300 Sequence detector. The equation used to determine relative transcript tative Egr binding sites were also identified in the promoters of abundance is 2^[21 3 ([IFNGR1 Ct 2 mean GAPDH Ct] 2 [mean unstimulated dCT])]. other constitutively expressed genes known to be repressed by Transcript half-life was calculated using the equation: half-life = (elapsed type I IFNs, suggesting Egr3 and Nab1 may play a general role in time*log2)/(log [beginning amount/ending amount]); SD was determined by pooling values from three separate experiments. negative regulation of myeloid cell gene expression. Chromatin immunoprecipitation Materials and Methods The chromatin immunoprecipitation (ChIP) experiments were performed Mice according to the protocol provided for the Active Motif ChIP Express kit (Active Motif, Carlsbad, CA). Briefly, after treatment with IFN-b,RAW C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, 2/2 264.7, EL4, and BMDMs were cross-linked with 1% methanol-free ME). IFNAR crossed to C57BL/6 (The Jackson Laboratory) for .10 6 formaldehyde for 7 min at room temperature. Fixed cells (7 3 10 in generations were described previously (14). Mice were housed in the 300 ml) were resuspended in kit lysis buffer plus protease inhibitors and National Jewish Health Biological Resource Center. The National Jewish incubated at 30 min at 4˚C. Cell nuclei were pelleted and resuspended in Health Institutional Animal Care and Use Committee approved all studies. 300 ml kit shearing buffer plus protease inhibitors. A Covaris S2 sonicator Cell culture and IFN-b treatment was used to shear the samples, using a 27-cycle treatment. To ensure that the shearing process consistently produced fragments of 200–500 bp, 5 ml To culture bone marrow–derived macrophages (BMDMs), cells were flushed of all supernatants was run on a 1% agarose gel (Supplemental Fig. 3A). from the femurs, tibias, and fibulas of mice and cultured for 6 d in bone An additional 10 ml supernatant was saved for use as total input DNA. All marrow macrophage medium (DMEM supplemented with 10% FBS, 1% samples were stored at 280˚C until use. Immunoprecipitations were per- sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin, 2-ME, plus formed with protein G magnetic beads overnight at 4˚C, using an estimated 10% L-cell conditioned media). Media components were Life Technolo- 7 mg sheared chromatin and Abs specific for pS5-RNA pol II (ab5131; gies (Carlsbad, CA). Fresh medium was added at day 3, and BMDMs were Abcam, Cambridge, MA), acetyl-histone H3 (39139; Active Motif), pan- used for experiments on day 7. RAW264.7 murine macrophage cells and acetyl-histone H4 (39925; Active Motif), total histone H3 (ab1791; Abcam) 3386 TRANSCRIPTIONAL REPRESSION BY TYPE I IFNs

Egr1 (number 4153; Cell Signaling Technology, Danvers, MA), Egr2 Stable knockdown of Egr and Nab family members (PRB-236P; Covance, Princeton, NJ), Egr3 (ab75461; Abcam), and Nab1 (NBP1-71838; Novus, Littleton, CO). Equal amounts of chromatin were Sigma-Aldrich Mission short hairpin RNA constructs Egr3 (TRCN number also immunoprecipitated using equivalent amounts of a control IgG Ab 96139), Nab1 (TRCN number 96134), and Nab2 (TRCN number 96349) (ab46540; Abcam). Following immunoprecipitation, beads were washed, were obtained from the University of Colorado Cancer Center (University of and the immune complexes were eluted with kit elution buffer. Reverse Colorado-Anschutz Medical Campus, Aurora, CO). To make stable cell cross-linking buffer was added to each eluted supernatant at 1:1, and the lines, each construct was linearized, and 1 mg was used for transfection of 3 6 samples and input DNA were heated for 1 h at 95˚C. After treatment with 5 10 RAW 264.7 cells. Transfected cells were selected using 5 mg/ml 10 mg/ml proteinase K for 1 h at 37˚C, samples were purified using Qiagen puromycin. The two independent Nab1-KD and Nab2-KD cell lines used PCR purification kit and then used for qPCR. The promoter primer sequences for experiments were obtained from two separate transfections. used to analyze chromatin immunoprecipitations for pS5-RNA pol II, acet- ylated histone H3, total histone H3, and acetylated histone H4 were sense, 59- GCAATTGTGTCCCTCGCGCAGGAATGGGCC-39, and antisense, 59-GC- Results TCGTCAAAGCTCCACTCCCGACC-39. The primer sequences used to Type I IFNs decrease surface expression of IFNGR1 on mouse analyze all Egr and Nab immunoprecipitations were sense, 59-CCTCAGG- and human macrophages CTAGTCCACCCCTTCTCC-39, and antisense, 59-GGAGGCGTGTCTTG- GCGGG-39. Real-time quantitative PCR was performed using an Absolute Consistent with our previous studies (14), we observed that cell qPCR Sybr Green PCR ROX Mix (Thermo, Waltham, MA) and an ABI Prism surface IFNGR1 staining was reduced at 6 h after IFN-b treatment 7900 Sequence detector. The equation used for determining percent input is (100 U/ml) in BMDMs from wt C57BL/6, but not IFNAR2/2, 2^(input Ct 2 immunoprecipitation Ct). The equation used for determining fold en- richment over isotype = 2^[21 3 ([IP Ct 2 input Ct] 2 [isotype Ct 2 input Ct])]. mice (Fig. 1A). The intensity of IFNGR1 staining on cells from IFNGR12/2 mice was similar to that of the secondary reagent

Luciferase constructs alone, demonstrating specificity of the stain (Supplemental Fig. Downloaded from b The wild-type (wt) ifngr1 promoter luciferase reporter construct (IFNGR1pr- 1A). The ability of IFN- treatment to reduce IFNGR1 staining luc) was created by amplification of the proximal portion of the ifngr1 pro- was also observed with RAW264.7 macrophages (Fig. 1B). How- moter from 22320 to +1 using C57BL/6 genomic DNA as template. Primers ever, downregulation of IFNGR1 was not seen in mouse EL4 were as follows: sense, 59-GGGGTACCAGACTAAGCCAATCCTGCCCC- T cells (Fig. 1B), even though they responded to IFN-b as judged ACC-39, and antisense, 59-CAGTCTCCACAGGGAGCGCGTCCTGAGCT- by upregulation of MHC class I (Supplemental Fig. 1B) and CGG-39. KpnI and XhoI restriction sites were included in the primers and used to clone the amplified sequence into the multiple cloning site of pGL3-Basic phosphorylation of STAT1 (Supplemental Fig. 1C). Increasing the http://www.jimmunol.org/ (Promega). The hygromycin resistance gene from pGL4.15[Hygro] (Promega) concentration of IFN-b used for stimulation did not further reduce was subsequently cloned into the BamHI and SalI sites of pGL3 to generate IFNGR1 downregulation for either BMDMs or RAW 264.7 cells IFNGR1pr-luc. For mutagenesis, the Stratagene QuikChange II XL site- (Supplemental Fig. 1D). We also observed similar reductions in directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) was used. a The primer set for mutagenesis of the Egr site in mEgr-luc was sense, 59- IFNGR1 staining when BMDMs were treated with IFN- (Sup- CCAACTTAGTGAAACTTCTCAGTACACGCAGCG-39, and antisense, plemental Fig. 1E). To facilitate statistical comparisons of cell 59-GGTTGAATCACTTTGAAGAGTCATGTGCGTCGC-39. The two inde- surface IFNGR1 staining across multiple experiments and time pendent IFNGR1pr-luc and mEgr-luc cell lines used for experiments were points, the MFIs of staining on IFN-b–treated cells were nor- obtained from two separate transfections. malized to those of mock-treated control cells. When normalized Creation of putative binding maps data from three independent experiments were plotted and ana- by guest on September 28, 2021 lyzed, the reduction in cell surface IFNGR1 staining in both wt To construct a putative transcription factor binding site map of the mouse BMDM and RAW264.7 cells was found to be significant as early and human ifngr1 promoters, the DNA sequences for the first 2320 bp were entered into the TFSearch program (40). Results were limited to consensus as 2 h poststimulation (hps) with IFN-b (Fig. 1C, 1D). These data sequences with a score of 85.0 or better according to the program’s al- indicate that both primary mouse macrophages and RAW264.7 gorithm. The presence and location of these binding sites were confirmed cells respond to type I IFNs by rapidly down regulating cell sur- using TESS (41) and ENCODE (42). face IFNGR1, although the reduction in IFNGR1 staining seen with RAW264.7 cells (40–50%) was consistently less than that Reporter cell lines and luciferase assay seen with wt C57BL/6 BMDMs (55–65%). 6 To make stable cell lines, 5 3 10 RAW 264.7 cells were transfected with To determine whether human myeloid cells also downregulate 1 mg linearized IFNGR1pr-luc or mEgr-luc plasmid by electroporation IFNGR1 in response to type I IFNs, cell surface IFNGR1 staining (250 V, 950 mF) using a Bio-Rad electroporator. Following electroporation, the cells were resuspended in 10 ml DM10 and plated at 100 ml/well in was evaluated in human THP-1 macrophage–like cells and in pri- a 96-well plate. The transfected cells were selected for using 250 mg/ml mary PBMCs. Similar to mouse myeloid cells, IFNGR1 staining hygromycin. To determine the luciferase activity, expanded cell lines was selectively and significantly reduced in THP-1 cells and 3 5 2 2 were plated at 1 10 in a 24-well plate and stimulated with 100 U/ml CD3 CD14+, but not CD3+CD14 , PBMCs at 6 hps with 100 IFN-b at various time points. Following stimulation, lysates were har- vested using lysis buffer from the Promega Luciferase Assay System. U/ml human IFN-b (Fig. 1E, 1F; gating shown in Supplemental Luminescence was measured with a Synergy 2 plate reader (Bio-Tek, Fig. 1F). The similar effect of type I IFNs on IFNGR expression Winooski, VT). in mouse and human myeloid cells is consistent with the known ability of type I IFNs to suppress activation of both mouse and Western blots human macrophages (43, 44) and the ability of IFN-b treatment to BMDM or RAW cell cultured monolayers were lysed in 100 ml13 SDS- ameliorate mouse and human neuroinflammatory diseases (45). In PAGE buffer (0.0625 M Tris-Cl [pH 6.8], 2% SDS, 10% glycerol, 5% 2- addition, the lack of IFNGR1 downregulation in mouse EL4 T cells ME, and 0.01% bromophenol blue) containing HALT protease inhibitors (Fig. 1B, 1D), mouse splenic CD3+ T cells (14), and human CD3+ (Thermo) at 13 concentration. Cell lysates were scraped from plates and frozen at 220˚C. The lysate volume corresponded to 3.5 3 105 cells for all PBMCs (Fig. 1E, 1F) suggests that this conserved response to type I Egr family members and 2 3 105 cell equivalents/lane for all other pro- IFNs primarily occurs in myeloid cells. teins. Proteins were transferred to nitrocellulose and probed with Abs to total STAT1 (number 9172; Cell Signaling Technology), STAT1pY701 (num- Type I IFNs silence transcription of ifngr1 ber 9167; Cell Signaling Technology), Egr1 (number 4153; Cell Signaling The observed reductions in cell surface IFNGR1 staining in mouse Technology), Egr2 (PRB-236P; Covance), Egr3 (ab75461; Abcam), Nab1 (NBP1-71838; Novus), and Nab2 (sc-22815; Santa Cruz Biotechnology, macrophages correlated with similar magnitude reductions in the Santa Cruz, CA). As loading control, blots were stripped and reprobed abundance of ifngr1 transcripts as measured by quantitative real- with an anti-actin Ab (MAB1501; Millipore, Billerica, MA). time RT-PCR (qPCR) (Fig. 2A, Supplemental 2A). The effects of The Journal of Immunology 3387 Downloaded from

FIGURE 1. Type I IFNs reduce cell surface IFNGR1 on mouse and http://www.jimmunol.org/ human macrophages. Cells were treated or not with 100 U/ml IFN-b and then stained to quantify cell surface IFNGR1. Histograms (A, B, E) il- lustrate reduced cell surface IFNGR1 staining typically seen on macro- phages after 6 h of IFN-b treatment (black lines). Control histograms included unstimulated cells (gray shading) and cells stained with sec- ondary reagent alone (dashed line). Graphs (C, D, F) depict MFI values normalized to those of the respective untreated cells (relative MFI = MFI from treated sample/average of MFI from untreated sample). Cells used 2/2 A C included live-gated C57BL/6 and B6.IFNAR mouse BMDMs ( , ), by guest on September 28, 2021 mouse RAW 264.7 macrophages and EL4 thymoma cells (B, D), and THP-1 cells and gated CD14+ or CD3+ human PBMCs (E, F). Each bar in the graphs represents the mean 6 SD of the pooled values for each condition from a total of at least three independent experiments. Statistical signifi- cance was determined using an unpaired t test: *p # 0.05; ***p # 0.001. FIGURE 2. Type I IFNs repress transcription of ifngr1. (A) Total RNA was isolated from BMDMs treated or not with 100 U/ml IFN-b or 1 mg/ml IFN-b on ifngr1 transcript abundance were significant as early as actinomycin D. Relative change in normalized expression between treated 2 hps. A similarly rapid decrease in transcript abundance was seen and untreated BMDMs was quantified by qRT-PCR. Graph depicts ifngr1 upon chemical inhibition of the transcription machinery using transcript abundance values normalized to those of the respective untreated inhibitory concentrations of actinomycin D (Fig. 2A). The acti- cells (relative ifngr1 transcript abundance = transcript abundance from nomycin D concentration used (1 mg/ml) did not affect BMDM treated sample/average abundance from untreated sample). ChIP assays for B C viability but was sufficient to block de novo expression of IFN-b pS5-RNA pol II ( ) and H3Ac and H4Ac ( ) in BMDMs treated or not b ifngr1 in BMDM treated with poly I:C (Supplemental Fig 2B). On the with 100 U/ml IFN- . Primers that amplify 100 bp within exon 1 of were used to quantify promoter-associated immunoprecipitated chromatin basis of the changes in transcript abundance over time, we cal- (B, inset). Graphs depict fold enrichment over isotype values normalized to culated the half-life of ifngr1 mRNA in the BMDM to be 2.74 h those of the respective untreated cells (relative fold enrichment over iso- 6 6 ( 0.257 SD) following IFN-b treatment, and 2.70 h ( 0.169 type = fold enrichment from treated sample/average fold enrichment from SD) following actinomycin D treatment. These results suggest that untreated sample). Each bar in the graphs represents the mean 6 SD of the IFN-b treatment rapidly and fully silences de novo transcription of pooled values for each condition from a total of at least three independent ifngr1. experiments. Statistical significance was determined using an unpaired t As an independent method to confirm whether IFN-b treatment test: *p # 0.05, **p # 0.01, ***p # 0.001. Representative data showing silenced new ifngr1 transcription, we used ChIP to evaluate ac- shearing efficiency and percent input graphs for each of these ChIP assays cumulation of active RNA pol II to a 100-bp region adjacent to the are in Supplemental Fig. 3. predicted transcriptional initiation start site (TSS) of the ifngr1 gene (Fig. 2B, inset). Chromatin isolated from BMDMs 0, 2, 4, qPCR (Fig. 2B). The data showed that IFN-b treatment signifi- and 6 hps with IFN-b was immunoprecipitated with an Ab specific cantly reduced association of activated RNA pol II with this region for a phosphorylated isoform of the RNA pol II complex (pS5- of the ifngr1 promoter by 2 hps. Consistent with the conclusion RNA pol II). This Ab specifically recognizes RNA pol II phos- that IFN-b prevents new ifngr1 transcription, the association of phorylated at 5 within the C-terminal domain heptapeptide activated RNA pol II and the ifngr1 promoter remained low for at repeat, a modification that is necessary for the initiation of tran- least 6 hps. scription (46). The relative abundance of promoter DNA immu- The silencing of ifngr1 transcription suggested that type I IFN noprecipitated with anti–pS5-RNA pol II was determined using stimulation might alter the epigenetic structure of the ifngr1 3388 TRANSCRIPTIONAL REPRESSION BY TYPE I IFNs promoter. Epigenetic changes associated with transcriptionally in- active condensed chromatin include deacetylation of residues in histones H3 and H4 (47, 48). We used Abs specific for lysine N-acetylation of histones H3 (H3Ac) and H4 (H4Ac) to conduct ChIP assays on BMDMs stimulated with IFN-b for0,2,4,and6h. Type I IFN stimulation induced a transient increase in H3Ac signal at 2 hps, presumably reflecting rapid changes in the structure of the promoter. However, starting at 4 hps, the stimulation significantly reduced acetylation of histones H3 and H4 at the ifngr1 promoter (Fig.2C).Takentogether,thepS5-RNApolII,H3Ac,andH4Ac ChIP results indicate that stimulation of primary macrophages and cell lines with type I IFNs induces a rapid and sustained silencing of ifngr1 transcription. An Egr site is required for silencing of transcription from the proximal ifngr1 promoter To determine whether the proximal ifngr1 promoter was respon- sive to transcriptional repression by type I IFNs, we stably trans- FIGURE 3. An Egr site the proximal ifngr1 promoter confers sensitivity fected RAW 264.7 macrophages with a luciferase construct to transcriptional silencing by type I IFNs. (A) Schematic of proximal Downloaded from containing the 2320-bp region from the proximal mouse ifngr1 region of mouse ifngr1 promoter. Black letters below Egr site represent promoter (IFNGR1pr-luc). The type I IFN–responsive region of DNA binding consensus sequence, and gray letters specify three inserted the mouse promoter contained three putative Sp1 binding sites point mutations. Black arrows denote Egr3 and Nab1 primers used in ChIP adjacent to a TATA box and a putative Egr binding site near the assays. (B) wt (IFNGR1pr-luc) and mutated (mEgr-luc) ifngr1 promoter ifngr1 transcriptional start site (Fig. 3A). Putative binding sites for constructs were stably transfected to generate RAW 264.7 luciferase re- porter cells. Cells were stimulated or not with 100 U/ml IFN-b. Graph

Sp1 and Egr1 were also present in the proximal region of the http://www.jimmunol.org/ human ifngr1 promoter (Supplemental Fig. 4A). A previous study depicts luciferase activity values normalized to those of the respective implicated Sp1 as a positive regulator of ifngr1 transcription (49), untreated cells (relative luc activity = luc activity from treated sample/ average of luc activity from untreated sample). Values were derived from and others suggested that Egr family members repress Sp1-dependent three separate experiments using at least two independently transfected transcriptional activity (26–29). We thus engineered a mutated IFNGR1pr-luc or mEgr-luc cell lines. Each bar in the graph represents the version of IFNGR1pr-luc with three point mutations at the puta- mean 6 SD, and statistical significance was determined using an unpaired tive Egr binding site (Fig. 3A). The selected mutations were pre- t test: *p # 0.05,; ***p # 0.001. viously shown to disrupt the ability of DNA to bind Egr1 protein (50). The IFNGR1pr-luc reporter cells experienced a rapid and significant decrease in luciferase activity following treatment with Nab proteins are known to interact with DNA-bound Egr family by guest on September 28, 2021 IFN-b, demonstrating a decrease in ifngr1 promoter activity (Fig members as corepressors of target gene expression (32, 33). We 3B). In contrast, the reporter activity in RAW 264.7 cells stably thus asked whether Egr3 binding to the ifngr1 promoter might transfected with the mutated construct (mEgr-luc) was not affected recruit Nab proteins to mediate silencing of ifngr1. Both Nab1 and by treatment with IFN-b. These data suggested that the presence of Nab2 showed detectable levels of protein expression in unstimu- a functional Egr binding site is essential for the silencing of tran- lated BMDMs (Fig. 4A). Stimulation with IFN-b modestly re- scription from the proximal ifngr1 promoter. duced Nab2 expression at late times (6 hps) but did not alter Nab1 protein amounts. ChIP assays were thus used to evaluate whether Egr3 and Nab1 are recruited to the proximal Egr site of the Nab1 was recruited to the ifngr1 promoter. By 4 hps, Nab1 was ifngr1 promoter in response to type I IFN stimulation detected at a region of the ifngr1 promoter containing the Egr site Previous studies reported expression of Egr1, Egr2, and Egr3 (Fig. 4C, Supplemental Fig. 4C). This result established that IFN-b mRNAs in BMDMs (51). We thus used immunoblots to investigate treatment of BMDMs stimulates the recruitment of Nab1 subse- expression of these Egr proteins in our BMDMs, and whether quent to recruitment of Egr3 (Fig. 4B) and at a time when there is IFN-b treatment affected such expression (Fig. 4A). Egr1 protein deacetylation of the ifngr1 promoter (Fig. 2C). Recruitment of was present at low or undetectable levels in the unstimulated cells Egr3 and Nab1 to the ifngr1 promoter also occurred in RAW 264.7 but was strongly induced within 2 hps with IFN-b. Egr2 protein cells following stimulation with IFN-b for 4 h (Fig. 4D). However, was present in unstimulated cells and at late times poststimulation Egr3 was not recruited to the ifngr1 promoter in EL4 cells (Sup- but appeared to undergo a transient depletion at 2 hps. Egr3 was plemental Fig. 4D), suggesting that these events selectively occur readily detected in unstimulated cells, and its expression was in myeloid cells as they silence ifngr1 transcription. unaffected by type I IFN stimulation. We next used ChIP to ask whether Egr1, 2, or 3 associated with the proximal Egr site in the Nab1 knockdown prevents ifngr1 promoter deacetylation and ifngr1 promoter before or after type I IFN stimulation of BMDMs. downregulation of IFNGR1 in response to type I IFN An initial percent input analysis revealed that Egr3 was the pri- stimulation mary Egr family member recruited to the ifngr1 promoter fol- Repeated attempts to knockdown Egr3 expression in RAW264.7 lowing type I IFN stimulation (Supplemental Fig. 4B). Moreover, cells failed, suggesting this factor may be important for macro- the kinetics of Egr3 recruitment to the proximal Egr site (Fig. 4B) phage viability. However, we were successful in generating knock- correlated well with loss of activated RNA pol II recruitment to downs of Nab1 and Nab2. Independent, stably transfected cell the ifngr1 transcriptional start site (Fig. 2B) and reduced ifngr1 lines were developed using constructs encoding short hairpin transcript abundance (Fig. 2A). These events all preceded the RNAs that targeted the and nab2 genes. These lines re- reduced cell surface IFNGR1 staining in IFN-b–treated BMDMs spectively showed large decreases in Nab1 (Nab1-KD) and almost (Fig. 1C). complete elimination of Nab2 (Nab2-KD) protein expression when The Journal of Immunology 3389 Downloaded from http://www.jimmunol.org/

FIGURE 5. Knockdown of Nab1 prevents downregulation of IFNGR1 and deacetylation of ifngr1 promoter by type I IFNs. (A) Representative FIGURE 4. Egr3 and Nab1 are recruited to the proximal Egr site of the immunoblots to determine expression of Nab1 and Nab2 proteins in RAW ifngr1 promoter in response to type I IFN stimulation. (A) Representative 264.7 cell lines with or without stable knockdown of Nab1 (Nab1-KD) or Western blots of Egr1, Egr2, Egr3, Nab1, and Nab2 to determine protein Nab2 (Nab2-KD). Anti-actin Ab was used to confirm equivalent protein expression in BMDMs stimulated or not with 100 U/ml IFN-b. Anti-actin loading. Control RAW264.7 and Nab1-KD (B) or Nab2-KD (C) cell lines Ab was used to determine that equivalent protein concentrations were

were treated or not with 100 U/ml IFN-b then stained to quantify cell by guest on September 28, 2021 loaded in each lane. ChIP assay for Egr3 (B) and Nab1(C) in BMDMs or in surface IFNGR1. ChIP assays for Nab1 (D) and H3Ac in Nab1-KD or RAW 264.7 cells (D) stimulated or not with 100 U/ml IFN-b. Primers Nab2-KD (E) cell lines stimulated or not for 6 h with 100 U/ml IFN-b. denoted in Fig. 3A were used for all qPCR analysis. Graphs depict fold Primers denoted in Fig.3A were used for Nab1 ChIP qPCR analysis, and enrichment over isotype values normalized to those of the respective un- primers denoted in Fig. 2B were used for H3Ac qPCR analysis. Graphs treated cells, calculated as in Fig. 2B. Each bar in the graphs represents the depict MFI (B, C) or fold enrichment over isotype (D, E) values normalized mean 6 SD of pooled values from a total of at least three independent to those of the respective untreated cells, calculated as in Figs. 1C and 2B, experiments. Statistical significance was determined using an unpaired t respectively. Each bar in the graphs represents the mean 6 SD of the test: *p # 0.05, **p # 0.01, ***p # 0.001. pooled values for each condition from a total of at least three independent experiments. Statistical significance was determined using an unpaired t compared with control RAW 264.7 cells (Fig. 5A). In response to test: *p # 0.05, **p # 0.01, ***p # 0.001. treatment with IFN-b, cell surface IFNGR1 staining was barely reduced in the Nab1-KD cells (Fig. 5B), whereas IFNGR1 stain- for the histone deacetylation associated with silencing of the ifngr1 ing in the Nab2-KD cells was reduced to a similar extent as seen promoter in response to type I IFN stimulation (Fig. 6). in control RAW 264.7 cells (Fig. 5C). These data established that Nab1, but not Nab2, plays a role in the suppression of IFNGR1 Discussion expression. Although IFN-b has been widely used in treatment of multiple Nab proteins were previously shown to interact with complexes sclerosis and is known to impair resistance to certain bacterial that actively deacetylate histones to silence gene expression (33, infections, the mechanisms through which type I IFNs suppress 39). We thus asked whether reducing the recruitment of Nab1 to inflammatory responses remain poorly understood. Our findings the ifngr1 promoter could prevent the histone deacetylation as- here provide further insight into this mystery by showing how type sociated with silencing of ifngr1 transcription. ChIP for Nab1 and I IFNs downregulate expression of the IFNGR. Reduced IFNGR H3Ac was performed on Nab1-KD and Nab2-KD cell lines. In expression is known to correlate with reduced myeloid cell re- contrast to Nab2-KD cells, recruitment of Nab1 to the infgr1 sponsiveness to the proinflammatory cytokine IFN-g (14), and thus promoter was not detectable in the Nab1-KD cells after 6 h of may itself contribute to the anti-inflammatory effects of type I stimulation with IFNb (Fig. 5D). This finding indicated that the IFNs. In addition, the mechanism suggested by our findings in this knockdown efficiency in the Nab1-KD cells was adequate to paper may also contribute to the silencing of other genes involved prevent detectable Nab1 recruitment and that Nab2 knockdown in the proinflammatory responses of myeloid cells. did not prevent this recruitment. Furthermore, this lack of Nab1 Transcripts for ifngr1 and ifngr2 are constitutively present in recruitment was associated with a failure of IFNb stimulation to a variety of tissues, and functional IFNGRs are constitutively induce deacetylation of histone H3 in Nab1-KD, but not Nab2- present at the surface of diverse cell types (2, 52, 53). However, KD, macrophages (Fig. 5E). Thus, Nab1 recruitment is necessary variations in ifngr1 and ifngr2 expression occur in lymphoid and 3390 TRANSCRIPTIONAL REPRESSION BY TYPE I IFNs

with Sp1 binding (26–28), and/or assembly of the RNA pol II transcriptional complex (30, 31). Hence, we speculate that the binding of Egr3 may initially impair new transcription of ifngr1 by blocking Sp1 binding to the promoter. Consistent with this model, decreased binding of Sp1 to the ifngr1 promoter was associated with decreased transcription of ifngr1 in THP-1 cells infected with M. tuberculosis (59), and in breast cancer cells (57). Prolonged silencing of active promoters often involves recruit- ment and activity of HDAC enzymes by DNA-bound repressors (60). We found that binding of Egr3 to the ifngr1 promoter pre- ceded recruitment of Nab1 and loss of acetylated histones H3 and H4. Nab family members have been shown to repress gene tran- scription by interacting with HDACs (33, 39), and our data reveal that Nab1 knockdown largely prevents deacetylation of histone H3 at the ifngr1 promoter and IFNGR1 downregulation. Thus, we speculate that the prolonged and complete silencing of ifngr1 in myeloid cells involves the formation of an Egr3/Nab1 complex that fosters the recruitment of HDACs to deacetylate the ifngr1

promoter (Fig. 6). Downloaded from Microarray analysis has shown that numerous basally active genes are rapidly repressed by type I IFN in BMDMs (9). The affected genes include several that code for proteins known to be associated with an activated macrophage state, such as ICAM-1, IL-1b, Jagged-1, and IL-12p40. The promoters for these four genes,

and numerous other genes repressed by type I IFNs in myeloid cells, http://www.jimmunol.org/ FIGURE 6. Model for silencing of ifngr1 transcription by type I IFNs. contain Egr sites. Thus, the downregulation of these genes may Schematic of observed and hypothesized (indicated by ?) events that use a similar mechanism to that proposed here for ifngr1.Inad- mediate silencing of ifngr1 transcription by type I IFNs. Model represents dition, such downregulation could arguably have anti-inflammatory factors present on the ifngr1 promoter in unstimulated BMDMs (A) and effects. For example, the induction of Jagged-1 has recently been following stimulation with type I IFNs for 2 h (B) or 4–6 h (C). shown to be important for enhancing the responsiveness to IFN-g (61), suggesting that its downregulation by type I IFN may syn- ergize with the suppressive effects of IFNGR downregulation. myeloid cells. Early studies established that the expression of Likewise, reductions in IL-12p40 would be expected to reduce the IFNGR2 is selectively downregulated in Th1-type T cells (54, 55), induction of IFN-g (and IL-17) production by T cells. Interest- by guest on September 28, 2021 presumably to protect them from killing by the IFN-g they pro- ingly, the ifngr1 silencing in breast cancer cells (which is not duce. IFNGR1 is not downregulated in T cells, but we recently known to require type I IFNs) was previously shown to involve the reported that both IFNGR1 and IFNGR2 are lost from the cell transcription factor AP-2a (57), rather than Egr3. A search of the surface of myeloid and B cells responding to type I IFNs (14). The ImmGen database showed that AP-2a is not expressed in immune loss of cell surface IFNGR in myeloid cells, which was associated cells (62), and our data here indicated that IFN-b stimulation with reduced abundance of ifngr1 (but not ifngr2) transcripts, failed to stimulate recruitment of Egr3 to the ifngr1 promoter in correlated with reduced responsiveness to IFNg and increased EL4 T cells. Thus, Egr3 appears to selectively participate in the susceptibility to infection by the bacterium L. monocytogenes silencing of ifngr1 transcription that is triggered by type I IFNs in (14). This previous work indicated that type I IFN stimulation myeloid cells. The observation that there are distinct mechanisms reduces both cell surface IFNGR1 and total cellular IFNGR1 in to permit repression of ifngr1 or ifngr2 transcription in myeloid, myeloid cells exposed to type I IFN (14). Moreover, the loss of lymphoid, and other cell types underscores the biological impor- cell surface IFNGR1 occurred with a half-life of ∼4 h, matching tance of regulating responsiveness to IFN-g, and raises the possibility the previously measured half-life of IFNGR1 protein (56). Si- of therapies that selectively increase or reduce such responsiveness lencing of ifngr1 transcription and the consequent reduction in cell in specific cell types. surface IFNGR1 has also been observed in breast cancer cells, It remains unclear how stimulation with type I IFNs induces the which enables the tumor cells to evade killing by IFN-g (57). rapid recruitment of Egr3/Nab1 to the ifngr1 promoter and why These findings together argue that constitutive transcription of only these family members are recruited. As shown in this paper, ifngr1 is necessary to maintain cell surface expression of the we failed to observe significant induction of Egr3 or Nab1 ex- IFNGR, and that silencing of new ifngr1 transcription is sufficient pression in response to type I IFN stimulation. We have also failed to reduce sensitivity of myeloid and other nonlymphoid cell types to see increased nuclear localization of Egr3 in stimulated mac- to IFN-g. rophages (data not shown). We thus hypothesize that recruitment Regarding the mechanism of ifngr1 silencing in myeloid cells, of Egr3 is triggered by phosphorylation or other posttranslational our studies here showed that an Egr binding region in the proximal modification of Egr3 or an associated factor. Multiple studies have ifngr1 promoter is responsive to type I IFNs. The type I IFN–re- demonstrated posttranslational modifications that alter the activity sponsive Egr site in the proximal ifngr1 promoter is near sites for of Egr1 in response to exogenous stimuli (63–66). In addition, one binding of the . Sp1 promotes transcrip- study suggests that different modifications to the phosphorylation tional activation through direct interaction with factors important state of Egr1 alter whether this factor acts as an inducer or re- for assembly of the RNA pol II complex (58), and has been shown pressor of transcription at the same promoter (65). Phosphoryla- to promote basal transcription of human ifngr1 (49). It has also tion or other modifications of Egr1 and 2 might also conceivably previously been established that Egr family members can interfere prevent their recruitment to the proximal ifngr1 promoter. Further The Journal of Immunology 3391 investigation is needed to resolve these issues and determine more 20. Abdel-Latif, M. M., H. J. Windle, K. A. Fitzgerald, Y. S. Ang, D. N. Eidhin, M. Li-Weber, K. Sabra, and D. Kelleher. 2004. Helicobacter pylori activates the precisely how type I IFN stimulation regulates the association of early growth response 1 protein in gastric epithelial cells. Infect. Immun. 72: Egr3 with the ifngr1 promoter. 3549–3560. Polymorphisms in the ifngr1 promoter are known to correlate 21. Cullen, E. M., J. C. Brazil, and C. M. O’Connor. 2010. Mature human neu- trophils constitutively express the transcription factor EGR-1. Mol. Immunol. 47: with susceptibility to diverse diseases such as Leishmaniasis, tu- 1701–1709. berculosis, leprosy, and hepatitis infections (67–70). Suppression 22. Droin, N. M., M. J. Pinkoski, E. Dejardin, and D. R. Green. 2003. Egr family of ifngr1 transcription by type I IFNs also correlates with impaired members regulate nonlymphoid expression of Fas ligand, TRAIL, and tumor necrosis factor during immune responses. Mol. Cell. 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