Responses ATF3 Is a Key Regulator of Macrophage

ATF3 Is a Key Regulator of Macrophage IFN Responses Larisa I. Labzin, Susanne V. Schmidt, Seth L. Masters, Marc Beyer, Wolfgang Krebs, Kathrin Klee, Rainer Stahl, Dieter This information is current as Lütjohann, Joachim L. Schultze, Eicke Latz and Dominic De of October 1, 2021. Nardo J Immunol 2015; 195:4446-4455; Prepublished online 28 September 2015; doi: 10.4049/jimmunol.1500204 http://www.jimmunol.org/content/195/9/4446 Downloaded from

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Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 64 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/195/9/4446.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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ATF3 Is a Key Regulator of Macrophage IFN Responses

Larisa I. Labzin,*,1 Susanne V. Schmidt,† Seth L. Masters,‡,x Marc Beyer,† Wolfgang Krebs,† Kathrin Klee,† Rainer Stahl,* Dieter Lutjohann,€ { Joachim L. Schultze,† Eicke Latz,*,‖,#,1 and Dominic De Nardo*,‡,x,1

Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-b expression, as well as the magnitude of IFN-b cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-b via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Downloaded from Additionally, we observed that ATF3 itself is a type I IFN–inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFNb, as well as the expression of genes downstream of IFN signaling. The Journal of Immunology, 2015, 195: 4446–4455. http://www.jimmunol.org/

ecognition of danger signals derived from bacteria and melanoma differentiation-associated protein 5, stimulator of IFN viruses by innate immune receptors leads to activation genes (STING), and DNA-dependent activators of IFNs, leads to R of specific transcriptional programs, culminating in the type I IFN induction (2). Production of type I IFNs is particularly production of potent inflammatory mediators, including proin- important for mounting a rapid antiviral response, including re- flammatory cytokines, chemokines, and type I IFNs (1). The ac- stricting viral replication and modulating adaptive immunity (3, 4). tivation of numerous innate immune receptors, including several Type I IFNs signal via the IFN-a/b receptor (IFNAR), which TLRs (TLR3, 4, 7, 8, and 9), retinoic acid–inducible gene I, induces the expression of IFN-stimulated genes (ISGs), encod- by guest on October 1, 2021 ing proteins with diverse antiviral functions (5, 6). Genome-wide transcriptional analysis of IFN-treated cells has revealed the ex- *Institute of Innate Immunity, University Hospital, University of Bonn, 53127 Bonn, Germany; †Life and Medical Sciences Institute, University of Bonn, 53115 Bonn, istence of hundreds of ISGs, of which only a few have been Germany; ‡Inflammation Division, Walter and Eliza Hall Institute of Medical Re- characterized to date (7–10). The production of type I IFNs by search, Parkville, Victoria 3052, Australia; xDepartment of Medical Biology, Uni- { pattern recognition receptors (PRRs) and the subsequent expres- versity of Melbourne, Parkville, Victoria 3010, Australia; Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, sion of ISGs are therefore critical for host protection, and, not ‖ 53127 Bonn, Germany; Department of Infectious Diseases and Immunology, Uni- surprisingly, mice and humans with defects in IFN responses are versity of Massachusetts Medical School, Worcester, MA 01605; and #German Cen- ter for Neurodegenerative Diseases, 53175 Bonn, Germany more susceptible to viral infections (11–13). 1 Inappropriate activation of PRRs can lead to excessive pro- L.I.L., E.L, and D.D.N. contributed equally to this work. duction of inflammatory mediators, which can damage local tis- Received for publication January 28, 2015. Accepted for publication August 24, 2015. sues and lead to systemic disease pathologies. High levels of type I This work was supported by a grant from the BONFOR Research Commission, IFN production are implicated in several inflammatory conditions University of Bonn (to D.D.N.), German Research Foundation Grants SFB645 and autoimmune diseases (e.g., systemic lupus erythematosus), in (toE.L.)andSFB670andSFB704(toJ.L.S.),the European Research Council InflammAct which patient leukocytes display an IFN signature of increased (to E.L.), and the Excellence Cluster ImmunoSensation (to E.L. and J.L.S.). ISG expression (14). Type I IFNs have also been shown to be Address correspondence and reprint requests to Prof. Eicke Latz or Dr. Dominic De Nardo, Institute of Innate Immunity, University Hospital, University of Bonn, Sig- immunosuppressive in various chronic viral and bacterial infec- mund Freud Strasse 25, 53175 Bonn, Germany (E.L.) or Inflammation Division, tions, mediated in part by their induction of IL-10 (3). Therefore, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, the magnitude and duration of signals emanating from immune VIC 3052, Australia (D.D.N.). E-mail addresses: [email protected] (E.L.) or [email protected] (D.D.N.) receptors must be tightly controlled at multiple steps, including The online version of this article contains supplemental material. at the receptor, signaling, transcriptional, posttranscriptional, and posttranslational levels (15). Activating transcription factor 3 Abbreviations used in this article: ATF3, activating transcription factor 3; BMDM, bone marrow–derived macrophage; CMA, 10-carboxymethyl-9-acridanone; DC, den- (ATF3) is a transcriptional modulator that can repress target genes dritic cell; HA, hemagglutinin; 25-HC, 25-hydroxycholesterol; iBMDM, immortal- by directly binding specific nucleotide motifs within promoter loci ized BMDM; IFNAR, IFN-a/b receptor; IRF, IFN regulatory factor; ISG, IFN- stimulated gene; LCMV, lymphocytic choriomeningitis virus; pppRNA, 59-triphos- (16). ATF3 is induced during TLR-dependent immune responses phate RNA; PRR, pattern recognition receptor; qPCR, quantitative real-time PCR; and negatively regulates numerous proinflammatory cytokines STING, stimulator of IFN genes; TSS, transcription start site; VSV, vesicular stoma- (e.g., IL-6, IL-12p40) and chemokines (e.g., MIP-1b), and, as such, titis virus; WT, wild-type. mice deficient in ATF3 are more susceptible to endotoxin shock Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 owing to excessive cytokine production (16–19). ATF3 can also www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500204 The Journal of Immunology 4447 modulate the expression of IFN-g in NK cells during the antiviral performed on cDNA using the Maxima SYBR Green/ROX qPCR Master response to murine CMV infection (20), and it has recently been Mix (Fermentas) on a 7900T thermocycler (Applied Biosystems). The shown to directly regulate TLR-induced IFN-b expression follow- mouse and human primer sequences used can be supplied upon request. Human ATF3 was examined using predeveloped probe/primer combina- ing its induction by an inhibitor of the class III lipid kinase, PIKfyve tions (ATF3; Hs00910173_m1 and HPRT1; Hs01003267_m1; Applied Bio- (21). Whereas the ability of ATF3 to repress proinflammatory cy- systems) with the TaqMan qPCR assay system (Applied Biosystems). tokine expression is firmly established, its role in regulating type I Expression of target genes was normalized to respective housekeeping IFN responses requires further investigation. genes. In this study, we show that in the absence of Atf3, primary ELISA mouse macrophages display significantly greater basal and PRR- b inducible IFN-b expression. We identify a new important en- Levels of IFN- in culture supernatants were measured using a custom- made ELISA protocol as described elsewhere (25). hancer site distal to the Ifnb1 (the gene encoding IFN-b) promoter and demonstrate that ATF3 binding to this regulatory region Cell lysis and immunoblotting appears to control the magnitude of IFN-b promoter activity. We Cells were lysed on ice with 13 RIPA buffer (20 mM Tris-HCl [pH 7.4], also report that type I IFN induces ATF3 in both human and 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, mouse immune cells and that a subset of inducible genes down- and 0.5% deoxycholate) supplemented with 0.1 mM PMSF, cOmplete stream of IFNAR signaling is further modulated in an ATF3- protease inhibitors, and PhosSTOP (Roche). Lysates were clarified by centrifugation at 13,000 3 g for 10 min at 4˚C before protein concentra- dependent manner. Finally, we show that ATF3 modulates the tion was measured by BCA assay (Pierce). Protein expression was mea- antiviral response by impacting viral replication. Our findings sured by immunoblotting as previously described (26). further demonstrate the role of ATF3 in controlling the IFN response of macrophages, and help to establish ATF3 as a key innate Luciferase assay Downloaded from immune regulator. The coding sequence of murine Atf3 was amplified (forward, 59- CTGTCTCGAGACCATGATGCTTCAACATCCA-39, reverse, 59-CTGT- CCCGGGTTAGCTCTGCAATGTTCC-39) and cloned into the pIRES2Ac- Materials and Methods GFP1 (Invitrogen) expression vector via XmaI and XhoI restriction sites Reagents with an additional insertion of a Kozak sequence in front of the start codon. The murine Ifnb1 promoter was amplified (forward, 59-CTGTCTCGAG-

Reagents included: ultrapure LPS (Escherichia coli 0111:B4), poly(I:C), http://www.jimmunol.org/ TTTCTCTTATAGTACACT-39,reverse,59-CTGTAGATCTGAGCTGCTTA- and R848 (Invivogen); human and murine IFN-a and IFN-b (R&D Sys- TAGTTGAT-39) and inserted into the promoterless pGL4.14 vector tems); human and murine IFN-g (ImmunoTools and PeproTech); anti- (Promega) directly in front of the firefly luciferase cassette (Luc2) using the ATF3 (C19; sc-188, Santa Cruz Biotechnology); anti–p-IFN regulatory XhoI and BglII restriction sites. Additionally, the potential enhancer region factor (IRF)3 (Ser396) (4D4G; 4947, Cell Signaling Technology); anti–b- and ATF3 binding site BS2 (mm9, chr4: 88182925-88183823) was amplified actin (926-42210) and secondary Abs (LI-COR Biosciences); anti-IFNAR1 (forward, 59-CTGTGGTACCTCTTACAAGGAAGAGGACGAGAGAAC- Ab (BioLegend); purified mouse IgG1 (Life Technologies); actinomycin 39,reverse,59-CTGTCTCGAGTCATTTCTGGGTATTCTG-39) and inserted D, BSA, anti-hemagglutinin (HA; H6908), and 10-carboxymethyl-9- in front of the Ifnb1 promoter via KpnI and XhoI. In parallel, a mutated acridanone (CMA; also known as 9-oxo-10(9H)-acridineacetic acid) BS2 was designed and synthetized (Integrated DNA Technologies). Insertion (Sigma-Aldrich); and 59-triphosphate RNA (pppRNA), which was syn- of the mutated BS2 was enabled via In-Fusion HD reaction (Clontech) thesized as described previously (22).

according to the manufacturer’s protocol. Resequencing of the pGL4.14 by guest on October 1, 2021 Cell culture reporter construct confirmed the directed insertion of the DNA fragment. HEK293T cells (InvivoGen) were plated at 25 3 105 cells per well in Primary bone marrow–derived macrophages (BMDMs) were obtained by a 96-well plate. Cells were transfected the following day using GeneJuice culturing bone marrow of 6- to 8-wk-old wild-type (WT) or Atf32/2 (Novagen) in OptiMEM with 50 ng IFN-b promoter firefly luciferase con- C57BL/6 mice in DMEM supplemented with 10% FBS, 10 mg/ml struct, 50 ng either ATF3 or an empty vector control plasmid (pIRES2Ac- ciprofloxacin, and 40 ng/ml recombinant human M-CSF (R&D Sys- GFP1), and finally an internal Renilla luciferase control vector pGL4.74 tems) for 6 d. Human PBMCs were purified from buffy coats over (Promega). The following day cells were stimulated with Lipofectamine a Ficoll density gradient (GE Healthcare). Human monocytes or plas- (Invitrogen)-transfected pppRNA (also known as IVT4). Cells were lysed macytoid dendritic cells (DCs) were further isolated using specific 16 h after stimulation in passive lysis buffer (Promega) and firefly luciferase negative selection kits (Miltenyi Biotec). Cell purity was .75% on av- was measured using luciferin (Promega), whereas Renilla luciferase was erage for isolated cell populations as determined by flow cytometry: measured using coelentrazine (Promega). The firefly signal was then nor- CD14+ monocytes (61D3; eBioscience) and CD123+ and CD303/BDCA- malized to the Renilla signal. 2+ plamacytoid DCs (AC145 and AC144; Miltenyi Biotec). Human cells were maintained in RPMI 1640 with 10% FBS and 10 mg/ml cipro- Microarray 2/2 2/2 floxacin. WT, Atf3 ,andIfnar1 immortalized BMDM (iBMDM) Cells were lysed in TRIzol reagent (Invitrogen) and total RNA isolation, cell lines were generated as previously described (23) and, along with quality control, and purification were performed as described (27). Gen- HEK293T cells, were maintained in DMEM with 10% FBS and 10 mg/ml eration of biotin-labeled cRNA was performed using the TargetAmp ciprofloxacin. Nano-g biotin-cRNA labeling kit for the Illumina system (Epicentre). cRNA (1.5 mg) was hybridized to MouseWG-6 v2.0 BeadChips (Illumina) and Generation of mouse macrophages expressing ATF3 scanned on an Illumina iScan device. A retroviral plasmid expressing a C-terminal HA-tagged version of ATF3 was generated by amplifying ATF3 (pCMV6-mATF3; NM_007498, MC201919, Data generation and bioinformatics analysis OriGene Tech) using specific primers (forward, 59-TTTTTGGCGCGCCT- Raw intensities of expression data were imported into BeadStudio 3.1.1.0 ATGATGCTTCAACATCCAGGCCAG-39, reverse 1, 59-GGGGACATCG- (Illumina) and exported as log2-transformed expression values into Partek TATGGGTAGCTCTGCAATGTTCCTTCTTTTATCTG-39, and reverse 2, Genomics Suite v6.6 (Partek). Batch effects were removed from normal- 59-AAAAAAGCGGCCGCTTCACGCGTAGTCGGGGACATCGTATGGG- ized data. Differentially expressed genes with a fold change $ 2(p # 0.05) TAGC-39) before being cloned into pRP_CMV-HA-IRES-mCherry with were included for further analysis. Identification of ISGs was performed 2/2 AscI and NotI. WT and Atf3 immortalized macrophages were subjected by screening differentially expressed genes against the Interferome data- to retroviral transduction with the retroviral plasmid above using a defined base v2.0 (28). protocol (24). Additionally, the empty pRP_CMV-HA-IRES-mCherry pa- rental vector was used to generate control cell lines. ATF3 binding analysis Quantitative real-time PCR Binding of ATF3 to promoters was predicted using Genomatix v3.2 (Supplemental Table III). For identification of ATF3 binding motifs in the RNA was isolated according to the manufacturers’ protocol (Qiagen) Ifnb1 and cis-regulatory regions, Vector NTI v10.3.0 was used with de- and synthesized into cDNA. Quantitative real-time PCR (qPCR) was generation settings (V$ATF.01) of 62.5%. 4448 REGULATION OF IFN RESPONSES BY ATF3

Chromatin immunoprecipitation sequencing analysis detected significantly higher basal expression of Ifnb1, the gene SRA files of used chromatin immunoprecipitation sequencing (ChIP-seq) encoding IFN-b (Fig. 1E). These findings led us to hypothesize data were converted to FASTQ files using fastq-dump 2.2.0 and subse- that in addition to regulating proinflammatory cytokines, ATF3 quently aligned with Bowtie against the mm9 reference genome using the may also be a negative regulator of type I IFN responses in best match options. Respective bedGraph files were either downloaded macrophages. from Gene Expression Omnibus or SAM files were converted by HOMER Production of type I IFNs from immune cells constitutes an into tag directories to perform peak calling and conversion into bedGraph files. Visualization of normalized tag counts for transcription factor and important part of host defense against infection and occurs fol- histone modification ChIP-seq data at the murine Ifnb1 locus and the 13 lowing the activation of several PRRs. To examine the role of genes encoding IFN-a were performed with the Integrative Genomics ATF3 in the context of PRR-driven IFN-b production, we com- Viewer (v2.3.34). pared responses between WT and Atf32/2 BMDMs following Quantification of secreted oxysterols activation of TLR4 (LPS), TLR3 [poly(I:C)], or STING (CMA). We saw significantly elevated Ifnb1 mRNA expression in Atf32/2 Cell supernatants were collected and subjected to gas chromatography– mass spectrometry–selected ion monitoring to determine levels of 25- and BMDMs in response to PRR activation (Fig. 2A–C), which was 27-HC. also observed in response to LPS over time (Fig. 2D). The increase in mRNA correlated with greater IFN-b protein release Lymphocytic choriomeningitis virus infection of BMDMs (Fig. 2E–H). Interestingly, whereas the kinetics of IFN-b mRNA 2 2 WT or Atf32/2 primary BMDMs were infected with lymphocytic chorio- induction remained unchanged between WT and Atf3 / meningitis virus (LCMV) clone 13 as indicated. For intracellular detection BMDMs (Fig. 2D), at the protein level IFN-b production was of LCMV nucleoprotein 24 h postinfection, cells were first stained with notably delayed in WT cells (Fig. 2H). Consistent with these a fixable viability dye (Life Technologies), treated with fixation and per- Downloaded from meabilization solutions (BD Biosciences), and then stained with an Ab observations, LPS-induced mRNA expression of the ISGs Irf7 2/2 specific to LCMV nucleoprotein (clone VL4). and Isg15 was greater in Atf3 BMDMs (Fig. 2I). However, no difference in Irf7 and Isg15 mRNA was observed between Vesicular stomatitis virus replicon luciferase assays 2 2 WT and Atf3 / BMDMs when directly stimulated with IFN-b 2 2 Control or ATF3-expressing Atf3 / iBMDM cells were infected with (Fig. 2J). This finding suggested that elevated production of IFN- vesicular stomatitis virus (VSV)*DG(Luc) replicon virus particles (29) as b by TLR4 activation (Fig. 2G, 2H) is likely to account for the indicated. Cells were lysed in passive cell lysis buffer (Promega) and firefly 2/2 http://www.jimmunol.org/ luminescence measured using a SpectraMax reader (Molecular Devices). increased expression of these ISGs in Atf3 cells. Consistent with this notion, elevated levels of Irf7 and Isg15 mRNA in Data deposition Atf32/2 BMDMscomparedwithWTBMDMswerelostinthe Microarray data are accessible at Gene Expression Omnibus under presence of an IFNAR blocking Ab (Fig. 2K). Taken together, GSE61055 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61055) these results indicate that ATF3 is important for regulating pro- and GSE44034 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44034). duction of IFN-b levels downstream of innate immune recep- ChIP-seq data are accessible under GSE36104 (http://www.ncbi.nlm.nih. tors and are in line with recent findings from Cai et al. (21) who gov/geo/query/acc.cgi?acc=GSE36104) (30), GSE55317 (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE55317) (31), GSE38379 (http:// observed that induction of ATF3 by a PIKfyve inhibitor sup- www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38379) (32), and pressed subsequent TLR-induced type I IFN production in by guest on October 1, 2021 GSE63339 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63339) RAW264.7 cells. (33). We next performed studies to assess the effects of ATF3 over- Statistical analysis expression on inducible IFN-b levels in macrophages. We generated Atf32/2 and WT iBMDMs overexpressing HA-tagged Data are typically presented as mean 6 SEM, where a p value #0.05 was considered significant as determined by an unpaired (mouse experiments) murine ATF3 or an empty vector control (Fig. 3A–C) and tested or paired (human experiments) two-tailed Student t tests, unless otherwise TLR4- or STING-induced IFN-b production in these cells. In described in individual figure legends. Analyses were performed with contrast to ATF3 deficiency, ATF3 overexpression resulted in ei- Prism (GraphPad Software), and for microarray data with Partek Genomics ther significant decreases or a trend toward reduced LPS- and Suite using ANOVA models. CMA-induced Ifnb1 in both Atf32/2 and WT iBMDMs (Fig. 3D, 3E). Consistent with the ability of ATF3 to limit IL-12p40 pro- Results duction in macrophages (16), we also observed a significant re- b ATF3 regulates IFN- production from mouse macrophages duction in LPS-induced Il12b (the gene encoding IL-12p40) and ATF3 is an inducible transcriptional repressor in innate immune a trend toward a decrease in CMA-mediated Il12b expression in cells that regulates the magnitude and duration of inducible cells overexpressing ATF3 (Fig. 3F). The findings that ATF3- proinflammatory gene expression. We performed transcriptome deficient macrophages displayed a basal IFN signature and in- analysis of resting WT and Atf3-deficient (Atf32/2)BMDMsto creased PRR-inducible IFN-b, whereas ATF3 overexpression led identify other potential pathways regulated by ATF3. As presented to reduced IFN-b induction, suggest that ATF3 is a key regulator in Fig. 1, this approach revealed a strong IFN signature of spon- of macrophage IFN-b production and downstream IFNAR re- taneous ISG expression in Atf32/2 BMDMs. Indeed, of the 50 sponses. genes most highly expressed in Atf32/2 BMDMs compared with b their WT counterparts, most appeared to be ISGs, including Ifit2, ATF3 directly regulates transcription of IFN- Irf7, Isg15, Ch25h, and Usp18 (Fig. 1A). We next examined the We next investigated how ATF3 modulates IFN-b production in genes that were expressed 2-fold greater in Atf32/2 BMDMs than response to activation of distinct PRRs. A common feature lead- WT cells (157 genes) for known ISGs (Fig. 1B) (28), revealing ingtoIFN-b induction downstream of numerous PRRs is the acti- 90 ISGs (57%), 33 of which were specific to type I IFN, 10 vation of the transcription factors IRF3 and IRF7 (34, 35). In specific to type II IFN, and 47 common to both type I and II IFNs plasmacytoid DCs, IRF7 is constitutively expressed and is the (Fig. 1C). We validated this general observation by qPCR, find- predominant IRF involved in Ifnb1 transcription, whereas in ing significant increases in the basal expression of several well- macrophages its expression is induced upon IFN stimulation. In characterized ISGs (Irf7, Isg15, Ch25h, and Usp18)inAtf32/2 contrast, IRF3 is constitutively expressed in macrophages and its BMDMs compared with WT cells (Fig. 1D). Of note, we also activation leads to the production of type I IFNs following PRR The Journal of Immunology 4449 Downloaded from http://www.jimmunol.org/

FIGURE 1. Spontaneous expression of ISGs in ATF3-deficient macrophages. (A–C) WT and Atf32/2 BMDMs analyzed by microarray; fold changes and 2/2 log2 expression values of the top 50 genes that were expressed more in Atf3 than in WT BMDM (A), schematic of microarray analysis and identification of ISGs (B), and an overview of ISGs expressed $2-fold in Atf32/2 BMDMs compared with WT cells (C). (D and E) WT and Atf32/2 BMDMs were analyzed for basal ISG (Irf7, Ch25h, Isg15, and Usp18)orIfnb1 mRNA expression by qPCR. Graphs show mean 6 SEM combined from four independent , experiments. *p 0.05. by guest on October 1, 2021 stimulation. Hence, we investigated whether ATF3 affected IRF3 revealing 11 ATF3 binding motifs at site 1 and 25 at site 2, sug- in mouse macrophages. However, we saw no obvious difference gesting that ATF3 binds at site 2 (Supplemental Fig. 1B, 1C, in basal IRF3 phosphorylation between WT and Atf32/2 BMDMs, Supplemental Table I). As a potential cis-regulatory element, or following LPS or poly(I:C) stimulation (Fig. 4A, 4B). There binding site 2 could be important for Ifnb1 gene expression. was also no difference in the amount of total IRF3 between WT Pivotal for the binding of transcription factors to defined en- and Atf32/2 BMDMs (Fig. 4A, 4B). This finding suggests that hancers is an open chromatin structure, which is achieved by ATF3, in keeping with its characterized role as a transcriptional posttranslational modifications (e.g., methylation and acetylation) modulator, may act downstream of IRF3 activation. We also found of histone proteins and the binding of pioneer transcription fac- that ATF3 deficiency did not affect LPS-induced Ifnb1, Ch25h,or tors, for example, PU.1 (36–39). Indeed, so-called poised Tnf mRNA stability, as assessed by mRNA degradation following enhancers, which are required for inducible gene expression, are actinomycin D treatment (Supplemental Fig. 1A). Because ATF3 defined by PU.1 binding and monomethylation of the histone H3 is known to bind DNA, we hypothesized that ATF3 may regulate at lysine 4 (H3K4me1) and acetlyation of histone H3 at lysine 27 IFN-b at the transcriptional level. We therefore investigated (H3K27ac) (32). We used available ChIP-seq data from BMDMs whether ATF3 can directly bind to the Ifnb1 promoter in resting stimulated for 4 and 24 h with LPS (32) to assess the enhancer BMDMs. First, we examined two independent ATF3 ChIP-seq classification of our novel ATF3 binding site 2 (Fig. 4D). Whereas datasets, including one we generated using WT and Atf32/2 untreated and activated macrophages showed similar H3K4me1 BMDMs (26, 30), for enrichment of ATF3 at sites proximal to marks at the designated binding sites 1 and 2, basal levels of PU.1 the Ifnb1 transcription start site (TSS). In both datasets a peak was and H3K27ac marks increased with prolonged LPS signaling at called distal to the TSS of Ifnb1 (from 214,200 to 215,175 bp; both sites. This was expected for site 1, which contains the well- termed site 2), whereas a site closer to the TSS (from +13 to 2467 characterized IFN enhanceosome (40) (e.g., IRF, NF-kB, and bp; termed site 1) was called only in the dataset not controlled by AP-1 binding sites); however, these data show that binding site 2 ChIP-seq analysis of Atf32/2 cells (Fig. 4C). Indeed, in our dataset is also a poised enhancer (Fig. 4D). This observation was further this region only showed a peak in Atf32/2 samples. Of note, exam- corroborated by detailed analysis of ChIP-seq data for the two ination of our ChIP-seq dataset identified no significant peaks in histone marks, H3K4me1 and H3K27ac, in resident tissue mac- the promoter loci (up to 220 kb) of the 13 genes encoding IFN-a rophages from five different tissues (Supplemental Fig. 1D) (33). (data not shown). As a second approach to assess the likelihood Both H3K4me1 and H3K27ac marks were found in all tissue of ATF3 binding to the Ifnb1 promoter and the distal binding site macrophages at binding site 2, supporting the hypothesis that site 2, we performed bioinformatic analysis of both identified sites, 2 is epigenetically accessible for transcription factors, and that 4450 REGULATION OF IFN RESPONSES BY ATF3

FIGURE 2. ATF3 reduces PRR-inducible IFN-b expression in mouse macrophages. (A–C and E–G) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml), poly(I:C) (1 mg/ml), or CMA (500 ng/ml) for 4 h and Ifnb1 mRNA was measured by qPCR (A–C) or IFN-b protein by ELISA (E–G). (D, H, and I) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml) as indicated before Ifnb1, Irf7, and Isg15 mRNA expression was analyzed by qPCR (D and I) and IFN-b protein by ELISA (H). (J) WT and Atf32/2 BMDMs were stimulated with IFN-b (1000 U/ml) as indicated and Irf7 and Isg15 mRNA were analyzed by qPCR. (K) WT and Atf32/2 BMDMs were treated with 1 mg/ml IFNAR blocking Ab or mouse IgG1 control Ab for 1 h before cells were stimulated for Downloaded from 6 h with 10 ng/ml LPS or 10 U/ml IFN-b and Irf7 and Isg15 mRNA were analyzed by qPCR. Graphs show mean 6 SEM combined from at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/

ATF3 may therefore act by inhibiting transcriptional activator to treatment with the different IFNs was demonstrated by exam- binding at this site. We further characterized these two poten- ining mRNA expression of the ISG Irf7 (Supplemental Fig. 1E). by guest on October 1, 2021 tial ATF3 binding sites by performing luciferase assays in Notably, ATF3 protein expression was induced to comparable HEK293T cells. To this end, we generated three murine Ifnb1 levels in BMDMs across several doses of IFN-b (Fig. 5C). The promoter constructs containing site 1 alone, sites 1 and 2, or site kinetics of Atf3 mRNA induction by IFN-b appeared delayed, 1 and a mutated version of site 2. Whereas all promoter cons- with significant increases first observed after 6 h (Fig. 5D). Ad- tructs were activated upon stimulation with the retinoic acid– ditionally, a similar, albeit more rapid, ATF3 induction profile inducible gene I agonist, pppRNA (also known as IVT4), inter- was observed in iBMDMs treated with IFN-b (Supplemental estingly the promoter construct containing both the WT site 1 Fig. 1F). Increases in expression of the ISG Irf7 demonstrated that and site 2 was activated significantly more than was the promoter these cells responded normally to IFN-b (Supplemental Fig. 1G). construct containing site 1 alone (Fig. 4E). Of note, cotrans- Whereas ATF3 induction in response to IFN-b was fully depen- fection of ATF3 significantly reduced activity of the Ifnb1 dent on Ifnar1, LPS-mediated ATF3 induction was only partially promoter construct containing WT sites 1 and 2, compared with IFNAR-dependent, particularly at later LPS time points (Fig. 5E). cotransfection of a control plasmid (Fig. 4F). However, cotrans- To date, the immune function of ATF3 has been primarily char- fection of ATF3 with the Ifnb1 promoter constructs containing acterized in mouse cells, where only one isoform is expressed. either WT site 1 alone or site 1 and a mutated site 2 showed no Humans, however, express six different isoforms of ATF3 (43), difference (Fig. 4F). We conclude that site 2 represents an im- some of which are thought to act as transcriptional activators portant regulatory element for controlling Ifnb1 expression and rather than repressors due to truncations in the C-terminal DNA- that ATF3 binds to this regulatory site to limit transcription and binding leucine zipper domain. We therefore investigated whether subsequent IFN-b production. the full-length repressive isoform of ATF3 was induced by IFNs in human monocytes using specific qPCR primers for detection. We Type I IFNs induce ATF3 expression in mouse and human found that low-dose type I IFNs significantly induced expression immune cells of full-length ATF3, whereas IFN-g treatment resulted in a more ATF3 is an early response gene, induced following activation of modest increase (Fig. 5F). CXCL10 was strongly induced by innate immune cells by a range of stimuli (41). As PEGylated all IFNs, demonstrating responsiveness to IFN treatment IFN-a was shown to induce ATF3 in human PBMCs (42), we (Supplemental Fig. 1H). Furthermore, we found that in addi- hypothesized that ATF3 may also be induced by IFN-b to act in tion to monocytes, IFN-b induced ATF3 and CXCL10 in both hu- a negative feedback loop. Hence, we examined ATF3 expression man plasmacytoid DCs and PBMCs (Fig. 5G–I, Supplemental Fig. upon type I (IFN-a and IFN-b) and type II (IFN-g) IFN treatment 1I–K). IFN-b induction of full-length ATF3 protein in human of BMDMs. We observed that type I IFNs induced ATF3 gene and monocytes from four individual donors was further confirmed using protein expression, whereas little or no induction was seen in an Ab that recognizes a sequence close to the C terminus (Fig. 5J). response to IFN-g (Fig. 5A, 5B). The responsiveness of BMDMs We also observed reduced expression of a smaller reactive protein The Journal of Immunology 4451 Downloaded from

FIGURE 3. Overexpression of ATF3 reduces PRR-mediated IFN-b 2/2 expression in mouse macrophages. (A–C) Atf3 or WT iBMDMs were http://www.jimmunol.org/ reconstituted with an empty vector control (ctrl) or with an HA-tagged version of murine ATF3. Atf3 mRNA expression was analyzed by qPCR (A and B) and ATF3, HA, and b-actin protein were analyzed by immunoblot; star indicates a nonspecific band (C). (D and F) Atf32/2 (red shading) or (E and F) WT cells (blue shading) were stimulated with LPS (100 ng/ml) or CMA (500 ng/ml) for 4 h and Ifnb1 or Il12b mRNA was analyzed by qPCR. Data show fold change relative to nonstimulated control cells. Graphs show mean 6 SEM combined from four independent experiments. *p , 0.05, ***p , 0.001. by guest on October 1, 2021 that likely represents the ATF3b isoform (containing an intact C terminus) (43), which remains to be functionally characterized. Taken together, these data demonstrate that ATF3 induction by type I IFNs is conserved between murine and human immune cells, potentially forming part of an IFN-b negative feedback loop. ATF3 directly regulates a subset of ISGs in response to IFN-b We next investigated the functional relevance of ATF3 induction by type I IFNs with respect to ISG expression. 25-Hydroxy- FIGURE 4. ATF3 downmodulates IFN-b expression by binding directly cholesterol (25-HC) is an oxysterol that displays potent antiviral 2 2 to its promoter. (A and B) WT and Atf3 / BMDMs were stimulated with activity (44, 45). Additionally, Ch25h, the gene encoding cho- LPS (100 ng/ml) or poly(I:C) (1 mg/ml) as indicated and p-IRF3, ATF3, lesterol 25-hydroxylase, which controls the production of 25- total IRF3, and b-actin were measured by immunoblot. Data are repre- HC, is classified as an ISG (46). Interestingly, ATF3 was iden- sentative of three independent experiments. (C and D) Schematic repre- tified as a negative regulator of Ch25h in response to foam sentation of the Ifnb1 locus (purple bar represents the TSS and exon). (C) cell formation and TLR4 stimulation (47). Consistent with this, ATF3 ChIP-seq peaks identified in dataset GSE36104 (orange bars); nor- 2 2 2 2 we observed that Atf3 / BMDMs expressed significantly more malized ChIP-seq signals in WT (light blue) and Atf3 / (gray) BMDMs Ch25h (Fig. 6A) and 25-HC (Fig. 6B) in response to LPS (GSE55317) with significant peaks are indicated by the red bar. Potential compared with WT cells. In contrast, we saw no difference in ATF3 binding sites are denoted as site 1 and site 2 (light red shading). (D) production of LPS-induced 27-HC (Supplemental Fig. 1L), an Visualization of the Ifnb1 locus in activated BMDMs with H3K4me1 oxysterol not under the control of Ch25h. Unlike the ISGs tested (orange), H3K27ac (blue), and PU.1 (green) marks. Gray bars indicate 2 2 significant peaks. (E and F) HEK293T cells were transfected with Ifnb1 earlier (Fig. 2K, 2L), direct IFN-b treatment of Atf3 / BMDMs promoter constructs alone (E) or in the presence of ATF3 or control led to significantly greater Ch25h gene expression compared plasmids (F) before 16 h stimulation with 20 ng/well transfected pppRNA with WT cells (Fig. 6C). The increase in Ch25h gene expression (IVT4). Firefly luciferase was measured and fold induction relative to 2/2 correlated with greater 25-HC release from Atf3 BMDMs nonstimulated cells is shown. Graphs show mean 6 SEM from three in- (Fig. 6D), confirming Ch25h as an ISG that is also a direct ATF3 dependent experiments. *p , 0.05, **p , 0.01. target gene. Furthermore, Atf32/2 BMDMs showed consistently higher Ch25h mRNA levels upon LPS or IFN treatment, even in other genes induced downstream of IFNAR signaling, we per- 2 2 the presence of an IFNAR blocking Ab (Fig. 6E), unlike Irf7 or formed transcriptome analysis of WT and Atf3 / BMDMs stim- Isg15 (Fig. 2K, 2L). To examine the possibility that ATF3 regulates ulated with IFN-b for 6 h. Using the model presented in Fig. 6F, 4452 REGULATION OF IFN RESPONSES BY ATF3 Downloaded from FIGURE 5. ATF3 is a type I IFN-inducible gene in mouse and human immune cells. (A and B) BMDMs were stimulated with IFN-b (1000 U/ml), IFN-a (1000 U/ml), IFN-g (1000 U/ml), or LPS (100 ng/ml) for 10 h before Atf3 mRNA was measured by qPCR (A) or ATF3 and b-actin protein were analyzed by immunoblot and quantified by densitometry (B). (C) BMDMs were stimulated with IFN-b at indicated doses (U/ml) for 10 h and ATF3 and b-actin protein were analyzed by immunoblot and quantified by densitometry. (D) BMDMs were stimulated with IFN-b (1000 U/ml) as indicated and Atf3 mRNA was measured by qPCR. (E)WTorIfnar12/2 iBMDMs were stimulated with LPS (100 ng/ml) or IFN-b (1000 U/ml) for indicated times before ATF3 and b-actin protein were measured by immunoblot and quantified by densitometry. (F) Human monocytes from four independent donors were stimulated overnight with IFN-b (10 U/ml) IFN-a (10 ng/ml) or IFN-g (10 ng/ml) before ATF3 mRNA was measured by qPCR. (G–I) Human monocytes, plas- http://www.jimmunol.org/ macytoid DCs, or PBMCs from four independent donors were stimulated overnight with IFN-b (1000 U/ml) before ATF3 mRNA was analyzed by qPCR. (J) Human monocytes from four separate donors (D1–4) were stimulated overnight with IFN-b (1000 U/ml) and ATF3 and b-actin protein were measured by immunoblot. Graphs show mean 6 SEM combined from at least three independent experiments (A–E) or four independent donors (F–I). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. we identified 34 IFN-b–inducible genes that were further induced in ATF3-dependent regulation of type I IFN responses is important the absence of Atf3 (Fig. 6G). Of note, several of these genes have for an appropriate antiviral response in macrophages.

known inflammatory functions, including the chemokines CCL3 by guest on October 1, 2021 and CCL12 (Supplemental Table II). We further examined the Discussion promoter regions of the 34 genes identified for conserved ATF3 We have demonstrated that ATF3 modulates basal and PRR- binding sites, discovering that 23 contained one or more of these inducible IFN-b levels in macrophages by directly regulating sites (Supplemental Table III). We next validated the increased Ifnb1 mRNA expression. These findings are consistent with a re- expression of identified ATF3 target genes Ccl12, Ccl3, Ifitm6, cent study showing that TLR-induced type I IFN levels were re- and Clec4e in Atf32/2 versusWTBMDMsupon6hIFN-b treat- duced by a PIKfyve inhibitor in RAW264.7 murine macrophage ment (Fig. 6H–K), using Ch25h as a positive control (Fig. 6L). As cells via induction of ATF3 (21). Immune cells can rapidly re- expected, no difference was observed for Irf7 (Fig. 6M). IFN-b– spond to low systemic concentrations of type I IFNs, which are induced Ccl3 and Ccl12 expression was also consistently higher constitutively maintained under homeostatic conditions by the over a time course in Atf32/2 BMDMs (Supplemental Fig. 1M). commensal microflora (48, 49). Indeed, low-level basal IFN-b These findings demonstrate that ATF3 can regulate a subset of expression induces IFNAR signaling via an autocrine loop and is genes induced in response to IFN-b. important for priming immune cells for rapid responses to microbial insults (3). Thus, ATF3 modulation of basal IFN-b pro- Expression of ATF3 modulates antiviral responses in duction represents an important threshold mechanism to ensure macrophages that appropriate responses to pathogens are elicited. In line with Production of type I IFNs and ISGs is important for mediating the ability of IFN-b to signal in an autocrine manner, we ob- host antiviral responses. We hypothesized that macrophages served higher basal and LPS-inducible ISG expression in Atf32/2 deficient in Atf3 would be more efficient at clearing viral in- BMDMs, potentially due to higher circulating levels of IFN-b in fections due to increased IFN-b and consequently increased ISG these cells (Figs. 1D, 2I). Indeed, when we treated Atf32/2 and expression. We therefore infected WT or Atf32/2 primary WT BMDMs with LPS, we found that increased Irf7 and Isg15 BMDMs with LCMV and assessed viral expression 24 h post- mRNA expression in Atf32/2 cells was due to increased IFN-b,as infection by measuring LCMV viral Ag in cells by flow this effect was blocked by the addition of an IFNAR blocking Ab cytometry. We found a marked reduction in LCMV viral repli- (Fig. 2K). The observation that Atf32/2 BMDMs display higher cation within Atf32/2 BMDMs compared with WT BMDMs basal ISG expression may be due to the use of M-CSF (also (Fig.7A).Finally,weinfectedAtf32/2 iBMDMs overexpressing known as CSF-1) in our cultures. M-CSF is the major growth ATF3 or an empty vector control with VSV replicon particles factor controlling the differentiation of macrophages from the encoding a firefly luciferase reporter [VSV*DG(Luc)] (29). bone marrow (50), and it is also known to induce low levels of Consistent with results from primary BMDMs, we observed a basal IFN-b production from macrophages (51). In the absence of significant increase in viral replication when cells expressed regulation by ATF3, M-CSF is likely to trigger more basal IFN-b ATF3 (Fig. 7B). Taken together, these data demonstrate that production, subsequently resulting in increased expression of The Journal of Immunology 4453

FIGURE 6. ATF3 directly regulates a subset of IFN-b–stimulated genes. (A–D) WT and Atf32/2 BMDMs were stimulated with LPS (100 ng/ml) (A and B) or IFN-b (1000 U/ml) (C and D)as indicated and Ch25h mRNA was analyzed by qPCR (A and C) or 25-HC in supernatants measured by mass spectrometry (B and D). (E) WT and Atf32/2 BMDMs were treated with 1 mg/ml IFNAR blocking Ab or mouse IgG1 control Ab for 1 h before cells were stimulated for 6 h with 10 ng/ml LPS or 10 U/ml IFN-b and Ch25h mRNA was analyzed by qPCR. (F and G) WT and Atf32/2 BMDMs (from three mice per Downloaded from genotype) were stimulated with IFN-b (1000 U/ml) for 6 h and analyzed by microarray. (F) Model for identifying ATF3-regulated ISGs. The 34 genes identiﬁed are shown in a heat map (red shading indicates expression as log2) http://www.jimmunol.org/ (G). (H–M) WT and Atf32/2 BMDMs were stimulated with IFN-b (1000 U/ml) for 6 h and Ccl12, Ccl3, Iﬁtm6,orClec4e mRNA was analyzed by qPCR. Graphs show mean 6 SEM combined from at least three independent experiments (A–C, E,andH–M)(*p , 0.05, **p , 0.01, ***p , 0.001) or average 6 SD (D) combined from two independent experiments. by guest on October 1, 2021

ISGs. Similar to Atf32/2 BMDMs, spontaneous basal ISG express IFN-b expression (57). Collectively, this suggests that ATF3 pression is also observed in macrophages deficient in other im- may regulate the magnitude of Ifnb1 expression by inducing a less portant IFN regulators, including TREX1, SAMHD1, and the transcriptionally active chromatin state, which is also reflected transcriptional repressor FOXO3 (52-55). by higher basal levels of Ifnb1 gene expression in Atf32/2 Considering the potential detrimental effects of excessive IFN-b macrophages. The importance of the regulatory element we have to the host, its production is tightly controlled, including on the termed site 2 for transcriptional regulation of Ifnb1 is underscored transcriptional level. For instance, cooperative binding of the transcription factors IRF3/7, NF-kB, and ATF-2/Jun to a highly conserved regulatory element within the Ifnb1 promoter, termed the enhanceosome, is required for optimal IFN-b induction (56). Additionally, virus infection induces nucleosome remodeling of the Ifnb1 promoter (via histone acetyl transferases, as well as the SWI/SNF chromatin remodeling complex), allowing for increased transcription (56). Consistent with the role of ATF3 as a transcriptional repressor, two recent studies suggested that ATF3 has binding sites within the Ifnb1 promoter (21, 30). Our ChIP-seq FIGURE 7. ATF3 regulates viral replication in macrophages by modu- A 2/2 dataset (26) has identified an ATF3 binding site at a regulatory lating IFN-b levels. ( ) WT and Atf3 BMDMs were infected with LCMV at indicated multiplicities of infection (MOI) for 24 h and infected element 15 kbp upstream of the Ifnb1 TSS, which showed sig- cells were measured for viral replication by flow cytometry. Graphs show nificant and specific enrichment of ATF3 under basal conditions average 6 SD of four technical replicates representative of two indepen- (Fig. 4C). ATF3 interacts with histone deacetylase 1 (16), which dent experiments. (B) Atf32/2 iBMDMs reconstituted with a control (Ctrl) can counteract the effects of histone acetyl transferases to main- or murine ATF3 were infected with VSV*DG(Luc) as indicated before tain a more closed chromatin conformation to limit transcription. luciferase activity was measured 6 h postinfection. Graph shows mean 6 Of note, histone deacetylase 1 has previously been shown to re- SEM combined from four independent experiments. *p , 0.05. 4454 REGULATION OF IFN RESPONSES BY ATF3 by its methylation and acetylation status, which suggests that it is vating inflammasomes (63), in line with the known immuno- indeed a poised enhancer in both LPS-stimulated BMDMs and suppressive activity of type I IFNs (3). Consistent with a role in in vivo in tissue macrophage populations (Fig. 4D, Supplemental dampening inflammation, we also found that ATF3 regulates the Fig. 1D). Furthermore, in luciferase reporter assays, addition of site expression of the IFN-inducible chemokines CCL3 and CCL12, 2 increased inducible Ifnb1 promoter activity (Fig. 4E), which was which are required for inflammatory monocyte recruitment (64). reduced when ATF3 was also present (Fig. 4F). These intriguing The overrepresentation of cell surface markers/receptors and findings suggest that other positive transcriptional regulators of chemokines (Supplemental Table II) in addition to Ch25h sug- IFN-b may bind this distal Ifnb1 enhancer (site 2), and potentially gests that ATF3 might be induced by IFN to limit subsequent be displaced upon ATF3 binding. Future studies that further ex- inflammation and modulate the adaptive immune response. amine the role of this new enhancer site and ATF3 will be of great Overall, our findings suggest that regulatory mechanisms interest. Interestingly, whereas ATF3 modulated the level of PRR- exertedonIFN-b and ISGs by ATF3 are important for an ap- inducible IFN-b mRNA expression in macrophages, it did not appropriate and balanced immune response: too little ATF3 ex- pear to affect its kinetics (Fig. 2D), which is likely due to the pression results in exaggerated IFN-b and ISG production, existence of additional regulatory mechanisms acting on IFN-b which can lead to chronic inflammation or immunosuppression, mRNA following immune activation (58). At the protein level, whereas too much ATF3 results in enhanced viral replication, however, we saw an obvious delay in IFN-b production in WT owing to repression of IFN-b and antiviral mediators. Our work compared with Atf32/2 BMDMs (Fig. 2H). reinforces the importance of ATF3 as a key player within the Previously, ATF3 was found to be IFN-a inducible in human gene regulatory networks of the immune system, and it highlights PBMCs (42). In the present study, we further show that ATF3 the potential for modulating ATF3 expression or activity thera- is induced in both mouse and human immune cells in response peutically to combat chronic viral infections. Moreover, further Downloaded from to both IFN-a and IFN-b. Although all type I IFNs are known investigation into the use of ATF3 inducers (e.g., HDL, PIKfyve to signal via the same receptor containing the IFNAR1 and inhibitors) in diseases involving unwarranted IFN production and IFNAR2 subunits, we observed differences in ATF3 induction by signaling, such as in systemic lupus erythematosus, would be of IFN-a and IFN-b (Fig. 5A, 5B). This may be due to observations great interest. made that suggest IFN-b can have differential effects on IFN-a on a variety of biological processes, including gene transcription Acknowledgments http://www.jimmunol.org/ (59–61). In general, induction of ATF3 is promiscuous, with We thank B.R.G. Williams (Monash Institute of Medical Research–Prince numerous studies showing that a range of stimuli (e.g., TLRs, Henry’s Institute, Clayton, VIC, Australia) and M. Ro¨cken (University of HDL, PIKfyve inhibitors) and cellular conditions (e.g., cellular Tubingen, Tubingen, Germany) for providing bones from Atf32/2 mice stress) can induce its expression (16, 18, 26, 41). This suggests (originally from T. Hai, The Ohio State University, Columbus, OH); that both the cell type and the specific context under which ATF3 P.J. Hertzog (Monash Institute of Medical Research–Prince Henry’s Insti- 2/2 is induced may dictate its functional role. Furthermore, our data tute) for bone marrow from Ifnar mice and helpful discussions; support the work presented by Whitmore et al. (18) showing that G. Zimmer (Institute of Virology and Immunology, Bern, Switzerland) TLRs that elicit IFN-b production (i.e., TLR3 or TLR4) can for VSV*DG(Luc) replicon particles; and D. Kalvakolanu (University of Maryland, College Park, MD) for J2 recombinant retroviruses. We ac- by guest on October 1, 2021 induce ATF3 expression in a biphasic manner (Fig. 5E) via an knowledge T. Cavlar and M. Schlee (University of Bonn) for providing early IFNAR-independent wave of transcription and via a sec- pppRNA and helpful discussions. We also acknowledge H. Theis, ond, delayed IFNAR-dependent wave. Whereas the first, acute M. Kraut, and S. Martin of the Life and Medical Sciences Institute, Bonn, wave is described for regulating proinflammatory cytokine pro- Germany; A. Kerksiek and B. Putschli of the University of Bonn; and duction (16), the second IFN-dependent induction of ATF3 was P. Langhoff of the Institute of Innate Immunity for technical support. not investigated until now. Although it is likely that IFN- mediated ATF3 expression constitutes an important component Disclosures of a negative feedback loop for regulating IFN-b,orfurther The authors have no financial conflicts of interest. downstream modulation of IFN-g expression (20), we have now discovered that IFN-b–mediated ATF3 is also important for controlling expression of a subset of genes downstream of References IFNAR signaling. Given that ATF3 expression was also robustly 1. Medzhitov, R., and T. Horng. 2009. Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9: 692–703. induced in human immune cells by type I IFNs (Fig. 5F–I), it is 2. Cavlar, T., A. Ablasser, and V. Hornung. 2012. Induction of type I IFNs by possible that a similar regulatory system exists in humans. Of the intracellular DNA-sensing pathways. Immunol. Cell Biol. 90: 474–482. ISGs we identified as direct ATF3 target genes, the most highly 3. Ivashkiv, L. B., and L. T. Donlin. 2014. Regulation of type I interferon responses. Nat. Rev. Immunol. 14: 36–49. induced was Ch25h. Cholesterol 25-hydroxylase (encoded by 4. Theofilopoulos, A. N., R. Baccala, B. Beutler, and D. H. Kono. 2005. Type I Ch25h) catalyzes the formation of 25-HC during cholesterol interferons (a/b) in immunity and autoimmunity. Annu. Rev. Immunol. 23: 307– metabolism, and ATF3 was previously found to directly regulate 336. 5. Schoggins, J. W. 2014. Interferon-stimulated genes: roles in viral pathogenesis. Ch25h expression (47). Additionally, separate studies have Curr. Opin. Virol. 6: 40–46. characterized Ch25h as an ISG, and 25-HC has been shown to 6. Sadler, A. J., and B. R. Williams. 2008. Interferon-inducible antiviral effectors. Nat. Rev. Immunol. 8: 559–568. elicit broad antiviral functions (44, 45). In this study, we show, 7. Schoggins, J. W., M. Dorner, M. Feulner, N. Imanaka, M. Y. Murphy, A. Ploss, to our knowledge for the first time, that ATF3 can regulate the and C. M. Rice. 2012. Dengue reporter viruses reveal viral dynamics in inter- expression of Ch25h following IFN signaling, a mechanism that feron receptor-deficient mice and sensitivity to interferon effectors in vitro. Proc. Natl. Acad. Sci. USA 109: 14610–14615. is likely important for maintaining an appropriate immune re- 8. Liu, S. Y., D. J. Sanchez, R. Aliyari, S. Lu, and G. Cheng. 2012. Systematic sponse to viruses. Indeed, a recent study found that whereas identification of type I and type II interferon-induced antiviral factors. Proc. production of 25-HC is advantageous during the acute stages of Natl. Acad. Sci. USA 109: 4239–4244. 9. Schoggins, J. W., S. J. Wilson, M. Panis, M. Y. Murphy, C. T. Jones, P. Bieniasz, influenza viral infection, its presence ultimately results in ex- and C. M. Rice. 2011. A diverse range of gene products are effectors of the type I cessive inflammation and severe tissue pathology via amplifi- interferon antiviral response. Nature 472: 481–485. 10. Zhang, Y., C. W. Burke, K. D. Ryman, and W. B. Klimstra. 2007. Identification cation of proinflammatory gene expression (62). Furthermore, and characterization of interferon-induced proteins that inhibit alphavirus rep- IFN-induced 25-HC was shown to broadly suppress IL-1–acti- lication. J. Virol. 81: 11246–11255. The Journal of Immunology 4455

11. Muller,€ U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, 39. Mancino, A., A. Termanini, I. Barozzi, S. Ghisletti, R. Ostuni, E. Prosperini, and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral K. Ozato, and G. Natoli. 2015. A dual cis-regulatory code links IRF8 to constitutive defense. Science 264: 1918–1921. and inducible gene expression in macrophages. Genes Dev. 29: 394–408. 12. Hwang, S. Y., P. J. Hertzog, K. A. Holland, S. H. Sumarsono, M. J. Tymms, 40. Panne, D., T. Maniatis, and S. C. Harrison. 2007. An atomic model of the in- J. A. Hamilton, G. Whitty, I. Bertoncello, and I. Kola. 1995. A null mutation in terferon-b enhanceosome. Cell 129: 1111–1123. the gene encoding a type I interferon receptor component eliminates anti- 41. Hai, T., C. C. Wolford, and Y. S. Chang. 2010. ATF3, a hub of the cellular proliferative and antiviral responses to interferons alpha and beta and alters adaptive-response network, in the pathogenesis of diseases: is modulation of macrophage responses. Proc. Natl. Acad. Sci. USA 92: 11284–11288. inflammation a unifying component? Gene Expr. 15: 1–11. 13. Sancho-Shimizu, V., R. Perez de Diego, E. Jouanguy, S. Y. Zhang, and 42. Taylor, M. W., W. M. Grosse, J. E. Schaley, C. Sanda, X. Wu, S. C. Chien, J. L. Casanova. 2011. Inborn errors of anti-viral interferon immunity in humans. F. Smith, T. G. Wu, M. Stephens, M. W. Ferris, et al. 2004. Global effect of PEG- Curr. Opin. Virol. 1: 487–496. IFN-a and ribavirin on gene expression in PBMC in vitro. J. Interferon Cytokine 14. Banchereau, J., and V. Pascual. 2006. Type I interferon in systemic lupus Res. 24: 107–118. erythematosus and other autoimmune diseases. Immunity 25: 383–392. 43. Hunt, D., G. Raivich, and P. N. Anderson. 2012. Activating transcription factor 3 15. Kondo, T., T. Kawai, and S. Akira. 2012. Dissecting negative regulation of Toll- and the nervous system. Front. Mol. Neurosci. 5: 7. like receptor signaling. Trends Immunol. 33: 449–458. 44. Liu, S. Y., R. Aliyari, K. Chikere, G. Li, M. D. Marsden, J. K. Smith, O. Pernet, 16. Gilchrist, M., V. Thorsson, B. Li, A. G. Rust, M. Korb, J. C. Roach, K. Kennedy, H. Guo, R. Nusbaum, J. A. Zack, et al. 2013. Interferon-inducible cholesterol- T. Hai, H. Bolouri, and A. Aderem. 2006. Systems biology approaches identify 25-hydroxylase broadly inhibits viral entry by production of 25-hydrox- ATF3 as a negative regulator of Toll-like receptor 4. Nature 441: 173–178. ycholesterol. Immunity 38: 92–105. 17. De Nardo, D. 2015. Toll-like receptors: activation, signalling and transcriptional 45. Blanc, M., W. Y. Hsieh, K. A. Robertson, K. A. Kropp, T. Forster, G. Shui, modulation. Cytokine 74: 181–189. P. Lacaze, S. Watterson, S. J. Griffiths, N. J. Spann, et al. 2013. The transcription 18. Whitmore, M. M., A. Iparraguirre, L. Kubelka, W. Weninger, T. Hai, and factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the B. R. Williams. 2007. Negative regulation of TLR-signaling pathways by acti- interferon antiviral response. Immunity 38: 106–118. vating transcription factor-3. J. Immunol. 179: 3622–3630. 46. Park, K., and A. L. Scott. 2010. Cholesterol 25-hydroxylase production by 19. Khuu, C. H., R. M. Barrozo, T. Hai, and S. L. Weinstein. 2007. Activating dendritic cells and macrophages is regulated by type I interferons. J. Leukoc. transcription factor 3 (ATF3) represses the expression of CCL4 in murine Biol. 88: 1081–1087. macrophages. Mol. Immunol. 44: 1598–1605. 47. Gold, E. S., S. A. Ramsey, M. J. Sartain, J. Selinummi, I. Podolsky, 20. Rosenberger, C. M., A. E. Clark, P. M. Treuting, C. D. Johnson, and A. Aderem. D. J. Rodriguez, R. L. Moritz, and A. Aderem. 2012. ATF3 protects against Downloaded from 2008. ATF3 regulates MCMV infection in mice by modulating IFN-g expression atherosclerosis by suppressing 25-hydroxycholesterol-induced lipid body for- in natural killer cells. Proc. Natl. Acad. Sci. USA 105: 2544–2549. mation. J. Exp. Med. 209: 807–817. 21. Cai, X., Y. Xu, Y. M. Kim, J. Loureiro, and Q. Huang. 2014. PIKfyve, a class III 48. Abt, M. C., L. C. Osborne, L. A. Monticelli, T. A. Doering, T. Alenghat, lipid kinase, is required for TLR-induced type I IFN production via modulation G. F. Sonnenberg, M. A. Paley, M. Antenus, K. L. Williams, J. Erikson, et al. of ATF3. J. Immunol. 192: 3383–3389. 2012. Commensal bacteria calibrate the activation threshold of innate antiviral 22. Goldeck, M., M. Schlee, G. Hartmann, and V. Hornung. 2014. Enzymatic synthesis immunity. Immunity 37: 158–170. and purification of a defined RIG-I ligand. Methods Mol. Biol. 1169: 15–25. 49. Kawashima, T., A. Kosaka, H. Yan, Z. Guo, R. Uchiyama, R. Fukui, D. Kaneko,

23. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock, Y. Kumagai, D. J. You, J. Carreras, et al. 2013. Double-stranded RNA of in- http://www.jimmunol.org/ K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate testinal commensal but not pathogenic bacteria triggers production of protective the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: interferon-b. Immunity 38: 1187–1197. 847–856. 50. Hamilton, J. A. 2008. Colony-stimulating factors in inflammation and autoim- 24. Stutz, A., G. L. Horvath, B. G. Monks, and E. Latz. 2013. ASC speck formation munity. Nat. Rev. Immunol. 8: 533–544. as a readout for inflammasome activation. Methods Mol. Biol. 1040: 91–101. 51. Moore, R. N., H. S. Larsen, D. W. Horohov, and B. T. Rouse. 1984. Endogenous 25. Roberts, Z. J., N. Goutagny, P. Y. Perera, H. Kato, H. Kumar, T. Kawai, S. Akira, regulation of macrophage proliferative expansion by colony-stimulating factor- R. Savan, D. van Echo, K. A. Fitzgerald, et al. 2007. The chemotherapeutic agent induced interferon. Science 223: 178–181. DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis. J. 52. Hasan, M., J. Koch, D. Rakheja, A. K. Pattnaik, J. Brugarolas, I. Dozmorov, Exp. Med. 204: 1559–1569. B. Levine, E. K. Wakeland, M. A. Lee-Kirsch, and N. Yan. 2013. Trex1 regulates 26. De Nardo, D., L. I. Labzin, H. Kono, R. Seki, S. V. Schmidt, M. Beyer, D. Xu, lysosomal biogenesis and interferon-independent activation of antiviral genes. S. Zimmer, C. Lahrmann, F. A. Schildberg, et al. 2014. High-density lipoprotein Nat. Immunol. 14: 61–71.

mediates anti-inflammatory reprogramming of macrophages via the transcrip- 53. Rehwinkel, J., J. Maelfait, A. Bridgeman, R. Rigby, B. Hayward, R. A. Liberatore, by guest on October 1, 2021 tional regulator ATF3. Nat. Immunol. 15: 152–160. P. D. Bieniasz, G. J. Towers, L. F. Moita, Y. J. Crow, et al. 2013. SAMHD1- 27. Xue, J., S. V. Schmidt, J. Sander, A. Draffehn, W. Krebs, I. Quester, D. De dependent retroviral control and escape in mice. EMBO J. 32: 2454–2462. Nardo, T. D. Gohel, M. Emde, L. Schmidleithner, et al. 2014. Transcriptome- 54. Behrendt, R., T. Schumann, A. Gerbaulet, L. A. Nguyen, N. Schubert, based network analysis reveals a spectrum model of human macrophage acti- D. Alexopoulou, U. Berka, S. Lienenklaus, K. Peschke, K. Gibbert, et al. 2013. vation. Immunity 40: 274–288. Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell- 28. Rusinova, I., S. Forster, S. Yu, A. Kannan, M. Masse, H. Cumming, R. Chapman, intrinsic antiviral response. Cell Reports 4: 689–696. and P. J. Hertzog. 2013. Interferome v2.0: an updated database of annotated 55. Litvak, V., A. V. Ratushny, A. E. Lampano, F. Schmitz, A. C. Huang, A. Raman, interferon-regulated genes. Nucleic Acids Res. 41: D1040–D1046. A. G. Rust, A. Bergthaler, J. D. Aitchison, and A. Aderem. 2012. A FOXO3- 29. Berger Rentsch, M., and G. Zimmer. 2011. A vesicular stomatitis virus replicon- IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses. based bioassay for the rapid and sensitive determination of multi-species type I Nature 490: 421–425. interferon. PLoS One 6: e25858. 56. Smale, S. T. 2010. Selective transcription in response to an inflammatory 30. Garber, M., N. Yosef, A. Goren, R. Raychowdhury, A. Thielke, M. Guttman, stimulus. Cell 140: 833–844. J. Robinson, B. Minie, N. Chevrier, Z. Itzhaki, et al. 2012. A high-throughput 57. Nusinzon, I., and C. M. Horvath. 2006. Positive and negative regulation of the chromatin immunoprecipitation approach reveals principles of dynamic gene innate antiviral response and b interferon gene expression by deacetylation. Mol. regulation in mammals. Mol. Cell 47: 810–822. Cell. Biol. 26: 3106–3113. 31. Krebs, W., S. V. Schmidt, A. Goren, D. De Nardo, L. Labzin, A. Bovier, T. Ulas, 58. Hertzog, P. J., and B. R. Williams. 2013. Fine tuning type I interferon responses. H. Theis, M. Kraut, E. Latz, et al. 2014. Optimization of transcription factor Cytokine Growth Factor Rev. 24: 217–225. binding map accuracy utilizing knockout-mouse models. Nucleic Acids Res. 42: 59. Platanias, L. C., S. Uddin, P. Domanski, and O. R. Colamonici. 1996. Differ- 13051–13060. ences in interferon a and b signaling. Interferon bselectively induces the in- 32. Ostuni, R., V. Piccolo, I. Barozzi, S. Polletti, A. Termanini, S. Bonifacio, teraction of the a and bL subunits of the type I interferon receptor. J. Biol. Chem. A. Curina, E. Prosperini, S. Ghisletti, and G. Natoli. 2013. Latent enhancers 271: 23630–23633. activated by stimulation in differentiated cells. Cell 152: 157–171. 60. Platanias, L. C., S. Uddin, and O. R. Colamonici. 1994. Tyrosine phosphoryla- 33. Lavin, Y., D. Winter, R. Blecher-Gonen, E. David, H. Keren-Shaul, M. Merad, tion of the a and b subunits of the type I interferon receptor. Interferon-b se- S. Jung, and I. Amit. 2014. Tissue-resident macrophage enhancer landscapes are lectively induces tyrosine phosphorylation of an a subunit-associated protein. J. shaped by the local microenvironment. Cell 159: 1312–1326. Biol. Chem. 269: 17761–17764. 34. Honda, K., A. Takaoka, and T. Taniguchi. 2006. Type I interferon gene induction by 61. de Weerd, N. A., J. P. Vivian, T. K. Nguyen, N. E. Mangan, J. A. Gould, the interferon regulatory factor family of transcription factors. Immunity 25: 349–360. S. J. Braniff, L. Zaker-Tabrizi, K. Y. Fung, S. C. Forster, T. Beddoe, et al. 2013. 35. Tanaka, Y., and Z. J. Chen. 2012. STING specifies IRF3 phosphorylation by Structural basis of a unique interferon-b signaling axis mediated via the receptor TBK1 in the cytosolic DNA signaling pathway. Sci. Signal. 5: ra20. IFNAR1. Nat. Immunol. 14: 901–907. 36. Barozzi, I., M. Simonatto, S. Bonifacio, L. Yang, R. Rohs, S. Ghisletti, and 62.Gold,E.S.,A.H.Diercks,I.Podolsky,R.L.Podyminogin,P.S.Askovich, G. Natoli. 2014. Coregulation of transcription factor binding and nucleosome oc- P. M. Treuting, and A. Aderem. 2014. 25-Hydroxycholesterol acts as an am- cupancy through DNA features of mammalian enhancers. Mol. Cell 54: 844–857. plifier of inflammatory signaling. Proc.Natl.Acad.Sci.USA111: 10666– 37. Ghisletti, S., I. Barozzi, F. Mietton, S. Polletti, F. De Santa, E. Venturini, 10671. L. Gregory, L. Lonie, A. Chew, C. L. Wei, et al. 2010. Identification and char- 63. Reboldi, A., E. V. Dang, J. G. McDonald, G. Liang, D. W. Russell, and acterization of enhancers controlling the inflammatory gene expression program J. G. Cyster. 2014. Inflammation. 25-Hydroxycholesterol suppresses interleukin- in macrophages. Immunity 32: 317–328. 1-driven inflammation downstream of type I interferon. Science 345: 679–684. 38. Heinz, S., C. Benner, N. Spann, E. Bertolino, Y. C. Lin, P. Laslo, J. X. Cheng, 64. Lee, P. Y., Y. Li, Y. Kumagai, Y. Xu, J. S. Weinstein, E. S. Kellner, C. Murre, H. Singh, and C. K. Glass. 2010. Simple combinations of lineage- D. C. Nacionales, E. J. Butfiloski, N. van Rooijen, S. Akira, et al. 2009. Type I determining transcription factors prime cis-regulatory elements required for interferon modulates monocyte recruitment and maturation in chronic inflam- macrophage and B cell identities. Mol. Cell 38: 576–589. mation. Am. J. Pathol. 175: 2023–2033. Sup. Figure 1

1.1 1.2 1.5 V$ATF A Atf3-/- Atf3-/- Atf3-/- B (fold ) 1.0 WT 1.0 WT WT 1.0 0.9 25 h (fold ) h 0.8 0.8 C 0.5 ti ve Tnf (fold )

ti ve Ifnb 1 0.6 a ti ve

a 0.7 a Re l

Re l 0.6 0.4 0.0 0 30 60 90 Re l 0 30 60 90 0 30 60 90 Actinomycin-D (min) Actinomycin-D (min) Actinomycin-D (min) Pos. (kpb) Chr4, mm9 C 88.168 88.170 88.172 88.174 88.176 88.178 88.180 88.182 88.184 Chr 4, mm9 H3K4me1 H3K27Ac Ifnb1 D 88.170kb 88.180kb 88.190kb GSE36104 Site 1 GSE55317 Site 1 Site 2 88.168.601 88.168.957

GSE36104 Kupffer 88.167.929 88.168.698

Ifnb1

Promoter

88.168.598 88.169.198 intestine arge degenerated Atf3 binding sites L 88.182.876 88.183.873 88.182.925 Site 2 88.183.823 GSE55317 Microglia

GSE36104 88.183.410 88.183.610 Macrophage Population E F G

50 Peritoneal *** 1.5 6 40 Irf 7

e 30 1.0 4

i 20 **** 0.5 2 Splenic Rela t Relative Irf 7 10 Relative A tf 3 *** 0 0.0 0 0 1 3 6 9 0 1 3 6 9 Ctrl Ifnb1 IFN IFN IFN IFN (h) IFN (h) H I J K Binding site 1 Binding site 2 Monocytes pDCs PBMCs 200 400 200

0 2000 0 0 1 * 0 1 1 1 L L L

* L C 150 300 150 C

C 1500 C X X X X C C C 100 * 1000 200 C 100 ti ve ti ve ti ve a ti ve a 50 a 100 500 a 50 Re l Re l Re l 0 0 0 Re l 0

Ctrl Ctrl IFN IFN IFN Ctrl IFNβ Ctrl IFN IFNβ L M WT Atf3–/– 10 Ccl12 50 Ccl3 2.5 WT –/– 8 40 2.0 Atf3 1.5 6 30 1.0 4 20 0.5 2 10 Relative mRNA 27-HC (ng/sample) 0.0 0 0 0 9 24 0 1 3 6 9 24 0 1 3 6 9 24 LPS (h) IFN (h) IFN (h) Supplementary Figure 1: (A) WT and Atf3–/– BMDMs were stimulated with LPS (100 ng/ml) for 3 h before Actinomycin-D (5 μg/ml) was added for indicated times and Ifnb1, Ch25h and Tnf mRNA expression measured by qPCR. Data was normalised to LPS stimulation alone for each genotype. Graphs show mean + SEM, of three independent experiments. (B,C) Potential ATF3 binding motifs within the Ifnb1 promoter. Representation of the degenerated ATF3 binding motif (B). Schematic representation of the Ifnb1 locus (exon visualized as blue bar) with degenerated ATF3 binding sites at the Ifnb1 promoter (including site 1) and 15 kb distal to the Ifnb1 promoter (including site 2) as identified in ATF3 ChIP-seq dataset GSE36104 (BMDC, orange bars) and data set GSE55317 (BMDM, red bar). Light green bars mark positions of the degenerated ATF3 binding motifs. (D) Visuali- zation of the Ifnb1 locus and the identified binding sites for ATF3 (red bars and shading) in tissue resident macrophages with H3K4me1 (orange) and H3K27ac (blue) marks as derived from dataset GSE63339. Grey bars indicate significant peaks. (E) BMDMs were stimulated with IFNβ (1000 U/ml), IFNα (1000 U/ml), IFNγ (1000 U/ml) for 10 h and Irf7 mRNA expression was measured by qPCR. Graph shows mean + SEM of four combined biological replicates (***p<0.001, ****p<0.0001). (F, G) WT iBMDMs were stimulated with IFNβ (1000 U/ml) for the indicated times before Atf3 and Irf7 mRNA expression was assessed by qPCR. Graph shows mean ± SEM of three combined biological replicates (H) Human monocytes from four independent donors were stimulated overnight with IFNβ (10 U/ml) IFNα (10 ng/ml) or IFNγ (10 ng/ml) and CXCL10 expression measured by qPCR. Graph shows mean ± SEM (*p<0.05). (I-K) Human monocytes, pDCs or PBMCs from four independent donors were stimulated overnight with IFNβ (1000 U/ml) and CXCL10 expression was analysed by qPCR. Graph shows mean ± SEM (*p<0.05). (L) WT and Atf3–/– BMDMs were stimulated with LPS (100 ng/ml) for indicated times and 27-HC in supernatants was measured by mass spectrometry. Graphs show mean + SEM of four independent experiments. (M) WT and Atf3–/– BMDMs were stimulated with IFNβ (1000 U/ml) for indicated times and gene expression analyzed by qPCR. Graphs show mean ± SEM of three combined biological replicates. Table S1: List of ATF3 binding motifs at binding sites 1 and 2 of the Ifnb1 locus. Genomic positions, original sequences and mutated ATF3 binding motifs at binding site 2 are shown. ATF3 binding sites (BS) in the IFNbeta promoter (Chr4, mm9) Start End Motif Distance from TSS Mutated motif TSS start 88168698 Promoter region 88169198 88168598

ATF3 BS 1 88168685 88168677 ttccatca 13 - ATF3 BS 2 88168689 88168681 tggcttcc 9 - ATF3 BS 3 88168700 88168692 cgacacca 2 - ATF3 BS 4 88168703 88168695 agccgaca -5 - ATF3 BS 5 88168724 88168716 ttgcctca -26 - ATF3 BS 6 88168733 88168725 tgacagcc -35 - ATF3 BS 7 88168746 88168738 tcactgca -48 - ATF3 BS 8 88168762 88168754 tgaaggaa -64 - ATF3 BS 9 88169038 88169030 tatcttca -340 - ATF3 BS 10 88169111 88169103 tgccatcc -413 - ATF3 BS 11 88169165 88169157 ggagctca -467 -

ATF3 binding sites (BS) in the novel ATF3 Binding site upstream of the IFNbeta promoter (Chr4, mm9) Start End Motif Distance from TSS Mutated motif TSS start 88168698 Novel ATF3 BS 88183873 88182876 ATF3 BS 1 88182898 88182889 ggacgagag -14200 tttgttggt ATF3 BS 2 88182921 88182912 tgcccgcac -14223 tttgttggt ATF3 BS 3 88183000 88182991 agaccacac -14302 tttgttggt ATF3 BS 4 88183057 88183048 tttcgccag -14359 tttgttggt ATF3 BS 5 88183176 88183168 tgacaact -14478 ctttgttg ATF3 BS 6 88183183 88183175 taacgtgt -14477 ggtcttgt ATF3 BS 7 88183210 88183202 ttgtgtca -14512 ctttgttg ATF3 BS 8 88183213 88183205 tgtcatcc -14515 tgttggtc ATF3 BS 9 88183257 88183248 ttacgcaac -14559 tttgttggt ATF3 BS 10 88183271 88183263 tgaggaaa -14573 ctttgttg ATF3 BS 11 88183274 88183266 ggaaatca -14576 tgttggtc ATF3 BS 12 88183288 88183279 ttgagttct -14590 tttgttggt ATF3 BS 13 88183384 88183375 tggcgtgtg -14686 tttgttggt ATF3 BS 14 88183427 88183418 tgtcttctt -14729 tttgttggt ATF3 BS 15 88183501 88183492 atgtggtta -14803 tttgttggt ATF3 BS 16 88183525 88183508 tgagaggtcacaggaca -14827 ttttgtgctggtcttgt ATF3 BS 17 88183548 88183538 ctgaaggcct -14850 tttgttggt ATF3 BS 18 88183653 88183645 ggaggcca -14955 ctttgttg ATF3 BS 19 88183662 88183653 ttgcagtca -14964 gtcttgtgc ATF3 BS 20 88183693 88183684 ttgtgctca -14995 tttgttggt ATF3 BS 21 88183744 88183735 ggaaggcac -15046 tttgttggt ATF3 BS 22 88183828 88183819 ctacctcac -15130 tttgttggt ATF3 BS 23 88183850 88183842 tgaaaaca -15152 ctttgttg ATF3 BS 24 88183857 88183847 acacatcaca -15159 ttggtcttgt ATF3 BS 25 88183873 88183870 tgactcga* -15175 ggtctcga

*ctcga(g) is part of XhoI restriction site for cloning into pGL4.14

Table S2 : Known functions of the 34 ATF3 target genes identified downstream of IFN sigaling

Gene symbol: Known function (not limited to): Ch25h This is a gene that is involved in cholesterol and lipid metabolism. CCL12 specifically attracts eosinophils, monocytes and lymphocytes. This chemokine is found predominately in lymph nodes and thymus Ccl12 under normal conditions, and its expression can be hugely induced in macrophages. It is thought to coordinate cell movements during early allergic reactions, and immune response to pathogens. This gene encodes a transmembrane adaptor protein that is expressed in antigen-presenting cells and is localized in the immunologic Scimp synapse. The encoded protein is involved in major histocompatibility complex class II signal transduction and immune synapse formation. The protein encoded by this gene is a member of the sialyltransferase family. Members of this family are enzymes that transfer sialic acid St3gal6 from the activated cytidine 5'-monophospho-N acetylneuraminic acid to terminal positions on sialylated glycolipids (gangliosides) or to the N- or O-linked sugar chains of glycoproteins. Low affinity receptor for N-formyl-methionyl peptides, which are powerful neutrophils chemotactic factors. Binding of FMLP to the receptor Fpr2 causes activation of neutrophils. Gm188 n/a 9030216K14Rik n/a Interacts with MSL1 and inhibits its activity on histone H4 'Lys-16' acetylation (H4K16ac). Binds the RELB promoter and activates its Nupr1 transcription, leading to the transactivation of IER3. The NUPR1/RELB/IER3 survival pathway may provide pancreatic ductal adenocarcinoma with remarkable resistance to cell stress, such as starvation or gemcitabine treatment The encoded protein, also known as macrophage inflammatory protein 1 alpha, plays a role in inflammatory responses through binding to the Ccl3 receptors CCR1, CCR4 and CCR5. Stk40 GO annotations related to this gene include protein serine/threonine kinase activity. An important paralog of this gene is TRIB3. Iﬁtm6 n/a This gene encodes a member of the immunoglobulin superfamily. The encoded protein is a lectin-like adhesion molecule that binds Siglec1 glycoconjugate ligands on cell surfaces in a sialic acid-dependent manner. This gene encodes a member of the GXGD family of aspartic proteases, which are transmembrane proteins with two conserved catalytic Sppl2a motifs localized within the membrane-spanning regions, as well as a member of the signal peptide peptidase-like protease (SPPL) family. Ier3 This gene functions in the protection of cells from Fas- or tumor necrosis factor type alpha-induced apoptosis. The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3- Wdfy1 phosphate (PtdIns(3)P)-containing membranes. 9930022F21Rik n/a This gene encodes a cell surface adhesion molecule that belongs to a family of adhesion/homing receptors. The gene product is required for Sell binding and subsequent rolling of leucocytes on endothelial cells, facilitating their migration into secondary lymphoid organs and inflammation sites. The protein encoded by this gene is a member of the Tyro3-Axl-Mer (TAM) receptor tyrosine kinase subfamily. This gene may be involved in Axl several cellular functions including growth, migration, aggregation and anti-inflammation in multiple cell types. Acts as a high affinity receptor for both nicotinic acid (also known as niacin) and (D)-beta-hydroxybutyrate and mediates increased Gpr109a adiponectin secretion and decreased lipolysis through G(i)-protein-mediated inhibition of adenylyl cyclase. This gene encodes a mitogen-activated protein kinase phosphatase that is a member of the dual specificity protein phosphatase subfamily. Dusp16 These phosphatases inactivate their target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. This gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common Mgl1 protein fold and have diverse functions, such as cell adhesion, cell-cell signalling, glycoprotein turnover, and roles in inflammation and immune response. This gene encodes a transcription factor which regulates genes involved in development and apoptosis. The encoded protein is also a Ets2 protooncogene and shown to be involved in regulation of telomerase. H2-Q8 n/a Plac8 PLAC8 (placenta-specific 8) is a protein-coding gene. GO annotations related to this gene include chromatin binding. This gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common Mgl2 protein fold and have diverse functions, such as cell adhesion, cell-cell signalling, glycoprotein turnover, and roles in inflammation and immune response. LOC676704 n/a This gene encodes a protein best known as a hematopoietic cell granule proteoglycan. Proteoglycans stored in the secretory granules of Srgn many hematopoietic cells also contain a protease-resistant peptide core, which may be important for neutralizing hydrolytic enzymes. Csprs n/a Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, Mmp14 such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. This gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signalling, glycoprotein turnover, and roles in inflammation and Clec4e immune response. The encoded type II transmembrane protein is a downstream target of CCAAT/enhancer binding protein (C/EBP), beta (CEBPB) and may play a role in inflammation. Idb2 n/a Participates in the innate immune response to microbial agents. Specifically recognizes diacylated and triacylated lipopeptides. Cooperates Tlr1 with TLR2 to mediate the innate immune response to bacterial lipoproteins or lipopeptides. Acts via MYD88 and TRAF6, leading to NF-kappa- B activation, cytokine secretion and the inflammatory response. Wfdc17 n/a This gene encodes a protein which is a member of the small GTPase protein superfamily. The encoded protein binds only GTP but has no Rnd3 GTPase activity, and appears to act as a negative regulator of cytoskeletal organization leading to loss of adhesion.

Table S3: Numbers of ATF3 binding motifs in promoter regions of ATF3 regulated genes.

SYMBOL No of Atf3 BS (V$ATF.01 + V$ATF.02) Scimp NA LOC676704 NA Dusp16 11 Siglec1 7 9930022F21Ri k 7 Sell 5 Axl 5 Mgl2 5 Sppl2a 3 Gm188 3 Ets2 3 Mgl1 3 H2-Q8 3 Mmp14 3 Rnd3 3 Idb2 3 Wdfy1 2 Stk40 2 Csprs 2 Ch25h 1 St3gal6 1 Ccl3 1 9030216K14R ik 1 Ifitm6 1 Srgn 1 Ccl12 0 Fpr2 0 Nupr1 0 Gpr109a 0 Ier3 0 Plac8 0 Clec4e 0 Wfdc17 0 Tlr1 0