gene expression in mouse macrophages

PU.1 and ICSBP control constitutive and IFN-�-regulated Tlr9gene expression in mouse macrophages

Kate Schroder,*,† Monika Lichtinger,‡ Katharine M. Irvine,* Kristian Brion,*,† Angela Trieu,*,†

Ian L. Ross,*,† Timothy Ravasi,§ Katryn J. Stacey,*,† Michael Rehli,‡ David A. Hume,*,†

and Matthew J. Sweet,*,†,��,1

*Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, and ��School ofMolecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia; †Cooperative ResearchCentre for Chronic Inflammatory Diseases, Australia; ‡Abt. fur Hamatologie und Internistische Onkologie, Klinikumder Universitat Regensburg, Germany; and §Scripps NeuroAIDS Preclinical Studies Centre and Department ofBioengineering, Jacobs School of Engineering, University of California San Diego, La Jolla, California, USA

Abstract: Macrophages are activated by unmethyl-ated CpG-containing DNA (CpG DNA) via TLR9.IFN-� and LPS can synergize with CpG DNA to en-hance proinflammatory responses in murine macro-phages. Here, we show that LPS and IFN-� up-regu-lated Tlr9 mRNA in murine bone marrow-derivedmacrophages (BMM). The ability of LPS and IFN-�to induce Tlr9 mRNA expression in BMM was depen-dent on the presence of the growth factor, CSF-1,which is constitutively present in vivo. However,there were clear differences in mechanisms of Tlr9mRNA induction. LPS stimulation rapidly removedthe CSF-1 receptor (CSF-1R) from the cell surface,thereby blocking CSF-1-mediated transcriptional re-pression and indirectly inducing Tlr9 mRNA expres-sion. By contrast, IFN-� activated the Tlr9 promoterdirectly and only marginally affected cell surfaceCSF-1R expression. An �100-bp proximal promoterof the murine Tlr9 gene was sufficient to confer basaland IFN-�-inducible expression in RAW264.7 cells.A composite IFN regulatory factor (IRF)/PU.1 siteupon the major transcription start site was identified.Mutation of the binding sites for PU.1 or IRF im-paired basal promoter activity, but only the IRF-binding site was required for IFN-� induction. ThemRNA expression of the IRF family member IFNconsensus-binding protein [(ICSBP)/IRF8] was co-regulated with Tlr9 in macrophages, and constitutiveand IFN-�-inducible Tlr9 mRNA expression was re-duced in ICSBP-deficient BMM. This study thereforecharacterizes the regulation of mouse Tlr9 expres-sion and defines a molecular mechanism by whichIFN-� amplifies mouse macrophage responses toCpG DNA. J. Leukoc. Biol. 81: 1577–1590; 2007.

Key Words: colony-stimulating factor-1 � inflammation � lipopoly-saccharide � interferon � IRF8 � Toll-like receptor

INTRODUCTION

The vertebrate immune system has evolved to recognize invari-ant pathogen-associated molecular patterns (PAMPs) for the

early detection and subsequent clearance of microbes. Onesuch PAMP is unmethylated CpG-containing DNA (CpGDNA), found in bacterial and viral genomes [1]. UnmethylatedCpG motifs within specific nucleotide contexts are recognizedby a TLR family member, TLR9 [2]. To date, 13 mammalianTLRs have been identified (TLR1–13), many of which havedemonstrated functions in innate immunity. TLR9 is located inthe endoplasmic reticulum in resting cells but upon cellularuptake of CpG-containing sequences, relocates to the lysoso-mal compartment to activate signaling [3–5]. The signal isrelayed through the MyD88 adaptor protein, members of theIL-1 receptor-associated kinase family, and TNF receptor-associated factor-6 (TRAF6) to promote I�B degradation andactivate JNK and p38 MAPK. This ultimately results in acti-vation of a number of transcription factors, including NF-�Band AP-1 and transcriptional control over genes directingimmune function (reviewed in refs. [6–9]).

In mice, Tlr9 is expressed by macrophages, B cells, myeloiddendritic cells (DCs), and plasmacytoid DCs (PDCs; reviewedin ref. [10]). A number of studies demonstrated an absence ofTLR9 expression in the monocyte/macrophage lineage in hu-mans [11–13], suggesting divergent regulation among the spe-cies. However, recent reports have claimed that TLR9 expres-sion was inducible by bacterial challenge [14] and IFN-� [15]in human myeloid cells. TLR9 stimulation of mouse B cellstriggers proliferation and IL-6 secretion and skews class switchrecombination to “Th1-like” Igs [16]. In human and mousePDCs, TLR9 ligation results in cellular differentiation andactivation to secrete Th1-polarizing cytokines such as Type IIFN, IL-12, and TNF [17, 18]. Mouse monocyte/macrophagesare activated by CpG DNA to release cytokines such as IL-12,IL-6, and TNF [2]. In the context of bacterial pathogen recog-nition, innate immune cells such as macrophages are likely to

1 Correspondence: Institute for Molecular Bioscience, University of Queens-land, St. Lucia, Brisbane 4072, Australia. E-mail: [email protected]

Received January 18, 2007; accepted February 12, 2007.doi: 10.1189/jlb.0107036

0741-5400/07/0081-1577 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 81, June 2007 1577

detect CpG DNA in concert with other bacterial products suchas LPS or proinflammatory cytokines such as IFN-�. LPS andIFN-� can amplify responses to CpG DNA [2, 19, 20]. Bacte-rial CpG DNA action can be mimicked by synthetic phosphodi-ester or phosphorothioate oligonucleotides [2], although phos-phorothioate oligonucleotides can also have CpG DNA-inde-pendent effects on cellular function [21–25]. CpG DNA, in theform of the phosphorothioate-stabilized oligodinucleotide, haspotential for clinical exploitation as a vaccine adjuvant and inthe treatment of cancer and infectious diseases.

Mice with targeted deficits in IFN-� signaling display com-promised innate and acquired immunity during CpG DNAstimulation [26] and are highly resistant to LPS-induced tox-icity [27]. This highlights the physiological significance ofIFN-� priming in vivo, as it suggests that IFN-� is normallyproduced during the response to these TLR agonists and func-tions to amplify the TLR-induced cellular responses. Manymechanisms have been elucidated for IFN-� priming of mac-rophage responses to LPS (reviewed in refs. [28, 29]) andinvolve the reciprocal cross-regulation of signaling moleculesbetween the IFN-� and LPS signaling pathways. These mech-anisms include induction of TLR4 and inhibition of LPS-induced TLR4 down-regulation, induction of MyD88 and themyeloid differentiation protein 2 adaptor molecule, potentia-tion of NF-�B activity, and transcription factor synergy at thepromoters of target genes. However, it is less clear how IFN-�primes macrophage responses to CpG DNA (reviewed in ref.[29]).

Although CpG DNA-regulated signaling events are well-characterized, information regarding regulation of the Tlr9gene remains limited. One report characterizing the humanTLR9 promoter [30] identified four promoter elements [cAMP-response element (CRE), 5� PU.1, 3� PU.1, and c/EBP sites],which conferred gene regulation in B cells. Another recentstudy identified some of the elements necessary for constitutiveand IFN-�-inducible regulation of the Tlr9 promoter in mousemacrophages [31]. This study found that a proximal promoterAP-1 site conferred constitutive expression, and a distal reg-ulatory region containing two IFN-stimulated response ele-ment/IFN regulatory factor (ISRE/IRF) sites, �2.4 kb up-stream of the transcription start site, contributed to basal andIFN-�-inducible Tlr9 expression. In the present report, we setout to define the mechanisms by which IFN-� and LPS primeresponses to CpG DNA. We show that LPS and IFN-� inducedTlr9 mRNA in mouse macrophages through divergent mecha-nisms. The induction of Tlr9 by IFN-� occurred at the level ofthe proximal promoter, and we identified regulators of basaland IFN-�-inducible Tlr9 expression. Definition of the keyelements of the mouse Tlr9 promoter provides insight intoexpression differences arising from evolutionary divergence ofthe promoter in mammals.

MATERIALS AND METHODS

General reagents

Recombinant mouse (rm)IFN-� (R&D Systems, Minneapolis, MN, USA) wasused at a final concentration of 500 pg/ml, and rmIFN-� (PBL BiomedicalLaboratories, Piscataway, NJ, USA) was used at 100 U/ml. LPS (from Salmo-

nella minnesota, Sigma Aldrich, St. Louis, MO, USA) was used at a finalconcentration of 10 ng/ml. Recombinant human (rh)CSF-1 (a gift from Chiron,Emeryville, CA, USA) was used at a final concentration of 1 � 104 U/ml (100ng/ml).

Cell culture

Specific, homogenous bone marrow-derived macrophages (BMM) were ob-tained by ex vivo differentiation from mouse BM progenitors in the presence ofrCSF-1 as described previously [32, 33]. All mice were C57Bl/6, specificpathogen-free and 6–8 weeks old at the time of BM collection. In experimentswith IFN consensus-binding protein (ICSBP)�/� versus wild-type (WT) BMM,matched numbers of female and male mice were used for BM collection. Malemice were used in all other experiments. BM cells were cultured in RPMI 1640(Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (heat-inacti-vated and endotoxin-tested; JRH Biosciences, Lenexa, KS, USA), 20 U/mlpenicillin and 20 g/ml streptomycin (Invitrogen), 2 mM L-glutamine (Glu-tamax-1, Invitrogen), and CSF-1 on bacteriological plastic (Sterilin, Stafford-shire, UK). CSF-1-replete media was replaced on Day 5, and cells werere-seeded at Day 6 onto tissue-culture plastic (Iwaki, Tokyo, Japan) in thepresence or absence of CSF-1. Cells were stimulated (e.g., with IFN-� or LPS)early on Day 7. The macrophage-like cell line RAW 264.7 was maintained onbacteriological plates in RPMI 1640 supplemented with 5% FBS, 20 U/mlpenicillin, 20 g/ml streptomycin, and 2 mM L-glutamine. Prior to experi-ments, cells were seeded onto tissue-culture plastic. All cells were maintainedin a 37°C incubator venting 5% CO2.

RNA purification and analysis

RNA extraction from mammalian cells or tissues was performed using RNeasykits (Qiagen, Valencia, CA, USA), genomic DNA was removed from RNApreparations using DNA-Free (Ambion, Austin, TX, USA), and cDNA wassynthesized using Superscript III (Invitrogen). These procedures were per-formed according to the manufacturer’s instructions. Negative control reactionswere performed for cDNA synthesis in the absence of the Superscript IIIenzyme.

Where possible, gene-specific PCR primer pairs were designed, such thatone of the primers overlapped an exon junction and produced an amplicon of�100 bp. The amplification of a single product was ensured by melt-curveanalysis for each primer pair. The sequences of the primer pairs used for cDNAquantitation are as follows: Tlr9 forward (F), 5�-AGGCTGTCAATGGCTCT-CAGTT-3�, reverse (R), 5�-TGAACGATTTCCAGTGGTACAAGT-3�; hypo-xanthine guanine phosphoribosyl transferase (Hprt) F, 5�-GCAGTACAGC-CCCAAAATGG-3�, R, 5�-AACAAAGTCTGGCCTGTATCCAA-3�; Icsbp F,5�-ACGAGGTTACGCTGTGCTCTG-3�, R, 5�-CACGCCCAGCTTGCATTT-3�;Ifn� F, 5�-CCACAGCCCTCTCCATCAAC-3�, R, 5�-TGAAGTCCGCCCTG-TAGGTG-3�; Irf4 F, 5�-AGCTCATCACAGCTCATGTGGA-3�, R, 5�-GCGGT-GGTAATCTGGAGTGGT-3�.

Gene expression was quantitated relative to Hprt using the PlatinumSybrGreen quantitative PCR (qPCR) system (Invitrogen) according to themanufacturer’s instructions, using an ABI Prism 7000 sequence detectionsystem (Applied Biosystems, Foster City, CA, USA). Each cDNA sample wasdetected in experimental triplicate. Negative control reactions were cycledalongside test samples to ensure the absence of contaminating genomic DNA.Data were analyzed using the ABI Prism software, and expression was deter-mined relative to Hprt mRNA. Transcript abundance (gene/Hprt) and SD werecalculated as recommended by Applied Biosystems. Unless otherwise stated,error bars in figures indicate the SD of triplicate cDNA quantitations in thesame thermal cycler run.

Surface CSF-1 receptor (CSF-1R) quantitation

Cells for flow cytometric quantification of surface CSF-1R expression wereprepared using prechilled solutions, and all centrifugation steps were carriedout at 4°C. Cells were harvested in lift buffer (1 mM EDTA, 0.1% azide inPBS), washed thoroughly, pelleted, and incubated in 50 l 2.4G2 hybridomamedium (rat anti-FcR mAb) for 20 min. Cells were pelleted and resuspendedin 100 l 0.1% BSA/0.1% azide in PBS (PBA) containing 4 g/ml ratantimouse CSF-1R (IgG1) antibody conjugated to PE or the rat IgG1-PEisotype control (Serotec, Oxford, UK). Samples were incubated for 45 min inthe dark before addition of 1 ml PBA. Cells were pelleted, washed again with

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1 ml PBA, and resuspended in 500 l PBA, and surface CSF-1R expressionwas quantitated using a FACSCalibur (BD Biosciences, San Jose, CA, USA)and CellQuest software (BD Biosciences).

5� RACE

The RACE PCR was performed as specified by the manufacturer (Invitrogen).Briefly, 5 g total spleen RNA was treated with DNase I and reverse-transcribed. The 5� end of mouse Tlr9 was PCR-amplified using the GeneR-acer forward primer and a Tlr9-specific reverse primer (5�-AGCCAGGAAG-GTTCTGGGCTCAA-3�, which binds to the second exon of the Tlr9 gene).Extension products were gel-purified and topoisomerase (TOPO)-cloned intopCR4-TOPO vector (Invitrogen). Clones were sequenced by automated dsDNAsequencing using the ABI PRISM BigDye sequencing kit and the ABI PRISM7000 sequence detection system (Applied Biosystems) at the Australian Ge-nome Research Facility (University of Queensland, St. Lucia).

DNA manipulation

The Tlr9 promoter was PCR-amplified from genomic DNA (primers: F, 5�-AACTCAACTCTGGGTAAAAGATGG-3�, R, 5�-GGTTCCATGAGTGTC-CCAAG-3�), and the 2158-bp PCR product was partially digested with BsaI,the DNA overhangs were blunted, and the fragment was digested further withXbaI. The blunt end of the restriction fragment was ligated into the SmaI siteof pGL2B, and the cohesive, XbaI-digested site was ligated into the compatibleNheI-digested site of pGL2B to form the plasmid pGL2B_Tlr9p_2 kb. The100-bp, proximal Tlr9 promoter inserts were PCR-amplified from thepGL2B_Tlr9p_2-kb plasmid using the same forward primer and WT or non-homologous reverse primers to introduce mutations (primers: F, 5�-GCTAGC-GACTATGCAAATGATGTGTGACTCAT-3�; WT Tlr9 promoter R, 5�-CA-GAGAATTGAGGAAGTGACACTTTCAC-3�; mutant IRF site Tlr9 promoterR, 5�-CAGAGAATTGAGGAAGTGTCTCTTTCAC-3�; mutant PU.1 site Tlr9promoter R, 5�-CAGAGAATTGAGGTTGTGACACTTTCAC-3�). PCR prod-ucts were blunted, digested with NheI, and inserted into the NheI/blunted BglIIsite of pGL2B. All constructs were sequenced before use.

Promoter activity analysis

RAW 264.7 cells were transfected with endotoxin-free plasmid preparations(plasmid maxi kit, Qiagen) by electroporation in 0.4 cm cuvettes. RAW 264.7cells (5�106) were electroporated (280 V/1 mFd, Gene-Pulser, Bio-Rad,Hercules, CA, USA) with 10 g plasmid in 250 l complete media supple-mented with 10 mM HEPES (Thermotrace, San Diego, CA, USA). For exper-iments in which the promoter activity of multiple plasmids with different sizeswas compared, separate transfections included 10 g of the largest plasmidand molar equivalents of the remaining plasmids. Cells were electroporatedimmediately after the addition of plasmid DNA and washed thoroughly withoutdelay after transfection to limit RAW 264.7 activation by the plasmid DNA[34]. After electroporation, the cells were divided into two to four wells of asix-well tissue-culture plate (Iwaki). Transfected cells were cultured for 24 hbefore cellular harvest or treatment with IFN-� or LPS. Cellular harvest andluciferase activity quantitation were performed using the high-sensitivity lu-ciferase reporter gene assay kit (Roche, Basel, Switzerland) according to themanufacturer’s instructions. Luciferase activity was normalized to the totalprotein concentration to give relative light units (RLU) per g. The totalprotein concentration of transfected cell lysates was determined by the Brad-ford assay kit (Bio-Rad).

EMSA

Nuclear extracts for EMSA were prepared by hypotonic lysis at 4°C. All bufferscontained phosphatase inhibitors (1 mM sodium vanadate, 1 mM sodiumpyrophosphate, 1 mM sodium molybdate, 10 mM sodium fluoride) and a 1�protease inhibitor cocktail (complete, mini, EDTA-free, Roche) in all steps ofnuclear extract preparation. Cells were harvested and washed twice in PBS.The pellet was resuspended in 1 ml per 10 � 106 cells Buffer A (10 mMHEPES, pH 7.6, 10 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT,0.5 mM PMSF). Buffer B [0.5 ml per 10�106 cells; 25 mM HEPES, pH 7.6,150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.6% Nonidet P-40 (NP-40), 2 mMDTT, 1 mM PMSF] was added, and the solution was vortexed immediately atlow speed for 30 s. The nuclei were pelleted and resuspended in 100 l per10 � 106 cells Buffer C (25 mM HEPES, pH 7.6, 350 mM NaCl, 1 mM EDTA,

1 mM EGTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF) and shaken vigorouslyfor 20 min. Cellular debris was removed by centrifugation, and nuclear extractswere aliquoted and stored at –80°C. Total protein in each extract was quan-titated and normalized before the binding reaction.

Oligonucleotide end-labeling was performed according to the manufacturer’sinstructions for T4 polynucleotide kinase (New England Biosciences, Beverly,MA, USA). MgCl2 was added to a final concentration of 5 mM, and strands wereannealed by heating the sample to 80°C for 2 min and allowing it to cool downslowly to room temperature. Probes were purified using a Nick Sephadexcolumn (Amersham Biosciences, Piscataway, NJ, USA). Complementary oli-gonucleotides used for ds probe preparation were as follows (bold depicts mutatednucleotides): WT Tlr9 promoter, 5�-GTAGTGAAAGTGTCACTTCCTCAAT-TCTCTGAGA-3�, 5�-TCTCAGAGAATTGAGGAAGTGACACTTTCACTAC-3�;mutant IRF site Tlr9 promoter, 5�-GTAGTGAAAGAGACACTTCCTCAAT-TCTCTGAGA-3�, 5�-TCTCAGAGAATTGAGGAAGTGTCTCTTTCACTAC-3�;mutant PU.1 site Tlr9 promoter, 5�-GTAGTGAAAGTGTCACAACCTCAAT-TCTCTGAGA-3�, 5�-TCTCAGAGAATTGAGGTTGTGACACTTTCACTAC-3�.

Nuclear extract proteins were bound to probe in a 10 l reaction containing20 mM HEPES, pH 7.9, 0.5 mM DTT, 2 mM EDTA, 40 mM KCl, 12%glycerol, and 1 g polyinosinic-polycytidylic (Sigma Aldrich), 0.04 pmolpurified probe, and 2 g nuclear extract. Binding reactions were incubated for30–60 min at room temperature. Reactions in which competitor probes wereadded included ten-, 50-, or 100-fold molar excess of unlabeled, ds competitorprobe (0.4, 2, or 4 pmol, respectively). The supershift reaction included 2 grabbit polyclonal antimouse PU.1 antibody (Santa Cruz Biotechnology, SantaCruz, CA, USA).

The Tris-glycine-EDTA gel system was used for size separation. Acrylamide(8%; 29:1) gels were cast by 0.1% w/v ammonium persulfate and 0.2%N,N,N�,N�-tetramethylethylenediamine in 0.15 M Tris-HCl, pH 8.8. Gels wereelectrophoresed in 1� 25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.2 mMEDTA (TGE buffer) at 100 V, fixed in 10% acetic acid for 15 min, dried, andexposed to X-ray film (Fuji, Tokyo, Japan).

Chromatin immunoprecipitation (ChIP) assay

Cells (20�106) in media were cross-linked by incubation for 10 min withformaldehyde to a final concentration of 1%. The reaction was quenched byaddition of glycine to a final concentration of 0.125 M. Unless otherwise stated,all further steps of ChIP were performed at 4°C with prechilled solutions. Afterwashing twice with PBS, the cells were harvested, resuspended in 1 ml lysisbuffer (85 mM KCl, 1 mM EDTA, 1% NP-40, 10 mM HEPES, pH 7.9, 1 mMPMSF, 1� Roche complete protease inhibitor), and incubated on ice for 15min. The nuclei were pelleted, resuspended in homogenization buffer (50 mMTris, pH 8.1, 1% SDS, 0.5% Empigen BB, 10 mM EDTA, 1 mM PMSF, 1�Roche complete protease inhibitor), and sonicated for 14 pulses of 45 sduration. The lysate was cleared by centrifugation for 10 min at 16,000 g, andpart of the supernatant was kept as 10% input for normalization. Lysate wasdiluted 2:3 with buffer (20 mM Tris, pH 8.1, 100 mM NaCl, 2 mM EDTA, 0.5%Triton X-100, 1� Roche complete protease inhibitor) and precleared with 25l salmon sperm DNA/Protein A agarose-50% slurry (Upstate Biotechnology,Lake Placid, NY, USA) per 200 l-diluted lysate by rotation for 30 min.Protein A agarose was removed by exclusion from a Millipore Ultrafree MCspin column. Precleared lysate (200 l) was incubated with 3 g antibody(rabbit polyclonal antimouse PU.1 antibody and IgG rabbit isotype control,Santa Cruz Biotechnology) and rotated overnight to allow binding. Complexeswere recovered by adding 50 l salmon sperm DNA/Protein A agarose-50%slurry and rotating for 2 h. Complexes were washed in Millipore Ultrafree MCspin columns by sequential application of wash buffer 1 (20 mM Tris/HCl, pH8.1, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), Wash Buffer2 (10 mM Tris/HCl, pH 8.1, 250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mMEDTA), Wash Buffer 3 (10 mM Tris/HCl, pH 8.1, 250 mM LiCl, 1% NP-40,1% deoxycholate, 1 mM EDTA), and two applications of 10 mM Tris-HCl, pH8.0, 1 mM EDTA. The complexes were recovered from protein A agarose byeluting with 200 l elution buffer (0.1 M NaHCO3, 1% SDS). NaCl was addedto eluates and 10% input samples to a final concentration of 190 mM, and thecross-links were reversed by incubation at 65°C for 5 h. Protein was digestedby incubation with 20 g proteinase K for a further 30 min at 65°C. DNA waspurified using a Qiaquick PCR purification column, and DNA was analyzed byqPCR as described above, using primer pairs that amplified the IRF/PU.1 site(F, 5�-TGCTCTTTCAGGGTAGGGACA-3�; R 5�-GGCGGCAGAGAATGAT-

Schroder et al. Regulation of mouse Tlr9 gene expression 1579

GTTC-3�) or the upstream PU.1 site (F, 5�-CATTGACCTTGGGCTGGAGT-3�;R, 5�-GGCAGACAGATAACCCCCTCA-3�) of the Tlr9 promoter.

RESULTS

IFN-� and LPS induce Tlr9 mRNA in mousemacrophages through divergent mechanisms

During pathogenic challenge, macrophages are likely to detectCpG DNA in combination with bacterial products such as LPSand proinflammatory cytokines such as IFN-�. As LPS andIFN-� can synergize with CpG DNA to activate proinflamma-tory gene expression in macrophages, we assessed whetherthese factors could influence Tlr9 mRNA expression in mac-rophages. A mechanism for priming TLR9-dependent re-sponses by IFN-� might be an up-regulation of TLR9 itself, asreported for human TLR4 [35, 36]. Indeed, IFN-� induced Tlr9expression approximately tenfold in the presence of CSF-1 inBMM (Fig. 1A). In the absence of CSF-1, basal expression ofTlr9 mRNA was elevated as reported previously [37, 38] andwas not significantly induced further by IFN-�. We have shownrecently that LPS induced a group of coregulated genes includ-ing Tlr9 in BMM only in the presence of CSF-1 [38], and we

confirmed that finding here (Fig. 1B). In the case of LPS, Tlr9mRNA induction was mediated by LPS-induced down-regulation of cell surface CSF-1R, which alleviates the repressiveeffect of CSF-1 on Tlr9 gene expression [38]. To determinewhether a similar mechanism accounted for induction byIFN-�, we compared the effects of LPS and IFN-� on cellsurface CSF-1R expression in BMM. IFN-� decreased surfaceCSF-1R expression only marginally (Fig. 1, C–F), whereas LPStriggered a rapid and sustained down-regulation of cell surfaceCSF-1R (Fig. 1, G–J) as expected. Such data suggest that analternative mechanism accounts for Tlr9 mRNA induction byIFN-�. In keeping with this hypothesis, Figure 2 shows thatIFN-� but not LPS induced Tlr9 mRNA in the murine macro-phage cell line RAW264.7, which expresses low levels of cellsurface CSF-1R [39]. By Northern blotting, two Tlr9 mRNAtranscripts can be detected [2], and we found that both tran-scripts were coregulated by CSF-1, LPS, and IFN-� (data notshown). It is interesting that in comparison with Tlr9, Tlr4induction by IFN-� in mouse BMM was modest (data notshown), despite published data suggesting this as a majormechanism for IFN-� priming of LPS responses in humanmonocytes and macrophages [35, 36].

Fig. 1. IFN-� and LPS induced Tlr9 expression through different mechanisms. BMM were differentiated and maintained overnight in the presence or absenceof CSF-1 before treatment with (A, C–F) IFN-� or (B, G–J) LPS. (A, B) Tlr9 expression relative to Hprt was measured by qPCR in experimental triplicate. Datapoints represent the average of triplicate quantitations SD. Profiles are representative of at least three independent experiments. (C–J) Surface CSF-1R wasstained using flow cytometry. Histogram overlays represent antibody (Ab) isotype-control staining (dashed lines) and CSF-1R staining before (unfilled histograms)or after (filled histograms) treatment with IFN-� or LPS. Profiles are indicative of two independent experiments.


IFN-� regulates Tlr9 expression at the promoterlevel in murine macrophages

To determine whether IFN-� regulates Tlr9 expression at thepromoter level, we isolated the murine Tlr9 promoter. We firstmapped the transcription start sites by 5� RACE (Fig. 3A).The most 5� start site (designated 1, eight out of 18 clones)was 88 bp upstream of the translation start ATG, and a secondmajor transcription start site was located at 29 (six out of 18clones). A minor transcription start was located at 32 (two of18 clones). 5� RACE analysis was verified independently usingcap analysis gene expression (data not shown) [40]. In transienttransfection analysis, an �100-bp proximal promoter (located–94 to 9 bp) displayed activity �300-fold greater than thepGL2B empty vector and had a similar activity to a 5�-ex-tended, �2 kb promoter construct (–1934 to 9 bp; Fig. 3B).As with expression of the endogenous gene, the Tlr9 promoterwas responsive to IFN-� and was induced threefold after 24 hIFN-� treatment (Fig. 3C). The IFN-� responsiveness wasapparent in the minimal 100-bp promoter construct, and arange of intermediate deletions between 100 bp and 2 kb hadno significant impact on constitutive or IFN-�-induced activity

(data not shown). The 100-bp proximal promoter is highlyhomologous in sequence to the human TLR9 promoter se-quence (Fig. 3A).

A composite IRF/PU.1 site is necessary forregulated Tlr9 expression in murinemacrophages

A recent study of the human TLR9 promoter identified PU.1,C/EBP, and CRE elements, which regulate gene expression[30]. Both of the PU.1-binding sites are completely conservedbetween mice and humans, and one PU.1 site overlaps themajor transcription start site in the mouse (Fig. 3A). This siteis adjacent to a consensus IRF half-site. Composite PU.1/IRFsites have been implicated in IFN-�-inducible gene expressionfor a number of genes (reviewed in ref. [41]), and the PU.1/IRFsite overlapping the mouse Tlr9 transcription start site has highsequence homology with such sites (Fig. 4A). Mutations in thePU.1 and IRF sites in the context of the 100-bp proximalpromoter were generated (Fig. 4B), and the effects of thesemutations on basal and IFN-�-induced promoter activity wereanalyzed (Fig. 4, C and D). Mutation of the PU.1 site reducedbasal promoter activity dramatically but did not affect induc-tion by IFN-� (Fig. 4, C and D). EMSA analysis confirmed thatPU.1 bound strongly to this site in the Tlr9 promoter and thatthe corresponding oligonucleotide with a mutation in the PU.1-binding region did not bind PU.1 (Fig. 5, A and B). Mutationof the IRF site did not affect PU.1 binding, suggesting thatPU.1 binding was independent of occupancy at the IRF site(Fig. 5B). ChIP analysis demonstrated further that PU.1 boundthis site in BMM in vivo (Fig. 5C) and also bound the otherconserved PU.1 site (–130 to –125) further upstream (Fig. 5D).Mutation of the IRF site also decreased basal promoter activity(Fig. 4C) and in contrast to mutation of the PU.1 site, attenu-ated early (6 h) induction of the Tlr9 promoter by IFN-� (Fig.4D). Delayed induction (24 h) did not require the IRF site.Despite the contribution of the IRF site for basal and IFN-�-inducible promoter activity, EMSA analysis using the PU.1/IRF site did not identify IFN-�-regulated binding proteins(Fig. 5A). We suggest that the EMSA conditions used did notmimic the in vivo situation, which occurs during IFN-� induc-tion of Tlr9 transcription. The IRF half-site, which contributesto IFN-� induction of the murine Tlr9 promoter, is not con-served in the human TLR9 promoter (Fig. 3A).

ICSBP regulates basal and IFN-�-inducible Tlr9expression in murine macrophages

Of the IRFs, only IRF4 (PU.1 interaction partner/lymphocyte-specific IRF/IFN consensus sequence-binding activated T cell)and ICSBP are known to interact with PU.1 to bind compositeIRF/PU.1 sites. The expression patterns of IRF4 and ICSBPacross IFN-� treatment of BMM and RAW 264.7 were inves-tigated to determine if either was coregulated with Tlr9 mRNA.qPCR analysis showed a strong correlation between the ex-pression of Icsbp and Tlr9 (Fig. 6, A and B). Like Tlr9, basalIcsbp mRNA expression was greatest in CSF-1-starved BMM,least in RAW 264.7, and intermediate in CSF-1-replete BMM.Similarly, Icsbp induction by IFN-� was greatest in RAW264.7and CSF-1-replete BMM and weak in CSF-1-starved BMM. By

Fig. 2. Tlr9 mRNA regulation in RAW 264.7 replicated BMM Tlr9 regulationby IFN-� but not LPS. The RAW 264.7 cell line was treated with (A) IFN-�or (B) LPS. Tlr9 expression relative to Hprt was measured by qPCR inexperimental triplicate. Data points represent the average of triplicate quan-titations SD. Profiles are representative of at least three independent exper-iments.


Fig. 3. Characterization of the mouse Tlr9 promoter. (A) Clustal W alignment of the human TLR9 promoter with the putative mouse Tlr9 promoter. Functional transcriptionfactor-binding sites, TATA box and transcription (C1), and translation (*ATG) start sites demonstrated for the human gene [30] are included. Potential transcription factor-bindingsites conserved between human and mouse are shown in dark gray. This includes the AP-1 site (–76 to –69) identified by others to contribute to basal activity of the mouse promoter[31]. Evolutionary, conserved regions (as identified by the Evolutionary Conserved Region browser, http://ecrbrowser.dcode.org) are shown in light gray. The major (1, 29) andminor (32) transcription start sites determined by 5� RACE for the mouse Tlr9 gene are annotated with arrows. Human and mouse Tlr9 gene sequences are numbered relativeto the most 5� (1) transcription start site for that species. (B and C) A 2-kb genomic sequence (–1937 to 9 bp) and a 100-bp 5� deletion (–94 to 9 bp) were compared forpromoter activity by transfection analysis of promoter-luciferase constructs. RAW 264.7 cells were transfected, and each transfection was split into multiple wells. One well washarvested at 24 h post-transfection for calculation of promoter basal activity. Cells in the other wells were left untreated or stimulated for a further 24 h with IFN-� before harvestat 48 h post-transfection. Each construct was electroporated in triplicate transfections within a single experiment. Luciferase activity was assayed to calculate RLU per g totalprotein. (B) Comparison of basal activities of the 100-bp and 2-kb Tlr9 promoters against the pGL2B control plasmid. The mean and SE were calculated from four independentexperiments. (C) Comparison of the inducible activities of the 100-bp and 2-kb Tlr9 promoters. Activity is expressed as average SD of RLU/g-fold induction by IFN-� overunstimulated cells from the same transfection. Data shown are representative of three independent experiments.


contrast, Irf4 was expressed poorly in these cells, and itsexpression was not induced by IFN-� in CSF-1-replete BMMor RAW264.7 (Fig. 6C), consistent with published data [43].Because of the apparent coregulation of Tlr9 and Icsbp expres-sion, we next investigated whether ICSBP contributes to basaland/or IFN-�-inducible Tlr9 mRNA expression in murine mac-rophages using ICSBP-deficient mice. Basal levels of Tlr9mRNA were reduced 18-fold in ICSBP-deficient macrophages(Fig. 7A). In addition, early but not late induction of Tlr9mRNA by IFN-� was impaired in ICSBP-deficient macro-phages (Fig. 7B). These results are consistent with the effect ofmutation of the IRF site in the Tlr9 promoter (Fig. 4D).

Type I IFNs induce Tlr9 expression in BMM

The observation that IRF site mutation or ICSBP deletionaffected early but not delayed Tlr9 mRNA induction by IFN-�indicated that a separate mechanism was responsible for thelate-stage induction. Given the time course, we hypothesizedthat an autocrine factor would be involved. Type I IFNs aremajor autocrine regulators of gene expression during macro-phage activation [44], and Figure 8A shows that IFN-� in-duced Ifn� expression in CSF-1-replete BMM and RAW264.7cells. We therefore assessed whether Type I IFN induced Tlr9expression in BMM. Figure 8B shows that IFN-� potently

induced Tlr9 expression and that this effect was, at least inpart, independent of ICSBP. Thus, Type I IFNs may contributeto the sustained induction of Tlr9 by IFN-�.

DISCUSSION

In this study, we set out to define pathways by which IFN-� andLPS prime macrophage responses to CpG DNA and in doingso, have comprehensively analyzed the regulation of the mu-rine Tlr9 gene. We found that transcriptional regulation of themouse Tlr9 gene is geared toward promoting macrophage rec-ognition and response to bacteria when in the presence ofextracellular “danger” signals provided by the host (IFN-� andIFN-�) and the invading pathogen (LPS). Our findings alsoprovide a molecular mechanism for the well-documented abil-ity of IFN-� to prime macrophage responses to CpG DNA [19].Although IFN-� and LPS induced Tlr9 mRNA, there werefundamental differences in the mechanisms used. LPS primar-ily induced Tlr9 mRNA by down-regulation of surface CSF-1Rand blockade of CSF-1 signaling (Fig. 1, G–J; ref. [38]),although LPS-induced IFN-� is also likely to contribute toregulation (Fig. 8B). Conversely IFN-� did not greatly regulatecell surface CSF-1R (Fig. 1, C–F) but up-regulated activity of

Fig. 4. The Tlr9 promoter contains a consensus IRF/PU.1 motif. (A) The mouse Tlr9 promoter contains a sequence (reverse-complemented in this figure)homologous to composite PU.1/IRF sites functional in the regulation of a number of genes [41, 42]. (B) The PU.1 and IRF sites of the predicted IRF/PU.1 motifwere mutated within the context of the 100-bp promoter for use in transfection analysis. (C and D) RAW 264.7 cells were transfected with promoter mutants, andeach transfection was split into three wells. One well was left untreated, and the other two wells were treated with IFN-� for 6 and 24 h. All wells were harvestedat 48 h post-transfection. Each construct was electroporated in triplicate transfections within a single experiment. Luciferase activity was measured and normalizedto total protein to calculate RLU per g. Data points represent the average of triplicate quantitations SD. Profiles are representative of at least three independentexperiments. (C) Basal promoter activities of Tlr9 promoter mutants depicted in B were determined at 48 h post-transfection. (D) IFN-�-inducible promoteractivities of Tlr9 promoter mutants depicted in B were determined at 48 h post-transfection.


the Tlr9 promoter (Fig. 3C). Indeed, IFN-� but not LPS in-duced Tlr9 mRNA in the RAW264.7 cell line (Fig. 2), whichexpresses low levels of CSF-1R [39]. The dramatic (�65-foldat 21 h) up-regulation of Tlr9 expression by IFN-� inRAW264.7 cells presented here contrasts with another report[31], which showed only slight induction (approximately three-fold at 24 h) and may reflect differences in cell culture proto-cols.

A number of studies have highlighted differences betweenmice and humans in the recognition of CpG motifs by TLR9.Human and mouse TLR9 have differences in the optimalCpG-containing sequence, which they recognize [13, 45]. How-ever, we showed recently that this difference is restricted tophosphorothioate-modified oligonucleotides and does not occurwith natural phosphodiester DNA [46]. Hence, human andmouse TLR9 have not evolved to recognize different DNAsequences during pathogen challenge. The expression patternsof TLR9 between mice and humans have also been reported to

be divergent. In particular, a number of studies suggest thatTLR9 is expressed in myeloid cells in mice but not humans [2,11–13]. This is still an area of debate, as the human/mousecomparisons are somewhat confounded by a lack of directlycomparable data; the human myeloid cells used for theseinvestigations are generally human monocytes, and the mousemyeloid cells often studied are myeloid DCs, peritoneal mac-rophages, BMM, or cell lines such as RAW264.7. We havereported previously that CSF-1 down-regulates Tlr9 expressionin murine macrophages [37], implying that mature murinetissue macrophages would be unlikely to express TLR9 andwould be unresponsive to CpG DNA. Indeed, we (unpublishedobservations) and others [47] have shown that resident orthioglycollate-elicited peritoneal macrophages are poorly re-sponsive to CpG DNA and express low levels of TLR9. This isin contrast to other publications and may reflect differentiallevels of circulating Type I and Type II IFNs in mice fromdifferent colonies or mouse strains; we show here that IFN-�

Fig. 5. The composite IRF/PU.1 site bound PU.1 in EMSA and ChIP. Nuclear extracts were prepared from IFN-�-stimulated RAW 264.7 and BMM maintainedin CSF-1 or starved of CSF-1 overnight. Nuclear extracts were incubated with a P32-labeled probe containing the Tlr9 promoter IRF/PU.1 site, and the samplewas size-fractionated on 8% polyacrylamide TGE gels and exposed to X-ray film. (A) Nuclear extracts from RAW 264.7 and CSF-1-replete or -starved BMM boundto the native IRF/PU.1 site in the Tlr9 promoter. Addition of a PU.1 antibody to the binding reaction supershifted (SS) the protein:probe complex. (B) EMSA studieswith unlabeled competitor probes confirmed PU.1 bound to the consensus PU.1 site. Nuclear extracts from RAW 264.7 cells stimulated with IFN-� for 10 h werebound to probe cocktails containing unvarying amounts of labeled probe but zero-, ten-, 50-, or 100-fold molar excess of unlabeled competitor probes. The WTcompetitor probe sequence was identical to the labeled probe. The mutant probes varied from the labeled probe in that the IRF or PU.1 half-sites were mutated(as in Fig. 4B). (A and B) Profiles are representative of six independent experiments using four independent preparations of nuclear extracts for each cell type.ChIP assay using an anti-PU.1 antibody to detect PU.1 binding to (C) the PU.1/IRF site (–12 to –1) or (D) the upstream PU.1 site (–130 to –125) of the Tlr9promoter. Data points correspond to the average SD of triplicate qRT-PCR quantitations within a single experiment. Data are representative of two independentexperiments comparing anti-PU.1 antibody to a matched isotype control antibody.


and IFN-� potently induced Tlr9 mRNA in mouse macro-phages (Figs. 1A and 8B), and the effect of IFN-� is alsoapparent in thioglycollate-elicited peritoneal macrophages (un-published observations).

Divergence in regulation of Tlr promoters between mice andhumans is well known. For example, although TLR4 is ex-pressed predominantly in myeloid cells and B cells in humansand mice, basal tissue expression between the species is quitedistinct. LPS induces TLR4 expression in human monocytes/macrophages but represses TLR4 expression in mouse macro-phages (reviewed in ref. [48]). This is despite highly homolo-gous promoter regions between the species. The human andmouse Tlr4 promoters resemble typical myeloid promoters inthat they are short, TATA-less, harbor multiple PU.1 sites, andcontain clustered transcription start sites [42, 48]. A recentcharacterization of the human TLR9 promoter showed thatPU.1, C/EBP, and CRE elements were important for expressionin the human B cell line, RPMI 8226. Our study analyzed themurine promoter in a murine macrophage cell line. As with theTlr4 promoter, there are likely to be differences in Tlr9 generegulation between humans and mice, as well as differencesbetween B cells and macrophages. Consequently, we found thatmany of the elements required for activity of the human pro-moter in B cells were dispensable for maximal promoter activ-

Fig. 7. Basal and IFN-�-inducible Tlr9 expression was compromised inICSBP-deficient BMM. BMM from WT and ICSBP-deficient (ICSBP�/�) micewere prepared and stimulated with IFN-� in the presence of CSF-1. Tlr9mRNA was measured by qPCR in experimental triplicate. The profiles de-picted represent the mean and SE calculated from quantitation of three inde-pendent RNA samples. (A) Tlr9 expression relative to Hprt in unstimulatedBMM; (B) Tlr9 expression relative to Hprt in IFN-�-stimulated BMM, ex-pressed as fold induction over basal expression within the genotype at thattime-point.

Fig. 6. Tlr9 and Icsbp were coregulated across CSF-1 and IFN-� stimulation.BMM were prepared and maintained overnight in the presence or absence ofCSF-1. RAW 264.7 cells and CSF-1-replete or -starved BMM were treatedwith IFN-� for 7 or 24 h. mRNAs for (A) Tlr9, (B) Icsbp, and (C) Irf4 werequantified by qPCR from the same cDNA time series in experimental tripli-cate. Data shown are the average SD of triplicate quantitations within a singleexperiment. Profiles are representative of at least two experiments usingindependent RNA samples.


ity in murine macrophages (Fig. 3). This included the 5� PU.1box (–130 to –125), which although it bound PU.1 in BMM(Fig. 5D), did not contribute significantly to basal promoteractivity as seen by transfection analysis of 5� promoter dele-tions in RAW264.7 (Fig. 3B). We did find that a conservedPU.1 site overlapping the murine transcription start site thatwas important for basal activity in human B cells and was alsorequired for optimal basal activity in murine macrophages (Fig.4C). It remains possible that the 5� and 3� PU.1 sites arefunctionally redundant, and occupancy at only one site isrequired for maximal basal expression. We also identified afunctional IRF half-site adjacent to the 3� PU.1 site in themurine promoter, which was not conserved in the humanpromoter (Fig. 3A). The IRF/PU.1 site and the upstream PU.1site identified in the present study were not identified in aprevious report [31], which demonstrated only that an AP-1site in the proximal promoter contributed to basal Tlr9 expres-sion in mouse macrophages. The binding of AP-1 to the prox-imal Tlr9 promoter was also confirmed in the present study(data not shown). Apart from the nonconserved IRF site, themouse and human Tlr9 promoters differ further; the mouse

promoter, unlike its human counterpart, lacks a TATA box andis structurally typical of myeloid promoters. Like many othermacrophage-specific promoters such as c-fms and FC�R1B[49, 50], the transcription start sites of Tlr9 cluster around thePU.1 site. The transcription initiation of TATA-less promotersis not well-characterized; however, there is evidence that atleast some TATA-less promoters form the preinitiation com-plex upon pyrimidine-rich initiator (Inr) motifs of consensusC/T

C/TA(1)NT/AC/T

C/T [51–53]. PU.1 has been shown to in-teract in vitro with transcription factor IID [54], a generaltranscription factor necessary for Inr-mediated transcription inthe absence of a TATA box. The sequence flanking the mouseTlr9 A 1 conforms to the Inr consensus, and the contiguousupstream sequence is a functional PU.1 site. This putative Inrmotif, perhaps in concert with PU.1, may interact with anInr-binding protein to tether the preinitiation complex to theDNA and drive transcription. Such differences in promoterarchitecture are likely to contribute to differential Tlr9 expres-sion in the myeloid compartment in mouse versus humans.

We confirmed that the IRF site in the murine proximalpromoter was required for basal activity and was partly re-quired for induction of promoter activity by IFN-� using trans-fection analysis with a mutant promoter construct. The possi-bility that the effect of this mutation on basal activity wasmediated by interference with PU.1 binding to the adjacent siteis unlikely, as the mutated IRF site could still bind PU.1 inEMSA analysis (Fig. 5B). The adjacent PU.1 site was dispens-able for IFN-� induction, and in fact, mutation of this sitemodestly enhanced IFN-� induction (Fig. 4D). It is possiblethat the Tlr9 promoter IRF/PU.1 site may be atypical in thatrather than acting in synergy, PU.1 binding prevents optimalIRF interaction at this site. This IRF site is distinct from theISRE/IRF sites identified by others in an upstream enhancerelement [31], and it should be noted that none of the Tlr9promoter constructs used in the present study contained theseenhancer sites. This suggests that the effect of IFN-� is medi-ated via the proximal promoter IRF/PU.1 site, acting alone orin combination with the upstream enhancer. IRF4 and ICSBPare the only IRFs reported to be able to bind PU.1 at compositeIRF/PU.1 sites. Although IRF4 and ICSBP are closely related,the phenotypes of the knockout mice demonstrated that theyperform nonredundant functions in vivo [55–58]. They canboth function as transcriptional activators or repressors, de-pending on cell type and sequence context. Typically, tran-scriptional repression occurs when heterodimeric complexes ofIRF4 or ICSBP interacting with other IRFs bind to ISRE.However, IRF4 or ICSBP interaction with PU.1 and perhapsother IRFs upon composite IRF/PU.1 sites tend to result inpromoter activation (reviewed in ref. [41]). The Icsbp, but notIrf4, mRNA expression pattern was coregulated with Tlr9mRNA (Fig. 6), and deletion of the Icsbp gene dramaticallyimpaired basal Tlr9 expression as well as early Tlr9 mRNAinduction by IFN-�, suggesting that ICSBP binds together withPU.1 at the composite IRF/PU.1 site. ICSBP is expressedprimarily by B cells and macrophages [59–61], and as in thisstudy, others have found that it is highly IFN-�-inducible atthe mRNA and protein level in macrophages [59, 62–64]. Infact, nuclear protein levels of ICSBP, but not IRF4, wereinduced 26-fold by IFN-� in RAW264.7, while nuclear levels

Fig. 8. IFN-�-dependent induction of Tlr9 was partially ICSBP-independent.(A) RAW 264.7 and CSF-1-replete BMM were left untreated or stimulated withIFN-� for 7 and 21 h. Expression of Ifn� mRNA relative to Hprt was measuredby qPCR. (B) BMM from WT and ICSBP-deficient (ICSBP�/�) mice wereprepared and stimulated with IFN-� in the presence of CSF-1. Tlr9 mRNA wasmeasured by qPCR and expressed as fold induction over basal expressionwithin the cell type or treatment. Data points represent the mean and SE

calculated from three experiments using independent RNA samples.


of PU.1 remained unchanged [61]. Furthermore, we foundexpression levels of ICSBP to be at least tenfold higher thanIRF4 in resting macrophages and at least 100-fold higher inIFN-�-stimulated macrophages at the mRNA level (Fig. 6).Despite this evidence for ICSBP in regulating Tlr9 expression,we were unable to detect ICSBP interaction with the Tlr9promoter or other reported targets by EMSA or ChIP assay inthis study (data not shown). This is not entirely unexpected, asfew reports demonstrate binding of an IRF/PU.1 complex toDNA by EMSA analysis using nuclear extracts from untrans-fected cells; instead, many used in vitro-translated protein[65–68] or nuclear extracts from transfected cells [69]. Thissuggests that native IRF/PU.1/DNA complexes are difficult toresolve for reasons of sensitivity or instability. The slightsequence deviation in the Tlr9 promoter IRF half-site from theconsensus (TGACA instead of TGAAA; Fig. 4A) may haveweakened the interaction between ICSBP and DNA and addedto difficulties in detecting the complex. Although our datasuggest that IFN-� activates the Tlr9 promoter via ICSBPinteraction with the IRF/PU.1 site, we cannot discount thepossibilities that ICSBP regulates Tlr9 expression indirectly(i.e., through mechanisms other than occupation of the Tlr9promoter) or that as found for other promoters [61, 70], IRF4may also bind PU.1 cooperatively at the IRF/PU.1 site. Furtherclarification of the roles of ICSBP and IRF4 in B cell-specificexpression of Tlr9 is required.

Unexpectedly that we found that CSF-1 down-regulatesexpression of Icsbp mRNA in BMM (Fig. 6B). This is likelyto provide a mechanism for CSF-1-mediated repression ofTlr9 expression and downstream, TLR9-dependent re-sponses [37]. This hypothesis is supported further by LPS

induction of Icsbp mRNA, specifically in CSF-1-repleteBMM but not in CSF-1-starved BMM or the CSF-1-unre-sponsive cell lines RAW264.7 and WEHI 231 (unpublishedobservations). In this way, the ICSBP/PU.1 complex mayorchestrate integration of incoming signals from the host andpotential pathogen for appropriate sensitization to CpG mo-tifs. In addition to its transcriptional regulator activities, arecent article suggested that ICSBP may participate moredirectly in TLR signaling, via interaction with TRAF6 [71].In light of the regulation of ICSBP by IFN-� and CSF-1presented in this report, ICSBP may also function to inte-grate signals from CSF-1 and IFN-� directly into the TLRsignaling pathway by modulating TRAF6 function.

The role of ICSBP in regulating Tlr9 gene expression hasimportant implications for functional studies of ICSBP-defi-cient mice. ICSBP�/� mice show increased susceptibility to arange of infections [55, 72–74], presumably as a result of theircompromised ability to respond to IFNs and execute macro-phage effector functions such as induction of gp91phox,gp67phox, inducible NO synthase, IL-18, and IL-1� [70, 75–77]. It is possible that part of the susceptibility to infections ismediated by deficiencies in basal and inducible Tlr9 expres-sion. In addition, a recent study by Tsujimura et al. [78] usingICSBP�/� mice implicated ICSBP as a signaling molecule,which relays TLR9-specific signaling in DCs. We cannot dis-count this possibility, but given the effect of ICSBP deletion onTlr9 expression, it is more likely that ICSBP is required forTlr9 expression, rather than for signaling. This requires furtherclarification.

It is somewhat surprising that induction of Tlr9 mRNA byIFN-� at later time-points was not affected by mutation of the

Fig. 9. Proposed model for Tlr9 promoter regulation. In the absence of signaling by Type I or II IFN, LPS, or CSF-1, the Tlr9 promoter is activated by ICSBP/PU.1,and Tlr9 mRNA is expressed. CSF-1 suppresses transcription of Icsbp mRNA, thereby suppressing Tlr9 mRNA expression. Macrophage stimulation with IFN-�in the presence of CSF-1 activates the Icsbp promoter directly via STAT1 interaction with an IFN-�-activated site [79], and Icsbp expression is induced. InCSF-1-starved macrophages, IFN-� is unable to induce Icsbp mRNA beyond its current maximal level. In addition to direct Icsbp promoter activation, it is possiblethat induction of Icsbp mRNA by IFN-� occurs via an additional mechanism, which involves blockade of CSF-1 action downstream of the CSF-1R. IFN-� inducesthe expression and subsequent autocrine activities of Type I IFN, which is able to further transactivate the Tlr9 promoter through ICSBP-independent mechanisms,possibly including activation of upstream enhancer elements [31]. IFNAR, IFN-� receptor; IFNGR, IFN-� receptor.


IRF site (Fig. 4D) or ablation of the Icsbp gene (Fig. 7B). Thetime course suggests that an autocrine factor is responsible forthis effect, and we provide evidence for the involvement ofIFN-� (Fig. 8), although this cannot be concluded definitively.An IFN-�-responsive element was mapped by others to anenhancer element �2.4 kb upstream of the transcription startsite [31], which was not contained in our promoter-reporterconstructs. This suggests that IFN-�-induced IFN-� may pos-itively regulate Tlr9 expression via the upstream enhancer aswell as an uncharacterized promoter-proximal element. A pro-posed mechanism for the IFN-�-mediated induction of Tlr9 viaICSBP and autocrine IFN-� is shown in Figure 9.

In summary, the induction of Tlr9 mRNA by IFN-� in mousemacrophages provides a mechanistic explanation for the prim-ing of CpG DNA responses by IFN-�. In contrast to inductionby LPS, IFN-� targets the proximal promoter to up-regulateTlr9. Our analysis of the murine Tlr9 promoter has also pro-vided further mechanistic knowledge of differential regulationof TLRs between mice and humans.

ACKNOWLEDGMENTS

M. J. S., K. J. S., and D. A. H. are supported by the NationalHealth and Medical Research Council (NHMRC) Project, grantnumbers 301211 and 301210. T. R. is supported by a researchgrant for the Scripps NeuroAIDS Preclinical Studies Center(SNAPS) from the National Institute of Mental Health (NIMH),grant number 2P30MH062261-07. M. L. and M. R. are sup-ported by a grant from the Deutsche Forschungsgemeinschaft(Re1310/7-1). We thank I. Horak for providing the ICSBP�/�

mice used in these experiments.

REFERENCES

1. Stacey, K. J., Young, G. R., Clark, F., Sester, D. P., Roberts, T. L., Naik,S., Sweet, M. J., Hume, D. A. (2003) The molecular basis for the lack ofimmunostimulatory activity of vertebrate DNA. J. Immunol. 170, 3614–3620.

2. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H.,Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., Akira, S. (2000) AToll-like receptor recognizes bacterial DNA. Nature 408, 740–745.

3. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K. A., Monks, B. G.,Knetter, C. F., Lien, E., Nilsen, N. J., Espevik, T., Golenbock, D. T. (2004)TLR9 signals after translocating from the ER to CpG DNA in the lyso-some. Nat. Immunol. 5, 190–198.

4. Leifer, C. A., Kennedy, M. N., Mazzoni, A., Lee, C., Kruhlak, M. J., Segal,D. M. (2004) TLR9 is localized in the endoplasmic reticulum prior tostimulation. J. Immunol. 173, 1179–1183.

5. Ahmad-Nejad, P., Hacker, H., Rutz, M., Bauer, S., Vabulas, R. M.,Wagner, H. (2002) Bacterial CpG-DNA and lipopolysaccharides activateToll-like receptors at distinct cellular compartments. Eur. J. Immunol.32, 1958–1968.

6. Sweet, M. J., Hume, D. A. (1996) Endotoxin signal transduction inmacrophages. J. Leukoc. Biol. 60, 8–26.

7. Akira, S. (2000) Toll-like receptors: lessons from knockout mice. Biochem.Soc. Trans. 28, 551–556.

8. Aderem, A., Ulevitch, R. J. (2000) Toll-like receptors in the induction ofthe innate immune response. Nature 406, 782–787.

9. Dobrovolskaia, M. A., Vogel, S. N. (2002) Toll receptors, CD14, andmacrophage activation and deactivation by LPS. Microbes Infect. 4, 903–914.

10. Wagner, H. (2004) The immunobiology of the TLR9 subfamily. TrendsImmunol. 25, 381–386.

11. Krug, A., Towarowski, A., Britsch, S., Rothenfusser, S., Hornung, V., Bals,R., Giese, T., Engelmann, H., Endres, S., Krieg, A. M., Hartmann, G.(2001) Toll-like receptor expression reveals CpG DNA as a unique mi-crobial stimulus for plasmacytoid dendritic cells which synergizes withCD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31,3026–3037.

12. Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R. W., Kastelein, R. A.,Bazan, F., Liu, Y. J. (2001) Subsets of human dendritic cell precursorsexpress different Toll-like receptors and respond to different microbialantigens. J. Exp. Med. 194, 863–869.

13. Bauer, S., Kirschning, C. J., Hacker, H., Redecke, V., Hausmann, S.,Akira, S., Wagner, H., Lipford, G. B. (2001) Human TLR9 confersresponsiveness to bacterial DNA via species-specific CpG motif recogni-tion. Proc. Natl. Acad. Sci. USA 98, 9237–9242.

14. Saikh, K. U., Kissner, T. L., Sultana, A., Ruthel, G., Ulrich, R. G. (2004)Human monocytes infected with Yersinia pestis express cell surface TLR9and differentiate into dendritic cells. J. Immunol. 173, 7426–7434.

15. Takeshita, F., Leifer, C. A., Gursel, I., Ishii, K. J., Takeshita, S., Gursel,M., Klinman, D. M. (2001) Cutting edge: role of Toll-like receptor 9 inCpG DNA-induced activation of human cells. J. Immunol. 167, 3555–3558.

16. Yi, A. K., Klinman, D. M., Martin, T. L., Matson, S., Krieg, A. M. (1996)Rapid immune activation by CpG motifs in bacterial DNA. Systemicinduction of IL-6 transcription through an antioxidant-sensitive pathway.J. Immunol. 157, 5394–5402.

17. Hochrein, H., O’Keeffe, M., Wagner, H. (2002) Human and mouse plas-macytoid dendritic cells. Hum. Immunol. 63, 1103–1110.

18. Wagner, H. (2002) Interactions between bacterial CpG-DNA and TLR9bridge innate and adaptive immunity. Curr. Opin. Microbiol. 5, 62–69.

19. Sweet, M. J., Stacey, K. J., Kakuda, D. K., Markovich, D., Hume, D. A.(1998) IFN-� primes macrophage responses to bacterial DNA. J. Inter-feron Cytokine Res. 18, 263–271.

20. An, H., Yu, Y., Zhang, M., Xu, H., Qi, R., Yan, X., Liu, S., Wang, W.,Guo, Z., Guo, J., et al. (2002) Involvement of ERK, p38 and NF-�B signaltransduction in regulation of TLR2, TLR4 and TLR9 gene expressioninduced by lipopolysaccharide in mouse dendritic cells. Immunology106, 38–45.

21. Pisetsky, D. S., Reich, C. (1993) Stimulation of in vitro proliferation ofmurine lymphocytes by synthetic oligodeoxynucleotides. Mol. Biol. Rep.18, 217–221.

22. Monteith, D. K., Henry, S. P., Howard, R. B., Flournoy, S., Levin, A. A.,Bennett, C. F., Crooke, S. T. (1997) Immune stimulation—a class effect ofphosphorothioate oligodeoxynucleotides in rodents. Anticancer Drug Des.12, 421–432.

23. Vollmer, J., Weeratna, R. D., Jurk, M., Samulowitz, U., McCluskie, M. J.,Payette, P., Davis, H. L., Schetter, C., Krieg, A. M. (2004) Oligode-oxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9dependent T helper type 2 biased immune stimulation. Immunology 113,212–223.

24. Baek, K. H., Ha, S. J., Sung, Y. C. (2001) A novel function of phospho-rothioate oligodeoxynucleotides as chemoattractants for primary macro-phages. J. Immunol. 167, 2847–2854.

25. Sester, D. P., Brion, K., Trieu, A., Goodridge, H. S., Roberts, T. L., Dunn,J., Hume, D. A., Stacey, K. J., Sweet, M. J. (2006) CpG DNA activatessurvival in murine macrophages through TLR9 and the phosphatidylino-sitol 3-kinase-Akt pathway. J. Immunol. 177, 4473–4480.

26. Van Uden, J. H., Tran, C. H., Carson, D. A., Raz, E. (2001) Type Iinterferon is required to mount an adaptive response to immunostimulatoryDNA. Eur. J. Immunol. 31, 3281–3290.

27. Car, B. D., Eng, V. M., Schnyder, B., Ozmen, L., Huang, S., Gallay, P.,Heumann, D., Aguet, M., Ryffel, B. (1994) Interferon � receptor deficientmice are resistant to endotoxic shock. J. Exp. Med. 179, 1437–1444.

28. Schroder, K., Hertzog, P. J., Ravasi, T., Hume, D. A. (2004) Interferon-�:an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75,163–189.

29. Schroder, K., Sweet, M. J., Hume, D. A. (2006) Signal integration betweenIFN� and TLR signaling pathways in macrophages. Immunobiology 211,511–524.

30. Takeshita, F., Suzuki, K., Sasaki, S., Ishii, N., Klinman, D. M., Ishii, K. J.(2004) Transcriptional regulation of the human TLR9 gene. J. Immunol.173, 2552–2561.

31. Guo, Z., Garg, S., Hill, K. M., Jayashankar, L., Mooney, M. R., Hoelscher,M., Katz, J. M., Boss, J. M., Sambhara, S. (2005) A distal regulatory regionis required for constitutive and IFN-�-induced expression of murine TLR9gene. J. Immunol. 175, 7407–7418.

32. Tushinski, R. J., Oliver, I. T., Guilbert, L. J., Tynan, P. W., Warner, J. R.,Stanley, E. R. (1982) Survival of mononuclear phagocytes depends on a


lineage-specific growth factor that the differentiated cells selectively de-stroy. Cell 28, 71–81.

33. Hume, D. A., Gordon, S. (1983) Optimal conditions for proliferation ofbone marrow-derived mouse macrophages in culture: the roles of CSF-1,serum, Ca2, and adherence. J. Cell. Physiol. 117, 189–194.

34. Stacey, K. J., Ross, I. L., Hume, D. A. (1993) Electroporation andDNA-dependent cell death in murine macrophages. Immunol. Cell Biol.71, 75–85.

35. Mita, Y., Dobashi, K., Shimizu, Y., Nakazawa, T., Mori, M. (2001) Toll-like receptor 2 and 4 surface expressions on human monocytes aremodulated by interferon-� and macrophage colony-stimulating factor.Immunol. Lett. 78, 97–101.

36. Bosisio, D., Polentarutti, N., Sironi, M., Bernasconi, S., Miyake, K., Webb,G. R., Martin, M. U., Mantovani, A., Muzio, M. (2002) Stimulation ofToll-like receptor 4 expression in human mononuclear phagocytes byinterferon-�: a molecular basis for priming and synergism with bacteriallipopolysaccharide. Blood 99, 3427–3431.

37. Sweet, M. J., Campbell, C. C., Sester, D. P., Xu, D., McDonald, R. C.,Stacey, K. J., Hume, D. A., Liew, F. Y. (2002) Colony-stimulating factor-1suppresses responses to CpG DNA and expression of Toll-like receptor 9but enhances responses to lipopolysaccharide in murine macrophages.J. Immunol. 168, 392–399.

38. Sester, D. P., Trieu, A., Brion, K., Schroder, K., Ravasi, T., Robinson,J. A., McDonald, R. C., Ripoll, V., Wells, C. A., Suzuki, H., et al. (2005)LPS regulates a set of genes in primary murine macrophages by antago-nizing CSF-1 action. Immunobiology 210, 97–107.

39. Fowles, L. F., Stacey, K. J., Marks, D., Hamilton, J. A., Hume, D. A.(2000) Regulation of urokinase plasminogen activator gene transcriptionin the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surfacereceptor. Biochem. J. 347, 313–320.

40. Nilsson, R., Bajic, V. B., Suzuki, H., di Bernardo, D., Bjorkegren, J.,Katayama, S., Reid, J. F., Sweet, M. J., Gariboldi, M., Carninci, P., et al.(2006) Transcriptional network dynamics in macrophage activation.Genomics 88, 133–142.

41. Marecki, S., Fenton, M. J. (2000) PU.1/interferon regulatory factor inter-actions: mechanisms of transcriptional regulation. Cell Biochem. Biophys.33, 127–148.

42. Matsuyama, T., Grossman, A., Mittrucker, H. W., Siderovski, D. P.,Kiefer, F., Kawakami, T., Richardson, C. D., Taniguchi, T., Yoshinaga,S. K., Mak, T. W. (1995) Molecular cloning of LSIRF, a lymphoid-specificmember of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). Nucleic Acids Res. 23, 2127–2136.

43. Vadiveloo, P. K., Vairo, G., Hertzog, P., Kola, I., Hamilton, J. A. (2000)Role of type I interferons during macrophage activation by lipopolysac-charide. Cytokine 12, 1639–1646.

44. Hartmann, G., Weeratna, R. D., Ballas, Z. K., Payette, P., Blackwell, S.,Suparto, I., Rasmussen, W. L., Waldschmidt, M., Sajuthi, D., Purcell,R. H., et al. (2000) Delineation of a CpG phosphorothioate oligode-oxynucleotide for activating primate immune responses in vitro and invivo. J. Immunol. 164, 1617–1624.

45. Roberts, T. L., Sweet, M. J., Hume, D. A., Stacey, K. J. (2005) Cuttingedge: species-specific TLR9-mediated recognition of CpG and non-CpGphosphorothioate-modified oligonucleotides. J. Immunol. 174, 605–608.

46. Yasuda, K., Kawano, H., Yamane, I., Ogawa, Y., Yoshinaga, T., Nish-ikawa, M., Takakura, Y. (2004) Restricted cytokine production frommouse peritoneal macrophages in culture in spite of extensive uptake ofplasmid DNA. Immunology 111, 282–290.

47. Rehli, M. (2002) Of mice and men: species variations of Toll-like receptorexpression. Trends Immunol. 23, 375–378.

48. Rehli, M., Poltorak, A., Schwarzfischer, L., Krause, S. W., Andreesen, R.,Beutler, B. (2000) PU.1 and interferon consensus sequence-binding pro-tein regulate the myeloid expression of the human Toll-like receptor 4gene. J. Biol. Chem. 275, 9773–9781.

49. Ross, I. L., Yue, X., Ostrowski, M. C., Hume, D. A. (1998) Interactionbetween PU.1 and another Ets family transcription factor promotes mac-rophage-specific basal transcription initiation. J. Biol. Chem. 273, 6662–6669.

50. Eichbaum, Q. G., Iyer, R., Raveh, D. P., Mathieu, C., Ezekowitz, R. A.(1994) Restriction of interferon � responsiveness and basal expression ofthe myeloid human Fc � R1b gene is mediated by a functional PU.1 siteand a transcription initiator consensus. J. Exp. Med. 179, 1985–1996.

51. Schumacher, M. A., Lau, A. O., Johnson, P. J. (2003) Structural basis ofcore promoter recognition in a primitive eukaryote. Cell 115, 413–424.

52. Weis, L., Reinberg, D. (1997) Accurate positioning of RNA polymerase IIon a natural TATA-less promoter is independent of TATA-binding-

protein-associated factors and initiator-binding proteins. Mol. Cell. Biol.17, 2973–2984.

53. Arman, M., Calvo, J., Trojanowska, M. E., Cockerill, P. N., Santana, M.,Lopez-Cabrera, M., Vives, J., Lozano, F. (2004) Transcriptional regulationof human CD5: important role of Ets transcription factors in CD5 expres-sion in T cells. J. Immunol. 172, 7519–7529.

54. Hagemeier, C., Bannister, A. J., Cook, A., Kouzarides, T. (1993) Theactivation domain of transcription factor PU.1 binds the retinoblastoma(RB) protein and the transcription factor TFIID in vitro: RB showssequence similarity to TFIID and TFIIB. Proc. Natl. Acad. Sci. USA 90,1580–1584.

55. Holtschke, T., Lohler, J., Kanno, Y., Fehr, T., Giese, N., Rosenbauer, F.,Lou, J., Knobeloch, K. P., Gabriele, L., Waring, J. F., et al. (1996)Immunodeficiency and chronic myelogenous leukemia-like syndrome inmice with a targeted mutation of the ICSBP gene. Cell 87, 307–317.

56. Tamura, T., Ozato, K. (2002) ICSBP/IRF-8: its regulatory roles in thedevelopment of myeloid cells. J. Interferon Cytokine Res. 22, 145–152.

57. Tsujimura, H., Tamura, T., Ozato, K. (2003) Cutting edge: IFN consensussequence binding protein/IFN regulatory factor 8 drives the developmentof type I IFN-producing plasmacytoid dendritic cells. J. Immunol. 170,1131–1135.

58. Wang, I. M., Contursi, C., Masumi, A., Ma, X., Trinchieri, G., Ozato, K.(2000) An IFN-�-inducible transcription factor, IFN consensus sequencebinding protein (ICSBP), stimulates IL-12 p40 expression in macrophages.J. Immunol. 165, 271–279.

59. Politis, A. D., Sivo, J., Driggers, P. H., Ozato, K., Vogel, S. N. (1992)Modulation of interferon consensus sequence binding protein mRNA inmurine peritoneal macrophages. Induction by IFN-� and down-regulationby IFN-�, dexamethasone, and protein kinase inhibitors. J. Immunol.148, 801–807.

60. Driggers, P. H., Ennist, D. L., Gleason, S. L., Mak, W. H., Marks, M. S.,Levi, B. Z., Flanagan, J. R., Appella, E., Ozato, K. (1990) An interferon�-regulated protein that binds the interferon-inducible enhancer elementof major histocompatibility complex class I genes. Proc. Natl. Acad. Sci.USA 87, 3743–3747.

61. Marecki, S., Atchison, M. L., Fenton, M. J. (1999) Differential expressionand distinct functions of IFN regulatory factor 4 and IFN consensussequence binding protein in macrophages. J. Immunol. 163, 2713–2722.

62. Fultz, M. J., Vogel, S. N. (1998) Analysis of the antagonist effect of IFN-�on IFN-�-induced interferon consensus sequence binding protein mes-senger RNA in murine macrophages. J. Inflamm. 48, 28–39.

63. Politis, A. D., Ozato, K., Coligan, J. E., Vogel, S. N. (1994) Regulation ofIFN-�-induced nuclear expression of IFN consensus sequence bindingprotein in murine peritoneal macrophages. J. Immunol. 152, 2270–2278.

64. Kantakamalakul, W., Politis, A. D., Marecki, S., Sullivan, T., Ozato, K.,Fenton, M. J., Vogel, S. N. (1999) Regulation of IFN consensus sequencebinding protein expression in murine macrophages. J. Immunol. 162,7417–7425.

65. Brass, A. L., Zhu, A. Q., Singh, H. (1999) Assembly requirements ofPU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo usingfused dimers. EMBO J. 18, 977–991.

66. Eklund, E. A., Jalava, A., Kakar, R. (1998) PU.1, interferon regulatoryfactor 1, and interferon consensus sequence-binding protein cooperate toincrease gp91(phox) expression. J. Biol. Chem. 273, 13957–13965.

67. Meraro, D., Gleit-Kielmanowicz, M., Hauser, H., Levi, B. Z. (2002)IFN-stimulated gene 15 is synergistically activated through interactionsbetween the myelocyte/lymphocyte-specific transcription factors, PU.1,IFN regulatory factor-8/IFN consensus sequence binding protein, and IFNregulatory factor-4: characterization of a new subtype of IFN-stimulatedresponse element. J. Immunol. 168, 6224–6231.

68. O’Reilly, D., Quinn, C. M., El-Shanawany, T., Gordon, S., Greaves, D. R.(2003) Multiple Ets factors and interferon regulatory factor-4 modulateCD68 expression in a cell type-specific manner. J. Biol. Chem. 278,21909–21919.

69. Sharma, S., Mamane, Y., Grandvaux, N., Bartlett, J., Petropoulos, L., Lin,R., Hiscott, J. (2000) Activation and regulation of interferon regulatoryfactor 4 in HTLV type 1-infected T lymphocytes. AIDS Res. Hum. Retro-viruses 16, 1613–1622.

70. Marecki, S., Riendeau, C. J., Liang, M. D., Fenton, M. J. (2001) PU.1 andmultiple IFN regulatory factor proteins synergize to mediate transcrip-tional activation of the human IL-1 � gene. J. Immunol. 166, 6829–6838.

71. Zhao, J., Kong, H.J., Li, H., Huang, B., Yang, M., Zhu, C., Bogunovic, M.,Zheng, F., Mayer, L., Ozato, K., et al. (2006) IRF-8/ICSBP is involved inTLR signaling and contributes to the cross talk between TLR and IFN-�signaling pathways. J. Biol. Chem. 281, 10073–10080.


72. Fehr, T., Schoedon, G., Odermatt, B., Holtschke, T., Schneemann, M.,Bachmann, M. F., Mak, T. W., Horak, I., Zinkernagel, R. M. (1997)Crucial role of interferon consensus sequence binding protein, but neitherof interferon regulatory factor 1 nor of nitric oxide synthesis for protectionagainst murine listeriosis. J. Exp. Med. 185, 921–931.

73. Scharton-Kersten, T., Contursi, C., Masumi, A., Sher, A., Ozato, K. (1997)Interferon consensus sequence binding protein-deficient mice displayimpaired resistance to intracellular infection due to a primary defect ininterleukin 12 p40 induction. J. Exp. Med. 186, 1523–1534.

74. Giese, N. A., Gabriele, L., Doherty, T. M., Klinman, D. M., Tadesse-Heath, L., Contursi, C., Epstein, S. L., Morse III, H. C. (1997) Interferon(IFN) consensus sequence-binding protein, a transcription factor of theIFN regulatory factor family, regulates immune responses in vivo throughcontrol of interleukin 12 expression. J. Exp. Med. 186, 1535–1546.

75. Eklund, E. A., Kakar, R. (1999) Recruitment of CREB-binding protein byPU.1, IFN-regulatory factor-1, and the IFN consensus sequence-bindingprotein is necessary for IFN-�-induced p67phox and gp91phox expres-sion. J. Immunol. 163, 6095–6105.

76. Xiong, H., Zhu, C., Li, H., Chen, F., Mayer, L., Ozato, K., Unkeless,J. C., Plevy, S. E. (2003) Complex formation of the interferon (IFN)consensus sequence-binding protein with IRF-1 is essential for murinemacrophage IFN-�-induced iNOS gene expression. J. Biol. Chem.278, 2271–2277.

77. Kim, Y. M., Kang, H. S., Paik, S. G., Pyun, K. H., Anderson, K. L.,Torbett, B. E., Choi, I. (1999) Roles of IFN consensus sequence bindingprotein and PU.1 in regulating IL-18 gene expression. J. Immunol. 163,2000–2007.

78. Tsujimura, H., Tamura, T., Kong, H. J., Nishiyama, A., Ishii, K. J.,Klinman, D. M., Ozato, K. (2004) Toll-like receptor 9 signaling activatesNF-�B through IFN regulatory factor-8/IFN consensus sequence bindingprotein in dendritic cells. J. Immunol. 172, 6820–6827.

79. Dosch, E., Zoller, B., Redmann-Muller, I., Nanda, I., Schmid, M., Viciano-Gofferge, A., Jungwirth, C. (1998) The genomic structure of the chickenICSBP gene and its transcriptional regulation by chicken interferon. Gene210, 265–275.


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