of April 20, 2017.This information is current as

p19 in MacrophagesSMAD-3 and ATF-2 in Expression of IL-23 Promoter Analysis Reveals Critical Roles for

Fahd Al-Salleeh and Thomas M. Petro

http://www.jimmunol.org/content/181/7/4523doi: 10.4049/jimmunol.181.7.4523

2008; 181:4523-4533; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/181/7/4523.full#ref-list-1

, 30 of which you can access for free at: cites 67 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Promoter Analysis Reveals Critical Roles for SMAD-3 andATF-2 in Expression of IL-23 p19 in Macrophages1

Fahd Al-Salleeh and Thomas M. Petro2

IL-23 p19/p40, produced by macrophages and dendritic cells, is critical for development of Th17 in several autoimmune diseases.In this study, bone marrow-derived (BMM) and splenic macrophages (SPM) from SJL/J mice, susceptible to autoimmune de-myelinating disease following Theiler’s virus (TMEV) infection, expressed IL-23 in response to TMEV. We identified potentialbinding sites for IFN response factor (IRF)-3 (nt �734 to �731), Sma- and Mad-related protein (SMAD)-3 (nt �584 to �581),activating transcription factor (ATF)-2 (nt �571 to �568), IRF-7 (nt �533 to-525), and NF-�B (nt �215 to �209) in the murinep19 promoter. The p19prom in the pGL3 promoter-reporter vector responded to TMEV or poly(I:C), a TLR3 agonist in theRAW264.7 macrophage cell line. Deletions upstream from the IRF-3 site and mutations at the IRF-3, SMAD-3, ATF-2, or NF-�B,but not the IRF-7, sites significantly reduced promoter activity. ATF-2 or SMAD-3, but not IRF-3, short-hairpin RNA reduced p19promoter activity and protein expression in RAW264.7 cells responding to TMEV. Chromosomal DNA immunoprecipitationassays revealed that SMAD-3 and ATF-2 bind to the endogenous p19 promoter in RAW264.7 cells and SJL/J SPM followingchallenge with TMEV. TGF-�1, which activates SMAD-3, was induced in RAW264.7 cells, BMM, and SPM by TMEV. Neutral-izing Ab to TGF-�1 eliminated TMEV-induced IL-23 production and SMAD-3 activation in RAW264.7 cells, BMM, and SPM.Activation of ATF-2 was JNK, but not p38 or ERK MAPK dependent. Inhibition of the JNK, but also the ERK MAPK pathwaysdecreased expression of p19. These results suggest that ATF-2 and SMAD-3 are transcription factors, which are, in addition toNF-�B, essential for IL-23 p19 expression. The Journal of Immunology, 2008, 181: 4523–4533.

C ytokines of the IL-12 family produced by macrophageand dendritic cell lineages are heterodimers. Therefore,each of these cytokines is encoded by different genes,

which must be expressed simultaneously, in order for an activeform of the cytokine to be secreted (1). IL-23, a member of theIL-12 cytokine family, consists of a p40 subunit coupled to a p19subunit (2). In contrast, IL-12 consists of the same p40 subunitcoupled to a p35 subunit (3). Despite the facts that p19 is a ho-mologue of p35 and also dimerizes with p40, IL-23 and IL-12 havedistinct functions in development of effector and memory CD4� Tcell subsets. IL-12 stimulates the development of the Th1 IFN-�-secreting CD4� T cell subset, whereas IL-23 stimulates develop-ment of the CD4� Th17 subset (4), which secretes IL-17, IL-17F,TNF-�, IL-6, and IL-22 during the adaptive immune response (2,5–7).

IL-12 has a role in the immune response to intracellular infec-tion (8), antiviral immunity (9), and anticancer immunity (10),whereas IL-17 has a role in antifungal immunity (11). Due to thepresence of p40, IL-12 was also suspected to play a role in thedevelopment of several T cell-mediated autoimmune diseases,such as diabetes, inflammatory bowel disease, multiple sclerosis(MS)3/experimental autoimmune encephalomyelitis, and collagen-

induced arthritis (12, 13). However, because of the discovery thatp40 is also part of IL-23, IL-12 has been exonerated, and it is nowaccepted that IL-23 has the essential role in the development of Tcell-mediated autoimmune diseases (2, 14). IL-23 appears to playa role in these autoimmune diseases by inducing development ofTh17 (5, 15, 16). Therefore, the mechanisms to control expressionof the p19 subunit of IL-23 must be elucidated.

Very little is known about the trigger for expression of IL-23p19 during autoimmune diseases such as MS. One hypothesis onthe development of T cell-mediated autoimmune diseases assertsthat certain viral epitopes that mimic self epitopes stimulate auto-immune CD4 T cell responses (17). Our corollary to that hypoth-esis is that the virus must also induce production of IL-23. Wehave shown that Theiler’s murine encephalomyelitis virus(TMEV), which infects macrophages and induces an MS-like dis-ease in SJL/J mice (18), triggers expression of both IL-23 subunitsfrom the mouse macrophage cell line, RAW264.7 (19, 20). How-ever, our understanding of the mechanisms for expression of IL-23is not complete.

Macrophage responses to viruses occur in part through TLRpathways (21). TLRs have extracellular domains consisting ofmultiple leucine-rich repeat elements and a cytoplasmic domainthat belong to the IL-1/Toll receptor family (22). Although TLRsthat recognize molecular structures unique to bacterial and fungalcells (TLR1, TLR2, TLR4, TLR5, and TLR6) are localized to theplasma membrane, TLRs that recognize viral and bacterial nucleicacids (TLR3, TLR7, and TLR9) are localized at endosomal mem-branes (23). We have shown that TLR3 and TLR7 contribute toIL-23 p19 expression during challenge of macrophages with

Department of Oral Biology and Nebraska Center for Virology, University of Ne-braska Medical Center, Lincoln, NE 68583

Received for publication March 14, 2008. Accepted for publication July 24, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a University of Nebraska Medical Center College ofDentistry Seed Grant and National Multiple Sclerosis Society Grant PP1423.2 Address correspondence and reprint requests to Dr. Thomas M. Petro, Departmentof Oral Biology, University of Nebraska Medical Center, 40th and Holdrege Avenue,Lincoln, NE 68583-0740. E-mail address: tpetro@unmc.edu3 Abbreviations used in this paper: MS, multiple sclerosis; ATF, activating transcrip-tion factor; BMM, bone marrow-derived macrophage; ChIP, chromosomal DNA im-

munoprecipitation; Ct, cycle threshold; IRF, IFN response factor; ORF, open readingframe; poly(I:C), polyinosine-polycytidylic acid; SCR, scrambled sequence; sh, short-hairpin; SMAD, Sma- and Mad-related protein; SPM, splenic macrophage; TMEV,Theiler’s murine encephalomyelitis virus.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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TMEV (19, 20). More recent promoter analyses have revealed, inboth macrophages and dendritic cells, that p19 expression is de-pendent on binding of c-Rel and RelA NF-�B to the proximal p19promoter (24, 25). However, cytokine gene expression usually in-volves multiple transcription factors. We have shown that p35gene expression is controlled by NF-�B and IFN response factor(IRF)-1 (26). Therefore, we hypothesize that additional transcrip-tion factors are required for IL-23 p19 expression. The presentinvestigation shows that besides a site for NF-�B, regulatory ele-ments for IRF-3, activating transcription factor (ATF)-2, and Sma-and Mad-related protein (SMAD)-3 at the p19 promoter are es-sential for promoter activity. However, activation of SMAD-3 andATF-2, but not IRF-3, is essential to p19 expression during theresponse to TMEV or TLR3 pathway activation.

Materials and MethodsMice, cells, virus, and reagents

Female SJL/J mice were purchased from Harlan Sprague-Dawley. Themouse macrophage cell line, RAW264.7 (American Type Culture Collec-tion), was grown in DMEM cell culture medium (Invitrogen) containing10% FBS (Invitrogen) and 50 �g/ml gentamicin (Invitrogen). The DAstrain of TMEV was obtained from K. Drescher (Department of MedicalMicrobiology and Immunology, Creighton University, Omaha, NE).TMEV was grown in BHK-21 cells to produce stocks with 1 � 107 PFU/ml. Macrophages were challenged with TMEV using a multiplicity of in-fection of 1. Loxoribine, an agonist of TLR7, and polyinosine-polycyti-dylic acid (poly(I:C)), an agonist of TLR3, were obtained from InvivoGen.SP 600125 (10 �M final concentration), an inhibitor of JNK MAPK; SB203580 (10 �M), an inhibitor of p38 MAPK; and U0126 (20 �M), aninhibitor of ERK MAPK, were obtained from Promega.

Isolation of splenic mononuclear cells

Spleens were extracted from female SJL/J mice and placed into cold RPMI1640 culture medium supplemented with glutamine, sodium pyruvate, 0.05mM 2-ME, and 10% FBS. Cells were dispersed using 70-�m meshscreens; washed in Dulbecco’s PBS; treated with erythrocyte-lysing re-agent containing 0.15 M NH4Cl, 1.0 M KHCO3, and 0.1 mM Na2EDTA;washed; and resuspended in cell culture medium. Cells were counted witha hemacytometer using trypan blue.

Isolation of bone marrow cells

Femurs were extracted from female SJL/J mice, severed at both ends, andusing a 25 G needle, 5 ml of cold RPMI 1640 culture medium supple-mented with glutamine, sodium pyruvate, 0.05 mM 2-ME, and 10% FBSwas flushed through the shaft. Cells were washed in Dulbecco’s PBS,treated with erythrocyte-lysing reagent, washed, and resuspended in RPMI1640 cell culture medium. Cells were counted with a hemacytometer usingtrypan blue.

Stimulation or challenge of macrophages

Splenic mononuclear cell population has been reported to contain �10%macrophages (27), located primarily in the red pulp, and �1% dendriticcells, located primarily in the white pulp (28), both of which are adherent.Therefore, enriched splenic macrophages (SPM) were derived by adding5 � 105 splenic mononuclear cells to individual wells of a 96-well tissueculture plate, incubating at 37°C for 48 h, and then washing away nonad-herent cells. Bone marrow-derived macrophages (BMM) were obtained byincubating 5 � 104 bone marrow cells/well of 96-well plates with 20 ng/mlmouse rGM-CSF (Invitrogen) added on days 1 and 4. On day 7, nonad-herent cells were removed and fresh RPMI 1640 culture medium wasadded. Adherent SPM and BMM were then challenged with TMEV in thepresence or absence of neutralizing anti-TGF-�1 (clone 9016; R&D Sys-tems). RAW264.7 cells were seeded into six-well plates at 1 � 106/mlculture medium. After 24 h, nonadherent cells were removed, and 1 ml ofculture medium was added. The adherent RAW264.7 cells were eitheruntreated (control), stimulated with poly(I:C) (50 �g/ml), loxoribine (200�M), LPS (500 ng/ml), poly(I:C) plus loxoribine, or challenged withTMEV. In one set of experiments, cells were untreated or pretreated for 30min before infection with SP 600125 (10 �M), SB 203580 (10 �M), andU0126 (at 20 �M) or 1 �l of DMSO carrier.

IL-23 p19 promoter analysis

The p19 promoter (based upon accession number NT 039502) was ampli-fied from genomic murine DNA by PCR using an upstream primer con-taining a SstI restriction site, 5�-CGAGCTCGAGGTTCTTAGCCAGCATTC-3�, and a downstream primer containing an XhoI restriction site, 5�-CCGCTCGAGCTTGTTCCCTGCTTCTCAGA-3�, and cloned into thepGL3-basic vector (p19prompGL3) (19). Sequential 5� deletions to removepotential promotor transcriptional regulatory elements (Fig. 2A) were alsogenerated by PCR using an upstream primer containing a SstI restrictionsite and a downstream primer containing an XhoI restriction site. Using aQuikChange II site-directed mutagenesis kit (Stratagene), nucleotides inthe IRF-3 site at bp �734 to �731 were mutated from ATTT to CCAC; inthe SMAD-3 site at bp �584 to �581 they were mutated from CAGAC toACAG; in the ATF-2 site at bp �571 to �568 they were mutated fromTGAG to GACT; in the IRF-7 site at bp �533 to �532 they were mutatedfrom TT to AG, and at bp �526 to �525 they were mutated from TT toCG; and in the NF- �B site at bp �215 to �214 they were mutated fromGG to TC, and at bp �210 to �209 they were mutated from CC to AA.The sequence of each insert was verified at the core facility of the BeadleCenter for Biotechnology, University of Nebraska. All plasmids were iso-lated using a Qiagen endo-free plasmid kit.

Transfections

Plasmids were transfected into the nucleus of RAW264.7 cells using theCell Line Nucleofector Kit (Amaxa Biosystem), according to manufactur-er’s instructions. Cells were transfected with 2 �g of the pGL3 reporterconstructs along with 0.02 �g of a pRL-SV40 reference construct thatconstitutively expresses Renilla luciferase. We routinely obtained 50–60%transfection efficiency, with little impact on cell viability. Following trans-fection, cells were seeded at 8.3 � 104 onto 96-well plates. After overnightculture, transfected cells were challenged with TMEV or stimulated with50 �g/ml poly(I:C) or 200 �� loxoribine. After 24 h, cells were lysed andluciferase activity was measured with Dual-Luciferase Reporter AssaySystem (Promega) using a Polarstar Optima luminometer (BMG Labtech).Luciferase activity from pGL3 was normalized to luciferase activity frompRL-SV40.

TGF-�1 quantitative real-time PCR

After challenge with TMEV or stimulation with poly(I:C) and loxoribine,RNA was extracted using the RNAeasy kit of Qiagen, according to themanufacturer’s instructions, as we have done previously (20). cDNAs wereprepared from 1 �g of RNA, as previously described (20). The sense/antisense primers used for quantitative PCR analyses were as follows: 5�-TACTGCCGCTTCTGCTCCCACT-3�/5�-GATGGCTTCGATGCGCTTCCGT-3� for TGF-�1 to yield a 124-bp product, and 5�-TTGTCAGCAATGCATCCTGCAC-3�/5�-ACAGCTTTCCAGAGGGGCCATC-3� forGAPDH to yield a 149-bp product. Quantitative real-time PCR was per-formed with the Platinum-SYBR Green I-UDG-quantitative PCR Super-Mix (Invitrogen) using an ABI Prism 7000 thermal cycler in which 1 �l ofcDNA was incubated at 50°C for 2 min and 95°C for 2 min, followed by40 cycles of 95°C for 15 s and 60°C for 30 s. Cycle thresholds (Ct) werenormalized to Ct of GAPDH for each cDNA, and was expressed by foldincrease using 2���Ct (29). Representative samples of quantitative real-time PCR products were run electrophoretically on an agarose gel to con-firm that a single PCR product was obtained.

ELISAs

IL-23 and TGF-�1 ELISA kits were obtained from eBiosciences. Ninety-six-well plates were coated with anti-mouse IL-23 p19 (clone G23-8) or 1.0�g/ml anti-mouse/human TGF-�1 (clone eBioTB2F) in coating buffer at4°C overnight. After five washes with PBS/0.05% Tween 20, each platewas blocked before addition of various concentrations of rIL-23, TGF-�1standards, samples, or acid-activated samples (for TGF-�1 assay). Theplates were incubated at room temperature for 3 h. After five washes withPBS/Tween 20, each plate was incubated with biotinylated anti-mouse IL-12/23 p40 (clone C17.8) or biotinylated anti-mouse/human TGF-�1 (cloneeBio16TFB) at room temperature for 1 h. After five washes, the plates wereincubated with avidin-HRP at room temperature for 30 min. The plateswere washed seven times and incubated with 3,3�,5,5� tetramethylbenzi-dine substrate/hydrogen peroxide solution. After adding 2 N H2SO4 stopsolution, IL-23 and TGF-�1 were measured by determining ODs at 450nm, with reference at 570 nm using an ELISA spectrophotometric platereader (PolarStar Optima).

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RNA interference

Expression vectors producing short-hairpin (sh) RNA against murineTLR3 (shTLR3) and TLR7 (shTLR7) were purchased from InvivoGen.Expression vectors producing shRNA against murine IRF-3 (shIRF-3),SMAD-3 (shSMAD-3), ATF-2 (shATF-2), or irrelevant scrambled se-quence (shSCR) were constructed by inserting shDNA sequences (pre-dicted to produce functional small interfering RNA from the SiRNA-Wizard program of InvivoGen) into the psiRNA-h7SK G1 expression vec-tor obtained from InvivoGen (Table I). Plasmids containing inserts wereisolated using a Qiagen endo-free plasmid kit. RAW264.7 cells were trans-fected with 2 �g of shRNA expression vectors using the Cell Line Nucleo-fector Kit (Amaxa). Transfected cells were seeded at 1 � 106 or 8.3 � 104

onto 6- or 96-well plates, respectively, and then stimulated with poly(I:C)or loxoribine or challenged with TMEV before Western blot analysis.

PAGE and Western blot analysis

To evaluate activation of IRF-3, SMAD-3, and ATF-2, RAW264.7 cellsafter challenge with TMEV or stimulation with poly(I:C) or loxoribinecells were lysed with protein-lysing reagent (Cell Signaling Technology),as we have done previously. To measure intracellular p19 protein,RAW264.7 cells were treated with brefeldin (10 �g/ml) to block proteinsecretion after 8 h of TMEV challenge or poly(I:C) and loxoribine stim-ulation, and then lysed at 24 h. The cell extracts were added to a samplebuffer, and protein concentrations were determined using the Bio-Rad pro-tein assay kit. Each sample containing equal amounts of protein was runthrough a 10% SDS, Tris-glycine-polyacrylamide gel, and transferredto a nitrocellulose membrane, as previously described (20). The primaryAbs used for immunoblotting include anti-IL-23 p19 Ab (R&D Sys-tems), anti-IRF-3 (Zymed Laboratories/Invitrogen), anti-phospho-IRF-3(Ser396; Upstate Biotechnology), anti-ATF-2, anti-phospho-ATF-2 (Thr69/

71), anti-SMAD-3, anti-phospho-SMAD-3 (Ser423/425; Cell Signaling Tech-nology), and anti-�-tubulin E7 (Developmental Studies Hybridoma Bank,University of Iowa, Department of Biological Sciences, Iowa City, IA).The membranes were then incubated with IRDye800-labeled (Invitrogen)or Alexa Fluor680-labeled (Rockland Immunochemicals) secondary Abs inblocking buffer for 1 h. The membrane was washed three times, and infra-red light emissions were detected with a Li-Cor Odyssey system.

Intracellular immunofluorescence

RAW264.7 cells were seeded in triplicate at 8.3 � 103 cells/well in a96-well plate. SPM and BMM were seeded at 5.0 � 104 cells/well in a96-well plate. Twenty-four hours after plating, cells were challenged withTMEV or stimulated, as described above, for the indicated time points.Cells were fixed in 3.7% formaldehyde for 20 min at room temperature andpermeabilized by four 5-min washes with 0.1% Triton X-100, and non-specific reactivity was blocked with Li-Cor Odyssey blocking buffer for1 h. Anti-phospho-IRF-3 (Ser396; Upstate Biotechnology) or anti-phospho-SMAD-3 (Ser423/425; Cell Signaling Technology) diluted in blocking buffer(1/100) was incubated with the cells overnight. Plates were then rinsedthree times with PBS-0.1% Tween 20 and incubated for 1 h with anti-rabbitIRDye800 (Rockland; 1:100) and Sapphire700 (1:1000), a nonspecific cellstain that accumulates in both the nucleus and cytoplasm of fixed cells.Plates were washed three times with PBS-0.1% Tween 20, and thenscanned with the Li-Cor Odyssey system. Fluorescence intensity at the800-nm channel was normalized to the fluorescence intensity at the 700channel.

Chromosomal DNA immunoprecipitation (ChIP)

ChIP assays were performed using the ChIP-It enzymatic digestion kit ofActive Motif, according to the manufacturer’s recommendation. Briefly,

RAW264.7 cells and splenic cells (2 � 107), unstimulated or challengedfor 6 h with TMEV, were cross-linked with 1% formaldehyde. Nuclei wereisolated into buffer containing protease inhibitors and PMSF using aDounce homogenizer and subjected to enzymatic digestion to yield to 300-to 1000-bp DNA fragments. Nuclei were immunoprecipitated with 3 �g ofspecific rabbit anti-IRF-3, anti-SMAD-3, or anti-ATF-2 (Cell SignalingTechnology) overnight at 4°C. Protein-DNA cross-links of both input andprecipitated DNA were reversed at 94°C for 15 min. Samples were treatedwith proteinase K before PCR analysis. Input and precipitated DNA wereamplified by primers encompassing the IRF-3, SMAD-3, and ATF-2 sitesin the promoter of p19: sense, 5�-ACCCGGGGAATGCCCTTACTTACTATTTCT-3�, and antisense, 5�-TCAAGGTTTATTCTTACCCAACCCCAGTC-3�. They were also amplified by primers designed away from the p19promoter within the p19 open reading frame (ORF): sense, 5�-GCTGGATTGCAGAGCAGTAATA-3�, and antisense, 5�-GCATGCAGAGATTCCGAGAGAG-3�, as a negative control in a 32-cycle PCR. Input DNA wasused as a positive control. The PCR products were analyzed by 1.8% aga-rose electrophoresis.

ResultsTMEV activates the IL-23 p19 promotor via TLR3 and TLR7

Previously, we reported that p19 expression in RAW264.7 cellsresponding to TMEV challenge is dependent on TLR3 and TLR7pathways (20). To confirm this requirement, RAW264.7 cells weretransfected with the p19 promoter reporter vector (p19prompGL3)that we previously described (19) and then challenged withTMEV, stimulated with the TLR3 agonist, poly(I:C) (50 �g/ml),or stimulated with the TLR7 agonist, loxoribine (200 �M). TMEVchallenge or poly(I:C), but not loxoribine stimulation, induced sig-nificant p19 promoter activity by 24 h (Fig. 1, A and B). To eval-uate further the requirement for TLR3 and TLR7, shTLR3 andshTLR7 expression vectors were transfected into RAW264.7 cellsalong with p19prompGL3. With a transfection efficiency of �50%,shTLR3 and shTLR7 significantly decreased expression of TLR3and TLR7 in RAW264.7 cells during TMEV challenge (20). Like-wise, transfection of either shTLR3 or shTLR7 significantly re-duced murine p19 promoter activity following TMEV challenge ofRAW264.7 cells (Fig. 1C). These data confirm that challenge ofRAW264.7 cells with TMEV induces p19 expression throughTLR3 and TLR7, but stimulation through TLR7 alone will notinduce p19.

IRF-3, SMAD-3, ATF-2, and NF-�B, but not IRF-7 binding sitesare essential for TMEV-induced IL-23 p19 promoter activity

Mise-Omata et al. (25) have shown that a putative site for NF-�Blocated at nt �215 in the murine p19 promoter is functional. Be-cause there is significant homology between the murine and humanp19 promoters (Fig. 2A), we used the MatInspector transcriptionfactor search program (30, 31) to identify additional transcriptionfactor binding sites in the murine p19 promoter. MatInspectoranalysis found potential sites for IRF-3 (nt �734 to �731), IRF-7(nt �533 to �525), ATF-2 (nt �571 to �568), SMAD-3 (nt �584

Table I. Sequences of nucleotides inserted into shRNA expression vectors

Target Double-Stranded Nucleotide Sequence Inserted into psiRNA-h7SK G1a

ATF-2 �5�-ACCTCGCAGAAGACTTGAGTTCACTATCAAGAGTAGTGAACTCAAGTCTTCTGCTT-3��5�-CAAAAAGCAGAAGACTTGAGTTCACTACTCTTGATAGTGAACTCAAGTCTTCTGCG-3�

IRF-3 �5�-ACCTCGTTGCGGTTAGCTGCTGACAATCAAGAGTTGTCAGCAGCTAACCGCAACTT-3��5�-CAAAAAGTTGCGGTTAGCTGCTGACAACTCTTGATTGTCAGCAGCTAACCGCAACG-3�

SMAD-3 �5�-ACCTCGGCTCCCTCACGTTATCTACTTCAAGAGAGTAGATAACGTGAGGGAGCCTT-3��5�-CAAAAAGGCTCCCTCACGTTATCTACTCTCTTGAAGTAGATAACGTGAGGGAGCCG-3�

SCR 5�-ACCTCGCCCTGAATCATCGACCTTATTCAAGAGATAAGGTCGATGATTCAGGGCTT-3�5�-CAAAAAGCCCTGAATCATCGACCTTATCTCTTGAATAAGGTCGATGATTCAGGGCG-3�

a Predicted to produce functional small interfering RNA from the SiRNAWizard program of InvivoGen.

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to �581), and NF-�B (nt �215 to �209) in the murine p19 pro-moter (Fig. 2A). There was 100% homology between the murineand human NF-�B sites (Fig. 2A), 41% homology in the region ofthe IRF-3 site, 78% homology at the SMAD-3 site, 57% homologyat the ATF-2 site, and 90% homology at the IRF-7 site. Therefore,5� deletion mutations and site mutations at each of these sites werecreated in p19prompGL3. The 5� deletions were made upstream ofand including the IRF-3, SMAD-3, ATF-2, IRF-7, and NF-�Bsites using PCR, and these PCR products were reinserted intopGL3. RAW264.7 cells transiently transfected with each of thesepromoter reporter constructs were challenged with TMEV or stim-ulated with poly(I:C) or loxoribine. The responsiveness of theIL-23 p19 promotor to TMEV or poly(I:C) declined significantly

A

B

C

FIGURE 1. A and B, Fold enhancement of luciferase activity gener-ated by RAW264.7 cells transfected with the p19 promoter in pGL3reporter vector in response to TMEV challenge (A) or stimulation withpoly(I:C) or loxoribine (B). IL-23 p19 promoter activity after TLR3 orTLR7 knockdown (C). RAW264.7 cells were transfected with emptyvector, shTLR3, shTLR7, or shTLR3 plus shTLR7. Cells were theneither uninfected (Control) or challenged with TMEV (T) for 24 h.Promoter activity was measured as firefly luciferase-dependent lumi-nescence of stimulated cells normalized to Renilla luciferase-dependentluminescence at 24 h after infection. Data are mean � SEM of fivesamples and a representative experiment from three experiments eval-uated by Student’s t test. Comparisons in which p values �0.05 wereconsidered significantly different are denoted (�).

FIGURE 2. A, Map of mouse compared with the human IL-23 p19 pro-moter region showing potential transcription factor binding sites (boxed) withthe site mutation locations in bold type. The cut sites for the deletional mutantsused in the present work are denoted by 2. B–E, The responsiveness of thep19 promoter to TMEV challenge (B and D) or stimulation with poly(I:C) orloxoribine (C and E) following 5� deletion mutations (B and C) or site muta-tions (D and E). The pGL3 basic reporter vector with p19 wild-type (WT)promoter, no promoter (empty) pGL3, mutated p19 promoters with 5� dele-tions (deleted 5� to the indicated sites), or mutated p19 promoters with muta-tions at the sites indicated were transfected into RAW264.7 cells along withthe pRL-SV40 Renilla luciferase reference vector. Promoter activity was mea-sured as firefly luciferase-dependent luminescence of stimulated cells normal-ized to Renilla luciferase-dependent luminescence in 24-h cell extracts dividedby values obtained from unstimulated cells normalized to Renilla luciferase.Data are mean � SE of five samples from a representative experiment repeatedat least three times. �, Indicates means that are significantly different from themean generated by the response of the wild-type promoter, p 0.05.

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upon elimination of the region upstream from nt �729, a regionthat contains the putative IRF-3 binding site (Fig. 2, B and C).Elimination of regions upstream from and including the SMAD-3,ATF-2, IRF-7, and NF-�B binding sites did not restore respon-siveness. These results suggest that the IRF-3 binding site in thep19 promoter is a positive regulatory element for p19 expression.However, the roles of the SMAD-3, ATF-2, and IRF-7 bindingsites were not clear. RAW264.7 cells were then transfected withthe wild-type p19prompGL3 or p19prompGL3 constructs in whichthe IRF-3, SMAD-3, ATF-2, or IRF-7 sites were mutated. Thesecells were then challenged with TMEV or stimulated withpoly(I:C) or loxoribine for 24 h. Mutations at the IRF-3, SMAD-3,ATF-2, or NF-�B binding sites significantly decreased the p19promoter response to TMEV or poly(I:C) (Fig. 2, D and E). Incontrast, mutation at the IRF-7 binding site did not affect the re-sponsiveness of the p19 promoter. These results suggest thatIRF-3, SMAD-3, ATF-2, and NF-�B are essential transcriptionfactors for expression of p19.

IRF-3, SMAD-3, and ATF-2 are activated during TMEVinfection or TLR3/7 pathway stimulation

The results to date suggest that TMEV stimulation of TLR3 andTLR7 pathways leads to activation of IRF-3, SMAD-3, and ATF-2for p19 expression. IRF-3, SMAD-3, and ATF-2 are transcriptionfactors that are each activated by hyperphosphorylation (9, 32–34).We have shown previously that TMEV challenge of RAW264.7

cells results in activation of ATF-2 (19). To determine whetherIRF-3 and SMAD-3 are also activated by TMEV challenge orstimulation with poly(I:C), the phosphorylation status of each tran-scription factor was determined by immunoblot analysis. Chal-lenge of RAW264.7 cells with TMEV or stimulation withpoly(I:C) plus loxoribine triggered phosphorylation of IRF-3within 90 min and phosphorylation of SMAD-3 and ATF-2 within30 min (Fig. 3). To confirm our results, we quantified phosphor-ylation of IRF-3 and SMAD-3 by measuring the intensity of in-tracellular immunofluorescence and phosphorylation of ATF-2 bymeasuring intensity of Western blot phospho-ATF-2 images. Theresults confirm that TMEV challenge and poly(I:C)/loxoribinestimulation triggered significant intracellular phosphorylation ofIRF-3 and SMAD-3 and stimulated ATF-2 phosphorylation, asmeasured by digital intensity of immunoblots compared with un-stimulated controls (Fig. 3).

SMAD-3 and ATF-2 are critical for IL-23 p19 promoter activity

To examine further the role of IRF-3, SMAD-3, and ATF-2 inIL-23 p19 promotor activity, we constructed plasmid vectorsthat express shRNA, which are predicted to significantly reduceexpression of each of these transcription factors. Transfectionof shIRF-3, shSMAD-3, or shATF-2 vectors into RAW264.7cells reduced total and activated IRF-3, SMAD-3, and ATF-2(Fig. 4) during challenge with TMEV compared with transfec-tion of a SCR shRNA vector. RAW264.7 cells transfected with

FIGURE 3. IRF-3 (A), SMAD-3 (B), and ATF-2 (C)activation in RAW264.7 cells following TMEV chal-lenge or poly(I:C)/loxoribine stimulation. Representa-tive Western immunoblots from three experiments ofphospho(p)- and total IRF-3 (A, left), phospho- and totalSMAD-3 (B, left), and phospho- and total ATF-2 (C,left) in cell lysates of RAW264.7 cells 30 min (SMAD-3and ATF-2) or 90 min (IRF-3) after challenge withTMEV, stimulation with poly(I: C) (50 �g/ml) plus lox-oribine (200 �M), or no stimulation (NIL). Mean digitalintensity of intracellular immunofluorescence of phos-pho-IRF-3 (A, right) or phospho-SMAD-3 (B, right) ormean digital intensity of membrane immunoblot phos-pho-ATF-2 (C, right) in cell lysates of RAW264.7 cells30 min (SMAD-3 and ATF-2) or 90 min (IRF-3) afterchallenge with TMEV or stimulation with poly(I:C)plus loxoribine. Mean digital intensity of intracellularimmunofluorescence was normalized to fluorescenceobtained with intracellular Sapphire700. Mean digitalintensities of phospho-ATF-2 immunoblots were nor-malized to digital intensities of tubulin immunoblots.Means � SEM are derived from three to five samplesper experiment of three separate experiments. �, Indi-cates means that are significantly different from the con-trol mean, p 0.05.

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shSMAD-3 or shATF-2 exhibited decreased p19 promoter ac-tivity 24 h after challenge with TMEV (Fig. 5A) or stimulationwith poly(I:C) (Fig. 5B) compared with cells transfected withshSCR. Despite a reduction in IRF-3 activation in shIRF-3-transfected RAW264.7 cells challenged with TMEV, p19 pro-moter activity was not significantly different from in TMEV-challenged cells transfected with control plasmid. In contrast,

transfection of shIRF-3 reduced IL-23 p19 promoter activityafter stimulation with poly(I:C)/loxoribine, but not enough toachieve significance (Fig. 5B). To confirm these data, intracel-lular p19 expression was measured by Western immunoblot inRAW264.7 cells that were transfected with shIRF-3, shSMAD-3,shATF-2, or shSCR, and then challenged with TMEV. TMEV-challenged RAW264.7 cells transfected with shSMAD-3 orshATF-2, but not shIRF-3, exhibited a decrease in p19 produc-tion compared with cells transfected with shSCR (Fig. 5C). Be-cause TMEV stimulation of RAW264.7 cells requires bothTLR3 and TLR7 (20), we decided to stimulate RAW264.7cells with poly(I:C) and loxoribine together to more closelymimic the TMEV challenge. Much like the TMEV challenge,poly(I:C)/loxoribine stimulated IL-23 p19 expression, whereasshSMAD-3 and shATF-2 significantly reduced the poly(I:C)/loxoribine-induced IL-23 p19 expression. Altogether, these datashow that activation of SMAD-3 and ATF-2 is critical for ex-pression of p19.

IRF-3, SMAD-3, and ATF-2 are present at the endogenousIL-23 p19 promoter following TMEV challenge

The data to date indicate that TMEV challenge of RAW264.7 cellsresults in activation of ATF-2, IRF-3, and SMAD-3, of whichATF-2 and SMAD-3 are required for p19 expression. To deter-mine whether these transcription factors are present at the endog-enous p19 promoter following challenge of RAW264.7 cells withTMEV, we used ChIP assays. As shown in Fig. 6A, using primers(Primer A) specific to the p19 promoter region flanking the IRF-3,SMAD-3, and ATF-2 sites, challenge of RAW264.7 cells withTMEV induced association of SMAD-3 and ATF-2 with the p19

FIGURE 4. Knockdown of total and phosphorylated IRF-3 (A),SMAD-3 (B), and ATF-2 (C) by shRNA expression vectors. RAW264.7cells were transfected with shIRF-3, shSMAD-3, shATF-2, or shSCR ex-pression vectors and then challenged with TMEV or left unchallenged(NIL). pIRF-3 and total IRF-3, pSMAD-3 and total SMAD-3, pATF-2 andtotal ATF-2, and tubulin were monitored by immunoblot.

FIGURE 5. Knockdown of SMAD-3 and ATF-2 activation reduces IL-23 p19 expression. A and B, RAW264.7 cells were transfected with plasmidsexpressing shIRF-3, shSMAD-3, shATF-2, or shSCR along with wild-type p19prompGL3 plus pRLSV40 (Renilla luciferase normalization vector). After 24 h,transfected cells were challenged with TMEV (T) (A) or poly(I:C) (IC) (B). Promoter activity was measured as firefly luciferase-dependent luminescenceof stimulated cells normalized to Renilla luciferase-dependent luminescence in 24-h cell extracts divided by values obtained from unstimulated cellsnormalized to Renilla luciferase. Data are mean � SE from representative of three experiments, n 7 (A), n 3 (B); �, indicates that the mean issignificantly different from the mean generated by the response in cells transfected with shSCR, p 0.05. C, RAW264.7 cells were transfected withplasmids expressing shIRF-3, shSMAD-3, shATF-2, or shSCR, and then challenged with TMEV or poly(I:C) plus loxoribine. After 8 h, cells were treatedwith brefeldin (10 �g/ml). After an additional 16 h, the cell lysates were collected, and 10 �g of cell lysate protein was subjected to PAGE and Westernimmunoblot using goat anti-IL-23 p19 IgG and then IRDye 680 monkey anti-goat IgG.

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promoter. Interestingly, IRF-3 was constitutively present at thep19 promoter in RAW264.7 cells, whereas TMEV challenge in-creased this association somewhat. Using primers specific to thep19 ORF as a negative control (Primer B) did not yield any de-tectable PCR products.

SJL/J mice are susceptible to TMEV-induced demyelinating au-toimmune disease in part because their macrophages can becomepersistently infected with TEMV (35). We hypothesize that theseTMEV-infected macrophages from SJL/J mice produce IL-23,thus contributing to the demyelinating autoimmune disease. Todetermine whether these transcription factors are present at theendogenous p19 promoter of primary SJL/J macrophages, we con-ducted ChIP assays on SPM from SJL/J mice that were challengedwith TMEV. Similar to RAW264.7 cells, TMEV challenge ofSPM induced association of SMAD-3 and ATF-2 with the endog-enous p19 promoter in SJL/J macrophages (Fig. 6B). Previously,we showed that IRF-3 is constitutively active and localized in thenucleus in SJL/J macrophages (36). Interestingly, in agreementwith that finding, we show in this study that IRF-3 is stronglyassociated with the p19 promoter in unchallenged SJL/J macro-phage (Fig. 6B). However, by 6 h after TMEV challenge, IRF-3could no longer be found associated with the p19 promoter. Alto-gether, these data confirm that activation of SMAD-3 and ATF-2induces their association with the IL-23p19 promoter, whereasIRF-3 is constitutively present at that promoter.

TGF-�1 is expressed by macrophages during challenge withTMEV or stimulation with poly(I:C) or loxoribine

Although it is clear that ATF-2 and NF-�B are activated followingviral infection of cells through TLR pathways, SMAD-3 is activatedonly through the TGF-� family of cytokines (37). Therefore, TGF-�1expression by RAW264.7 cells and primary macrophages was eval-uated by RT-PCR and ELISA following TMEV challenge, poly(I:C),or loxoribine stimulation. Within 6 h, significant TGF-�1 mRNA ex-pression was induced in RAW264.7 cells challenged with TMEV

(Fig. 7A) or stimulated with poly(I:C) (Fig. 7B). UnchallengedRAW264.7 cells and especially BMM produced measurable TGF-�1(Fig. 7C). In contrast, unchallenged SPM did not produce measurableTGF-�1. Nevertheless, challenge of BMM, SPM, and RAW264.7cells with TMEV induced significant TGF-�1 protein secretion com-pared with unchallenged cells.

Because SMAD-3 is phosphorylated within 30 min after TMEVchallenge of RAW264.7 cells, but detectable TGF-�1 mRNA is in-duced between 3 and 6 h, we challenged SPM, RAW264.7 cells, andBMM with TMEV in the presence or absence of neutralizing Ab toTGF-�1 and then measured secreted IL-23 protein by ELISA, acti-vation of SMAD-3 by intracellular immunofluorescence, and intra-cellular p19 protein by Western immunoblot. All three cell types pro-duced low, but detectable levels of IL-23 constitutively. However,IL-23 production from SPM and BMM of SJL/J mice, as well asRAW264.7 cells, was induced in response to TMEV challenge,whereas neutralizing anti-TGF-�1 caused a significant reduction inTMEV-induced IL-23 in SPM and BMM, and nearly a significant

FIGURE 6. IRF-3, SMAD-3, and ATF-2 associate with the endoge-nous p19 promoter. ChIP assay of the p19 promoter in RAW264.7 cells(A) and splenic macrophages (B). RAW264.7 cells or splenic cells at2 � 107 were left unstimulated (NIL) or challenged with TMEV. After6 h, nuclei were immunoprecipitated with anti-IRF-3, anti-SMAD-3,anti-ATF-2, or nonspecific IgG. DNA in the precipitate was identifiedby PCR using sense primers starting at nt �760 and antisense primersstarting at nt �490 of the p19 promoter (Primer A) or primers specificto the p19 ORF (Primer B). PCR products were electrophoresed in anethidium bromide 1.8% agarose gel.

FIGURE 7. RAW264.7 cells (A–C), bone marrow-derived macro-phages (C), and splenic macrophages (C) express TGF-�1 in response toTMEV challenge (A and C), poly(I:C), or loxoribine (B) stimulation. Real-time PCR of TGF-�1 in RAW264.7 cells (A and B). A total of 1 � 106

RAW264.7 cells was unchallenged (control), challenged with TMEV (A),or stimulated with poly(I:C) or loxoribine (B) for 3, 6, and 24 h. C, ELISAof TGF-�1 secreted from RAW264.7, SPM, and BMM 24 h after challengewith TMEV. Bar graphs represent mean � SEM of three to five samplesfrom one of three experiments. �, Indicates that the mean is significantlydifferent from control, p 0.05.

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reduction in RAW264.7 cells ( p 0.06) (Fig. 8A). TMEV challengeof BMM and SPM increased SMAD-3 phosphorylation within 30min (Fig. 8B), whereas neutralizing anti-TGF-�1 prevented TMEV-induced SMAD-3 activation. Likewise, TMEV challenge ofRAW264.7 cells induced p19 protein expression, whereas neutraliz-ing anti-TGF-�1 prevented this expression of p19 (Fig. 8C). There-fore, TGF-�1 is most likely responsible for SMAD-3 activation andIL-23 expression in TMEV-challenged macrophages.

ATF-2 activation for IL-23 p19 expression is through the JNK,but not the p38, MAPK pathway

Previously, we showed that ATF-2 is activated following TMEVchallenge of RAW264.7 cells. ATF-2 is activated by phosphory-

lation through either the JNK, ERK, or p38 MAPK pathways (38),all of which are activated following TMEV infection ofRAW264.7 cells or SJL/J macrophages (36, 39, 40). To determinewhich of the MAPKs are responsible for the ATF-2 activation thatis required for p19 expression, RAW264.7 cells were pretreatedwith SB203580 (10 �M), which inhibits activation of componentsdownstream of the p38 pathway; U0126 (20 �M), which inhibitsactivation of ERK; SP 600125 (10 �M), which inhibits JNKMAPKs; or 1 �l of DMSO, which was used to dissolve the inhib-itors. Inhibition of either the ERK or p38 MAPK had no effect onATF-2 activation in response to TMEV challenge of RAW264.7cells (Fig. 9A). In contrast, inhibition of the JNK MAPK pathwayreduced activation of ATF-2 in response to TMEV. Previously, wehave seen that ERK is important in p19 promoter activity (19). Wenext determined the effect of the MAPK inhibitors on p19 expres-sion using Western immunoblot. TMEV induced expression ofp19, and 1 �l of DMSO increased that induction (Fig. 9B). Inhi-bition of either the ERK or JNK, but not the p38, MAPK pathwaysreduced expression of p19 in response to TMEV to below thatfound with TMEV alone or TMEV with DMSO (Fig. 9B). There-fore, ATF-2 activation for p19 expression in response to TMEVchallenge of RAW264.7 cells occurs through the JNK MAPKpathway, but both the ERK and JNK inhibitors decreased TMEV-induced and DMSO-induced p19 expression.

DiscussionTwo recent reports have shown that following its activation,NF-�B binds to the proximal p19 promoter to participate in p19expression in response to TLR activation (24, 25). The results ofthe present investigation clearly show that besides NF-�B, activa-tion of SMAD-3 and ATF-2 transcription factors is also involvedin p19 expression by macrophages challenged with TMEV. Theinteraction of multiple transcription factors has been documentedfor other genes. IFN-� expression requires coordinated binding ofthe transcription factors ATF-2/c-Jun, IRF-3, IRF-7 (41), andNF-�B to the well-studied IFN-� virus-inducible enhancer (34,42). Likewise, we have shown that expression of the promoteractivity for the p35 subunit of IL-12 requires both IRF-1 andNF-�B for optimal expression (26, 43). In the present study, weshow that the transcription from the p19 promoter involvesSMAD-3, ATF-2, and NF-�B.

FIGURE 8. IL-23 secretion, pSMAD-3 activation, and intracellular p19protein from SPM, RAW264.7 cells, or BMM challenged with TMEV andtreated with neutralizing Ab to TGF-�1. A, SPM, RAW264.7 cells, andBMM in 96-well plates were left unchallenged or challenged with TMEVin the presence or absence of neutralizing anti-TGF-�1 for 24 h, and thenIL-23 ELISAs were performed on supernatants. Data (n 5) are mean �SEM representative of two experiments. �, Indicates means that are sig-nificantly different from the mean generated in response to TMEV chal-lenge without anti-TGF-�1, p 0.05. B, SPM and BMM were left un-challenged or challenged with TMEV in the presence or absence ofneutralizing anti-TGF-�1 for 30 min and then analyzed for intracellularpSMAD-3 using intracellular infrared immunofluorescence. Data (n 3)are mean � SEM representative of two experiments. �, Indicates meansthat are significantly different from the mean generated in response toTMEV challenge without anti-TGF-�1, p 0.05. C, RAW264.7 cells wereleft unchallenged or challenged with TMEV in the presence or absence ofneutralizing anti-TGF-�1. After 8 h, cells were treated with brefeldin, andafter an additional 16 h, cell lysates were collected and analyzed for in-tracellular IL-23p19 using Western immunoblot.

FIGURE 9. Phospho-ATF-2 (A) and IL-23 p19 protein (B) in TMEV-challenged RAW264.7 cells pretreated with p38, ERK, and JNK MAPKinhibitors. A, Western immunoblot of phospho-ATF-2 and tubulin at 30min in 1 � 106 RAW264.7 cells pretreated with SP 600125 (SP; 10 �M),SB 203580 (SB; 10 �M), U0126 (U; 20 �M), or 1 �l of DMSO carrierbefore challenge infection with TMEV. B, Western immunoblot of intra-cellular p19 and tubulin at 24 h in 1 � 106 RAW264.7 cells pretreated withSP 600125, SB 203580, U0126, or 1 �l of DMSO carrier before challengewith TMEV. After 8 h of TMEV, cells were treated with brefeldin.

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However, activation of SMAD-3 in the macrophages used inthis study is not directly related to TMEV challenge or poly(I:C)stimulation. SMAD transcription factors are activated by TGF-�and other members of the TGF-� family (44, 45). TGF-� bindingto the TGF-� type II receptor induces formation of a complex withthe type I receptor, which then leads to phosphorylation ofSMAD-2 and -3 and their association with SMAD-4. The activatedSMAD-2/3/4 complex translocates to the nucleus and directly par-ticipates in TGF-�-dependent transcriptional activation by bindingto SBEs, such as the one located within the p19 promoter (46, 47).We have shown in this study that as early as 6 h following TMEVchallenge or poly(I:C) stimulation, TGF-�1 expression is induced.Nevertheless, it appears that RAW264.7 cells and BMM producesome TGF-�1 constitutively. We show in this work that neutral-ization of TGF-�1 blocks SMAD-3 phosphorylation and preventsTMEV-induced IL-23 production by macrophages.

However, SMADs are also phosphorylated by MAPKs. ERKMAPKs, which are activated by macrophages challenged withTMEV, appear to have a negative impact upon nuclear localizationof SMADs by additional phosphorylation of SMADs (48). In con-trast, p38 and JNK MAPK phosphorylation of SMAD-3 enhancesits nuclear localization (49). Therefore, we can conclude thatTMEV-challenged macrophages express substantially more TGF-�1, exhibit more activated SMAD-3, and express activatedMAPKs, which regulate SMAD-2/3/4 in positive and negativemanners.

Like SMAD-3, ATF-2 is activated directly by TMEV challengeby way of TLR3 and TLR7 pathway stimulation of MAPKs (50).ATF-2 is a member of the ATF/CREB family transcription factorand binds to the cAMP response elements of promoters. MAPKs,p38, ERK, and JNK can phosphorylate and thus activate ATF-2(51, 52). On ATF-2, activated JNK phosphorylates Thr69/Thr70/Ser90, activated p38 phosphorylates Thr69/Thr70, whereas acti-vated ERK phosphorylates Thr70 (38). Our results show that themost important MAPK activation of ATF-2 leading to p19 expres-sion in response to TMEV is JNK MAPK. However, p38 MAPKsare also activated by TGF-� through the TGF-� receptor and TGF-�-activated kinase-1 (53). In addition, ATF-2 expression and ac-tivation are induced by TGF-� (53). Therefore, TGF-� receptorsignaling could have led to ATF-2 activation in response to TMEVchallenge.

At the enhancesome of the IFN-� promoter, activated ATF-2associates with c-Jun (54). However, ATF-2 has also been reportedto bind directly to SMAD-3/4 complexes and is phosphorylated byTGF-� signaling through TGF-�-activated kinase-1 and p38 (53).This indicates that ATF-2 is a common nuclear target of the TGF-�/SMAD signaling and can form stable complexes with SMAD-3.It is interesting that the SMAD- and ATF-2 (cAMP responseelement)-binding elements are separated by 8 nt in the p19 pro-moter, suggesting that SMAD-3 and ATF-2 may be part of anenhancer complex for p19 expression. The present results show forthe first time that IL-23 p19 gene expression requires such coop-eration between ATF-2 and SMAD-3.

To our surprise, down-regulation of IRF-3 did not affect pro-moter activity or expression of IL-23 p19 protein, even though theputative IRF-3 site was important to p19 expression and IRF-3 isbound to this site. IRF-3 is activated by way of the TLR3 pathwayby phosphorylation of Ser396 (55). However, additional phospho-rylations through the PI3K/Akt pathway are required for completeactivation, binding to the CBP protein, and association with pro-moter regions (55). We have shown previously that IRF-3 is con-stitutively active and localized to the nucleus in SJL/J macro-phages, which are susceptible to TMEV-induced demyelinatingdisease, but not in macrophages from B10.S mice, which are re-

sistant to TMEV-induced demyelinating disease (36). ChIP assayspresented in this study agree with that observation, in that IRF-3exhibits strong constitutive association with the endogenous p19promoter in SJL/J macrophages and to some extent RAW264.7cells. Interestingly, IRF-3 association with the endogenous p19promoter was absent 6 h after TMEV challenge in SJL/J macro-phages. We have seen unstimulated SJL/J macrophages express ahigh background of p19 (our unpublished data) and IFN-� (40)mRNA. These results suggest that because of constitutive activa-tion of IRF-3, SJL/J macrophages may be primed for a quick burstof p19 mRNA expression compared with other strains of mice,therefore rendering SJL/J mice susceptible to demyelinatingdisease.

The constitutive association of IRF-3 with the p19 promotermay be one reason that knockdown of IRF-3 expression had littleeffect on p19 promoter activity. However, IRF-3 is only one mem-ber of an IRF family of transcription factors, IRF-1 to IRF-9 (re-viewed in Ref. 56). IRFs (57) share a highly conserved N-terminalDNA binding domain, which binds directly to IFN-stimulated re-sponse elements or IFN response elements, such as that found inthe p19 promoter (58) (reviewed in Ref. 57). Several IRF familyproteins play essential roles in induction of type I IFNs (34) andproinflammatory cytokines (59). It is plausible that in the knock-down of IRF-3 due to shIRF-3, other IRFs compensated for IRF-3.Therefore, the IRF-3 binding site is required for p19 promoteractivity, but other IRFs may possibly bind to this site in lieu ofIRF-3.

In addition to ATF-2 and SMAD-3, NF-�B cRel and RelA sub-units have been previously shown to be essential for p19 expres-sion (24, 25). The NF-�B family members, RelA (p65), RelB,cRel, p50/p105, and p52/p100, form homo- and heterodimers (60,61). Dimers of NF-�B p50 with p65 or cRel, or of p52 with RelBare located in the cytoplasm in an inactive complex with I�B. TLRstimulation leads to phosphorylation of I�Bs, release of NF-�B,and I�B ubiquitination and proteasomal degradation (62, 63). Bothreports have shown that a mutation of proximal �B site abolishesIL-23 p19 promotor activity in dendritic cells and macrophages.The present study confirms these findings in RAW264.7 cells andextends these findings by showing the involvement of other tran-scription factors, ATF-2 and SMAD-3, in IL-23 p19 expression.

TMEV infection of macrophages in certain strains of mice, suchas SJL/J, leads to a demyelinating disease much like MS. Giventhe facts that many studies point to the involvement of macro-phages and IL-23 in the pathology of MS, establishing that TMEV-induced cellular signaling leads to IL-23 expression by macro-phages is crucial to understanding the mechanism by which virusinfections could possibly lead to MS. The goal of the present studywas to determine some of the mechanisms by which TMEV-in-fected macrophages induce expression of IL-23 at the transcrip-tional level. Because the RAW264.7 macrophage cell line ex-presses most TLRs and becomes chronically infected with TMEV(20, 64), these cells are ideal to investigate innate antiviral cellularsignaling for expression of IL-23. Although TLRs play an impor-tant role in the innate immune responses against RNA viruses, it ispossible that other innate antiviral response pathways are involved.Retinoic acid-inducible gene I and melanoma differentiation-asso-ciated gene 5 pathways, which are cytoplasmic, are also activatedduring RNA virus infection of macrophages. Activation of retinoicacid-inducible gene I (65) and melanoma differentiation-associatedgene 5 (66, 67) pathways, which detect cytoplasmic viral RNA,triggers activation of NF-�B and IRF-3/7, which cooperate in in-duction of type I IFN. It is unknown whether these cytoplasmicpathways also lead to expression of IL-23.

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DisclosuresThe authors have no financial conflict of interest.

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