The Journal of Immunology

CpG-B Oligodeoxynucleotides Inhibit TLR-Dependentand -Independent Induction of Type I IFN in Dendritic Cells

Yi C. Liu,*,1 Reginald C. Gray,*,†,1 Gareth A. D. Hardy,* John Kuchtey,*,2

Derek W. Abbott,* Steven N. Emancipator,* and Clifford V. Harding*,†,‡

CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN-ab) in dendritic cells (DCs). CpG-AODNs are

more efficacious than CpG-B ODNs for induction of IFN-ab. Because IFN-ab may contribute to autoimmunity, it is important to

identify mechanisms to inhibit induction of IFN-ab. In our studies, CpG-B ODN inhibited induction of IFN-ab by CpG-A ODN,

whereas induction of TNF-a and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN-ab was observed in FLT3

ligand-inducedmurine DCs, purifiedmurinemyeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-BODN inhibited induction

of IFN-ab by agonists ofmultiple receptors, includingMyD88-dependent TLRs (CpG-AODN signaling via TLR9, orR837 or Sendai

virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA

signaling via a cytosolic pathway).CpG-BODNdid not inhibit the IFN-ab positive feedback loop second-wave IFN-ab, because IFN-

ab–induced expression of IFN-abwas unaffected, andCpG-B inhibition of IFN-abwasmanifested in IFN-abR2/2DCs, which lack

the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced first wave IFN-a4 and IFN-b. Chromatin

immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN-a4 and IFN-b promoters was induced by

CpG-A ODN but not CpG-B ODN. Moreover, CpG-A–induced association of IFN regulatory factor 1 with these promoters was

inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFN-ab that

selectively inhibits induction of IFN-ab downstream of multiple receptors and may provide targets for future therapeutic inhibition

of IFN-ab expression in vivo. The Journal of Immunology, 2010, 184: 3367–3376.

To induce innate immunity and trigger adaptive immunity,ssDNA or CpG oligodeoxynucleotides (ODNs) signalthrough TLR9 (1–5). Inmice, TLR9 is widely expressed by

APCs, including myeloid dendritic cells (mDCs), plasmacytoidDCs (pDCs), macrophages, and B cells. In humans, TLR9 expres-sion is restricted to pDCs and B cells (4–6).There are three classes of CpG ODNs, class A (also known as D-

type), classB (also knownasK-type), and class CODNs (1, 3, 4, 7, 8).CpG-BandCpG-CODNsstimulateBcells efficiently to produce IgMand IL-6, whereas CpG-A ODNs are weak inducers of B cell re-sponses. CpG-A and CpG-C ODNs are more efficacious than CpG-BODNsfor inducing type I IFN (IFN-ab) (1, 7–9), particularly inpDCs(1, 3, 4, 7, 9–11), although CpG-B ODNs are as potent for the in-duction of low levels of IFN-ab (9). All three classes of ODNs induce

DCs tomature and produceTNF-a (1, 3, 10). Thus, CpG-A andCpG-

B ODNs have distinct effects, and CpG-C ODNs combine properties

of CpG-A and CpG-B ODNs.Differences in CpG ODN induction of IFN-ab may depend on

differences in spatial localization of different ODNs within the cell as

well as differences in signaling in different cell types. CpG-A ODNs

localize to endosomes in pDCs and lysosomes inmDCs;CpG-BODNs

localize to lysosome-associated membranes in both pDCs and mDCs

(12, 13). In pDCs,CpG-A induction of IFN-ab is IFN regulatory factor

(IRF) 7-dependent,whereas inmDCs,CpG-AandCpG-B induction of

IFN-b is independent of IRF7, but dependent on IRF1 (14, 15).IFN-ab is induced by CpG-AODNs, agonists that signal via other

TLRs (dsRNA via TLR3, LPS via TLR4, and ssRNA and antiviral

agents via TLR7) and agonists that signal via non-TLRs (dsRNA

via melanoma differentiation-associated gene-5 or retinoic acid-

inducing gene I [RIG-I], and ds break-DNA [dsB-DNA] via a cyto-

solic pathway) (16–19). IFN-ab includes 13 functional isoforms of

IFN-a andoneof IFN-b that all signal througha single transmembrane

receptor (IFN-abR) to induce key immunoregulatory functions that

affect both innate and adaptive immunity. Signaling by TLRs and

other receptors initially leads to induction of IFN-b and IFN-a4,

which induce autocrine or paracrine signaling to trigger a positive

feedback loop in which IFN-b and IFN-a4 induce more IFN-ab, in-

cluding other forms of IFN-a, amplifying the IFN-ab response and

ultimately inducing a number of IFN-ab–stimulated genes.Currently, CpG ODNs demonstrate potential in preclinical and

clinical studies as vaccine adjuvants and as therapies for allergies and

cancers.Autoimmunediseases, suchas systemic lupus erythematosus

andmultiple sclerosis, however,maybe exacerbated byCpG-induced

IFN-ab (20). In addition, host DNA may contribute to pathogenesis

of autoimmune diseases, including rheumatoid arthritis (20). Thus,

further studies are needed to examine themechanisms by which CpG

*Department of Pathology, †Department of Pharmacology, and ‡Center for AIDSResearch, Case Western Reserve University, University Hospitals Case Medical Cen-ter, Cleveland, OH 44106

1Y.L. and R.C.G. shared first authorship.

2Current address: Vanderbilt Eye Institute, Vanderbilt University, Nashville, TN.

Received for publication September 21, 2009. Accepted for publication January 20,2010.

This work was supported byNational Institutes of Health Grants AI034343, AI035726,and AI069085 to C.V.H.

Address correspondence and reprint requests to Dr. Clifford V. Harding, Departmentof Pathology, Wolstein 6534, Case Western Reserve University, 2103 Cornell Road,Cleveland, OH 44106-7288. E-mail address: cvh3@cwru.edu

Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; CIITA, classII MHC transactivator; DC, dendritic cell; dsB-DNA, ds break-DNA; FLT3L, FLT3ligand; IFN-ab, type I IFN; IRF, IFN regulatory factor; ISGF3, IFN-stimulated genefactor 3; mDC, myeloid dendritic cell; mPDCA-1, murine plasmacytoid dendritic cellAg-1; NC, normalized copy number; ODN, oligodeoxynucleotide; PBST, PBS with0.1% Tween 20; pDC, plasmacytoid dendritic cell; qRT-PCR, quantitative real-timePCR; RIG-I, retinoic acid-inducing gene I; SeV, Sendai virus.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903079

ODNs induce IFN-ab and to develop agents to inhibit induction of

IFN-ab by self- and nonself-DNA.Our studies describe selective inhibition of IFN-ab induction by

CpG-B ODNs. High concentrations of CpG-B ODNs specifically in-hibited the induction of IFN-ab by agonists of TLR9 and other re-ceptors without inhibiting other responses (e.g., induction of TNF-aand IL-12p40). This inhibition was evident as early as 3 h and fora period of at least 48 h in all cell types tested: FLT 3 ligand (FLT3L)-induced murine bone marrow-derived DCs, purified murine mDCs,purifiedmurine pDCs, and human PBMCs. This mechanism inhibitedinduction of IFN-ab by agonists of a range of receptors, includingMyD88-dependent TLRs (TLR9, TLR7), aMyD88-independent TLR(TLR3), and non-TLR signals (e.g., a cytosolic pathway for signalingby dsB-DNA and perhaps RIG-I–like proteins) (19), suggesting thatthe mechanism affected downstream signaling at a point where sig-naling from these receptors converges for the induction of IFN-ab. Onthe other hand, inhibition was upstream of mechanisms involved inIFN-ab positive feedback signaling (second-wave IFN-ab). Fur-thermore, chromatin immunoprecipitation (ChIP) demonstrated thatCpG-B ODN inhibited association of IRF1 with IFN-b and IFN-a4promoters. Our results indicate a novel pathway for transcriptionalregulation that results in inhibition of first-wave IFN-ab.

Materials and MethodsAbs and reagents

CpGODNs includedCpGODN-A2216 (59-ggGGGACGATCGTCg ggg gG-39), CpGODN-A2336 (59-ggGGACGACGTCGTGggg ggG-39), CpGODN-B2006 (59-tcg tcg ttt tgt cgt ttt gtc gtt-39),CpGODN-B1668 (59-tcc atg acg ttc ctgatg ct-39),CpGODN-B1826 (59-tcc atg acg ttc ctg acg tt-39), andnon-CpGODN-B2138 (59-tcc atg agc ttc ctg agc tt-39). Lowercase letters inODNsequences referto nucleotides for which the 39 internucleotide linkage is phosphorothioate-modified, and uppercase letters refer to standard phosphodiester-linked nucleo-tides. ODNs contained phosphorothioate-modified linkages at the 59 and 39 ends(A class) or throughout (B class) to resist nuclease degradation. ODNs wereprovided by Coley Pharmaceutical Group (Wellesley, MA), purchased fromInvivoGen (San Diego, CA) or described elsewhere (21, 22), and synthesized byMWG-Biotech (High Point, NC). Sendai viruses (SeVs) (ATCC strain 52-SeVand Cantell strain C-SeV) were purchased (from American Type Culture Col-lection, Manassas, VA, and Charles River Laboratories,Wilmington, MA, re-spectively), grown in chicken eggs, and inactivated with b-propiolactone aspreviously detailed (23, 24). LPS (ultra pure Escherichia coli), imiquimod (R-837), ssRNA40, anddsB-DNAwerepurchased fromInvivoGen.ODNsandotheragonists were dissolved in endotoxin-tested (#0.05 units) PBS (Cambrex, EastRutherford, NJ) or sterile cell-culture water (Sigma-Aldrich, St. Louis, MO).Recombinant mouse IFN-aA (rIFN-a) and IFN-b (rIFN-b) were purchasedfrom PBL InterferonSource (Piscataway, NJ). Abs to IRF1 (sc-640), IRF3 (sc-9082), IRF7 (sc-9083), a-tubulin (sc-5286), and Ku-70 (sc-9033) were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA).

Murine cell culture and media

Standard medium was RPMI 1640 with L-glutamine and glucose (HyCloneLaboratories, Logan, UT) with 10% heat-inactivated FCS (HyClone), 50 mM2-ME (Sigma-Aldrich), 1 mM sodium pyruvate (HyClone), and penicillin-strep-tomycin (HyClone). Mice were housed under specific pathogen-free conditions.DCs were prepared from femur and tibia bone marrow cells of C57BL/6 mice(TheJacksonLaboratory,BarHarbor,ME),MyD882/2mice [generousgifts fromS. Akira (2, 11) on a C57BL/6 background], IFN-abR2/2A129 mice on a 129/SvEvbackground (B&KUniversal, Grimston,Aldbrough,U.K.), or 129S6/SvEvwild-type mice (Taconic Laboratories, Germantown, NY). Bone marrow cellswere cultured at 106 cells/ml in six-well tissue culture- grade dishes for 8–10 d inrecombinant mouse FLT3L-Ig fusion protein (1 mg/ml, BioExpress, West Leb-anon,NH).MediumandFLT3L-Igwere replenished ondays 3 and6.Onday8or9, nonadherent cells were removed, pelleted, resuspended, and counted. FLT3L-induced DCs contain a mixture of mDCs and pDCs and were used directly inexperimentsorused topurifypDCs (CD11c+,B220+,murinepDCAg[mPDCA]-1+, CD11b2) and mDCs (CD11c+, CD11b+, mPDCA-12, and B2202) by mag-netic beads and the MACS system (Miltenyi Biotec, Auburn, CA) as previouslydescribed (9). Briefly, pDCswere purified by positive selection using amixture ofanti-B220 beads and anti–mPDCA-1 beads (Miltenyi Biotec), whereas mDCswere purified by negative selection with the same bead mixture followed by

positive selection with CD11b beads. These methods produce mDCs and pDCswith purities of.95%, as previously described (9).

Human PBMC cultures

Blood was collected with lithium heparin anticoagulant. PBMCs were sep-arated by density gradient centrifugation with endotoxin-free Ficoll-paque(GE Healthcare, Pittsburgh, PA). Cells were cultured in RPMI 1640 sup-plemented with 2 mM L-glutamine, 10% human male AB serum (GeminiBio-Products, West Sacramento, CA), and 10 mM penicillin and strepto-mycin at 53 106 cells/well in 48-well flat-bottom non-tissue culture-treatedplates with or without CpG ODN-A2336 and/or CpG ODN-B2006. Super-natants were harvested 48 h after addition of ODN and frozen for subsequentIFN-ab quantification by ELISA.

ELISA and quantitative real-time PCR

DCswere plated at 7.5–103105 cells/well in flat-bottom96-well plates or 23106 cells/well in 48-well non-tissue culture-treated plates in standard mediumand incubated for 3–48 h with or without CpG ODN and/or other agonists.Plates were centrifuged to pellet cells, and supernatants were removed andeither assessed immediately or stored at280˚C. Supernatantswere diluted andtested by ELISA for mouse IFN-a or IFN-b (PBL InteferonSource) or mouseTNF-aor IL-12p40(BDBiosciences, San Jose,CA).For quantitative real-timePCR (qRT-PCR), RNAwas isolated from DCs using an RNeasy Plus mini kit(Qiagen, Valencia, CA). Cells were pelleted at 4˚C and resuspended in RLTlysis buffer (Qiagen). Total RNAwas extracted following on-column DNasedigestion using RNeasy Plus mini columns (Qiagen) and collected in RNase-free water. Yield was determined by OD. Oligo(dT)-primed reverse tran-scription of RNA into cDNAwas performed with Superscript II First-StrandSynthesis kit (Invitrogen, Carlsbad, CA), and 5% of the product was used foreach qRT-PCR sample using PCR buffer with hot start Invitrogen Taq poly-merase,Bio-RadSYBRgreen detection, and theBio-Rad iCycler fluorescencedetection system (Bio-Rad, Hercules, CA). All conditions were tested intriplicate. Primer pairs from murine gene sequences were as follows: for totalIFN-a (sense, 59-ATG GCTAGR CTC TGT GCT TTC CT-39 and antisense,59-AGG GCT CTC CAG AYT TCT GCT CTG-39), IFN-b (sense, 59-CATCAA CTATAA GCAGCT CCA-39 and antisense, 59-TTC AAG TGG AGAGCAGTT GAG-39), GAPDH (sense, 59-AAC GAC CCC TTC ATT GAC-39and antisense, 59-TCC ACG ACATAC TCA GCA C-39), CD40 (sense, 59-ACT GAC AAG CCA CTG CAC AG-39 and antisense, 59-CTT GCT GGTGCA GTG TTG TC-39), CD80 (sense, 59-TCG GCG CAG TAATAA CAGTCG-39 and antisense, 59-CCC ACATGG ACAGAG AAG AAC-39), CD86(sense, 59-CTC TTT CAT TCC CGG ATG GTG-39 and antisense, 59-CTAGGCTGATTCGGCTTCTTG-39), and class IIMHC transactivator (CIITA)(sense, 59-ACG CTT TCT GGC TGG ATT AGT-39 and antisense, 59-TCAACG CCAGTC TGA CGA AGG-39). Primers were previously described (9,25, 26) or designed using Clone Manager Suite v7.11 and Primers Designersv5.11 (Scientific & Educational Software, Cary, NC). A BLAST search wasthen performed to verify specificity.

Western blot analysis of whole cell lysates and nuclear andcytoplasmic fractions

DCswere incubated in 12-well tissue culture-treated disheswith orwithout CpGODNfor various periods at 37˚C. Subsequent stepswere performed at 4˚Cunlessotherwise stated. Cells were washed in PBS with protease inhibitor mixture(P8340,Sigma-Aldrich;1:200), 1mMNaF,1mMPMSF,and10nMcalyculinA.To prepare whole cell lysates, the cells were pelleted and resuspended in 160 mlRIPA lysis buffer (50 mMTris-Cl, 150mMNaCl, 2 mMEDTA, 1%Nonidet P-40, 0.5%Na-deoxycholate, and 0.1%SDS [pH7.4]) with protease inhibitors andmixedona rotator for1h.Sampleswerecentrifugedat16,0003g for10min, and150 ml supernatant was collected as whole cell lysates. Alternatively, to isolatenuclear and cytoplasmic fractions, the cells were pelleted and resuspended for10 min in 1 ml Nuclei EZ Prep Lysis Buffer (N3408, Sigma-Aldrich) with pro-tease inhibitors. After centrifugation at 500 3 g for 5 min, supernatants wereremoved as cytoplasmic fractions. Pellets were resuspended in 1 ml Nuclei EZPrep Lysis Buffer (Sigma-Aldrich) with protease inhibitors for 10 min to washaway residual cytoplasmic material, and the nuclei were collected by centrifu-gation at 500 3 g for 5 min. For SDS-PAGE, aliquots of whole cell lysates orcytoplasmic and nuclear fractions containing equal quantities of total proteinwere added to reducing sample buffer (Thermo Fisher Scientific,Waltham,MA;39000), subjected to electrophoresis on 8% SDS polyacrylamide gels, andtransferred onto polyvinylidene difluoridemembranes.MembraneswerewashedinPBSwith0.1%Tween20 (PBST), incubated for 1hat room temperature in5%nonfat milk in PBST, incubated overnight at 4˚Cwith primaryAb in 3%BSA or5% nonfat milk in PBST, washed, incubated for 1 h at room temperature withHRP-labeled secondary Ab (Amersham Biosciences, Piscataway, NJ), and de-veloped with the ECL detection kit (Amersham Biosciences, Amersham, U.K.).

3368 CpG-B ODNs INHIBIT TYPE I IFN IN DENDRITIC CELLS

ChIP assays

DCswere plated at 8–103 106/well in six-well plates in standardmedium andincubated for 3 h with or without CpG ODN. ChIP assays were performed aspreviously described (27).Cellswere fixed in 1%paraformaldehyde for 10minat room temperature. Nuclei were isolated using Nuclei EZ Prep Lysis Buffer(Sigma-Aldrich). Nuclei were then incubated in nuclear lysis buffer (50 mMTris-Cl [pH8.1], 10mMEDTA, and1%SDS)on ice for 10min, and chromatinDNAwas sheared to ∼500 bp average size with a sonicator (Misonix S-3000,Cup horn 431B, Misonix, Farmingdale, NY). Sheared lysates were spun at16,0003 g for 10 min at 4˚C. The supernatant was harvested, diluted 10-foldwith immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100,1.2mMEDTA, 16.7mMTris-Cl, and 167mMNaCl [pH8.1]), preclearedwithsalmon sperm DNA/protein A agarose beads (Millipore, Billerica, MA; 16-157), and incubatedwithanti-IRF1 (SantaCruz, sc-640x) or anti-acetyl histoneH4 (Millipore,06-866)Ab for 4h at4˚C.Ab-boundchromatinwasprecipitatedwith salmon sperm DNA/protein A agarose beads. Beads were washed fivetimes with immunoprecipitaton washing buffer (100 mM Tris-Cl, 500 mMLiCl, 1% Nonidet P-40, and 1% deoxycholic acid [pH 8]) and eluted withelution buffer (50 mMNaHCO3 and 1% SDS). Crosslinking was reversed byincubation in 300 mM NaCl overnight at 67˚C, and chromatin DNAwas pu-rified via phenol/chloroform extraction. Chromatin recoverywas quantified byqPCR with primers for murine IFN-a4 promoter (sense 59-GGG AGG GTATTCCGAAAGG-39; antisense 59-TCTGGGCTGTGGGTT TGAG-39) ormurine IFN-b promoter (sense 59-GCT CCA GCA ATT GGT GAA AC-39;antisense 59-CAAAGGCTGCAGTGAGAATG-39). Primers were designedusing Clone Manager Suite v9.0 (Scientific & Educational Software). ABLAST search was then performed to verify specificity.

Statistical analysis

Data for qRT-PCR were analyzed based on normalized copy number (NC),defined as 1000 3 (NC of mRNA of interest)/(NC of GAPDH). The foldinduction was computed as (NC of treated cells)/(NC of untreated cells).Unless otherwise stated, data points show themean of triplicate sampleswithSD, and p values were calculated with two-tailed Student t test. ELISAfigures show pooled data from three independent experiments, each of whichincluded independent duplicate samples for each condition, resulting in sixvalues for calculation of mean and SD. Statistical analysis was performedwith SigmaStat version 3.1 (SysTat Software, Chicago, IL) for two- or three-way ANOVA, and p values were obtained by Student-Newman-Keulsmethod. In all figures, error bars represent SD; where not shown, they aresmaller than the symbol size.

ResultsHigh concentrations of CpG-B ODNs inhibit CpG-A–inducedproduction of IFN-ab by murine DCs

Although CpG-AODNs and CpG-B ODNs have similar potency forinduction of IFN-ab, CpG-A ODNs are much more efficacious inthis regard (9). CpG-B ODNs demonstrate a biphasic dose-responsecurve in which the induction of IFN-ab declines with increasingCpG-B ODN concentrations above 0.1 mM (9, 10). This biphasicresponse is unique to the induction of IFN-ab, because high con-centrations of CpG-B ODNs effectively induce TNF-a and IL-12p40in both splenicDCs and FLT3L-inducedDCs (10). It has been unclearwhether CpG-B ODNs simply lack activity possessed by CpG-AODNs that is required for efficacious induction of IFN-ab or if higherconcentrations of CpG-B ODNs actively induce a mechanism thatinhibits induction of IFN-ab.Our studies addressed the hypothesis that high concentrations

of CpG-B ODNs may specifically inhibit induction of IFN-ab.DCs were incubated with CpG-A ODN and/or CpG-B ODN.Because our previous studies established consistent patterns ofactivity for the different ODN classes with multiple CpG-A andCpG-B ODNs [(9) and data not shown], our current studies fo-cused on representative ODNs, including CpG ODN-A2336, CpGODN-A2216, CpG ODN-B1668, CpG ODN-B1826, CpG ODN-B2006, and non-CpG ODN-B2138.DCs from FLT3L-induced mouse bone marrow cultures (contain-

ing pDCs andmDCs)were stimulated for 24hwithCpGODN-A2216or CpG ODN-B1826 at varying concentrations (Fig. 1A), and super-natants were assessed by ELISA for production of IFN-a. At higher

concentrations (.0.1 mM), CpG-A ODN was more efficacious thanCpG-B ODN for induction of IFN-ab. CpG ODN-B1826 inducedIFN-ab expression only at lowerODNconcentrations (0.01–0.3mM)with a biphasic dose-response curve and decreasing induction of IFN-ab at higher ODN concentrations. These results confirm that CpG-AODN and CpG-B ODN have similar potency, but CpG-A ODN hasmuch higher efficacy, consistent with prior observations (9).To explore the impact of CpG-B ODN on induction of IFN-ab,

FLT3L-induced DCs were incubated with a maximal dose (3 mM) ofCpG ODN-A2336 and varying concentrations of CpG ODN-B1668(Fig. 1A). Interestingly, induction of IFN-a by CpG-A ODN was in-hibited by CpG-B ODN at concentrations as low as 0.03 mM, andstrong inhibition was observed in the range of 0.1–0.3 mM, mirroringthe concentrations of CpG-BODNatwhich the downturn occurs in thebiphasic CpG-B ODN dose curve for induction of IFN-a. In sub-sequent experiments, DCs were incubated with CpG ODN-A2336(3 mM), CpG ODN-B1668 (30 nM or 3 mM), non-CpG ODN-B2138(30 nM), or mixtures containing both CpG ODN-A2336 and CpGODN-B1668 (both ODNs at 3 mM). Supernatants were assessed forIFN-a, IFN-b, and IL-12p40 by ELISA (Fig. 1B–D). Consistent withFig. 1A,CpGODN-B1668 induceda low level of IFN-ab at 30nM,butat 3 mM, it induced little or no IFN-ab, whereas 3 mM CpG ODN-A2336 induced IFN-ab effectively (Fig. 1B, 1C). As observed in Fig.1A, whenDCswere exposed to both CpG-AODNandCpG-BODNat3mM, the presence of CpG-BODN inhibited the induction of IFN-abby CpG-A ODN (Fig. 1B, 1C). Importantly, this effect was CpG se-quence-specific, as non-CpG-B ODN exerted significantly less in-hibition of IFN-a induction by CpG-AODN than the CpG-BODN. Incontrast, induction of IL-12p40 (Fig. 1D) andDCmaturation (assessedby induction of mRNA for CD40, CD80, and CD86 and reduced ex-pressionofmRNAforCIITA;Fig. 2)were effectively inducedbyCpG-A ODN, CpG-B ODN, or the combination of both ODNs at 3 mM.These data suggest that the inhibitory mechanism does not occur at thelevel of TLR-ligand binding or signaling components that are commonto all TLR9 responses and that the inhibitory mechanism selectivelyinvolves induction of IFN-ab.MyD882/2DCs failed to produce eitherIFN-ab or IL-12p40 in response to CpGODN (Fig. 1B–D), consistentwith thedependenceofTLR9signalingonMyD88. PurifiedmDCsandpDCs showed similar responses, where induction of IFN-ab by CpGODN-A2336 was inhibited by 3mMCpGODN-B1668 (Fig. 1E, 1F),despite some possible differences in the mechanisms of IFN-ab in-duction in the two cell types (28). In conclusion, CpG-B ODN (3mM)induces a novel inhibitory mechanism that inhibits the induction ofIFN-ab by CpG-AODN.

CpG-B ODN inhibits induction of IFN-a by CpG-A ODN inhuman PBMCs

Althoughmice and humans respond similarly to CpGODNs, speciesto species variability exists (5, 6). Therefore, studies were performedto confirm that CpG-B ODNs also inhibit induction of IFN-ab inhuman cells. We tested the effect of 3 mM CpG ODN-B2006 (aCpG-B ODN that is active for human cells) on the ability of CpGODN-A2336 to induce IFN-a expression by human PBMCs.PBMCs were incubated for 48 h with 3 mM CpG-A ODN, 3 mMCpG-B ODN, or a combination of both ODNs at 3 mM. Super-natants were assessed for IFN-a by ELISA. CpG ODN-B2006failed to induce IFN-a and inhibited induction of IFN-a by CpGODN-A2336 (Fig. 3). These data indicate that CpG-B ODN caninhibit the induction of IFN-a in human as well as mouse cells.

CpG-B ODNs inhibit induction of IFN-a mRNA within 3 h

To elucidate the mechanisms involved in CpG-B–mediated inhi-bition of IFN-ab expression, we used qRT-PCR to assess transcrip-tion of IFN-b and IFN-a genes at an early timepoint after the addition

The Journal of Immunology 3369

of CpGODN. FLT3L-DCs were incubated with CpG-A and/or CpG-BODNs (3mM).By3h,CpG-AODN induced transcription of IFN-band IFN-a genes, and this induction was inhibited by CpG-B ODN(Fig. 4). CpG-A also induced IFN-b and IFN-a mRNA at 6, 12, 24,and 48 h, and this induction was inhibited by CpG-B ODN at all ofthese time points (data not shown). These data indicate that CpG-BODN induces a mechanism to inhibit induction of IFN-b and IFN-agene transcription within 3 h of exposure to ODN.

CpG-B ODN inhibits induction of IFN-ab by multiple TLRs andother receptors, including MyD88-dependent andMyD88-independent receptors

Inhibition of IFN-ab induction by CpG-B ODN could occur at dif-ferent points along the signaling pathway for induction of IFN-ab byCpG-AODN. In principle, CpG-BODNs could competewith CpG-AODNs for binding to TLR9, affect signaling mechanisms upstream ofinitial induction of first-wave IFN-b and IFN-a4, or inhibit the auto-crine/paracrine IFN-ab positive feedback loop that produces second-wave IFN-ab. To investigate these possibilities, we tested the effect of

CpG-B ODN on induction of IFN-ab by agonists of several differentreceptors with varying downstream signal transduction molecules,including TLRs other than TLR9 (some MyD88-dependent, someMyD88-independent) and non-TLRs (MyD88-independent).FLT3L-inducedDCs or purified pDCs ormDCswere incubated for

24 h with agonists of various receptors with or without 3 mM CpGODN-B1668. CpG-B ODN inhibited induction of IFN-ab by in-fectious (Fig. 5A) or inactivated (data not shown) strain 52-SeV; in-fectious SeV signals through the cytosolic receptors RIG-I ormelanoma differentiation-associated gene-5 as well as TLR3 andTLR7, whereas inactivated SeV signals only through TLR7. More-over, CpG-B ODN inhibited responses to the MyD88-dependentTLR7 agonist R837 (Fig. 5D), a MyD88-independent TLR3 ago-nist (polyinosinic:polycytidylic acid; Fig. 5E, 5F), and a TLR-independent, MyD88-independent agonist that signals through aputative cytosolic receptor [dsB-DNA (16–19) ; Fig. 5E, 5F]. Finally,CpG-B ODN inhibited LPS-induced IFN-a protein in FLT3L-DCs(Fig. 5G); LPS signals through TLR4 via MyD88-dependent and-independent pathways. Non–CpG-B negative control ODN did not

FIGURE 1. CpG-B ODN at high concentration

inhibits CpG-AODN-induced production of IFN-ab by

murineDCs. FLT3L-inducedDCs (A–D), purifiedpDCs

(E), or purified mDCs (F) were incubated for 24 h with

CpG ODN. Supernatants were assessed by ELISA for

IFN-a, IFN-b, or IL-12p40. A, IFN-a was measured

postincubation of FLT3L-induced DCs with varying

doses of CpG ODN-A2216 and/or CpG ODN-B1826.

CpG ODN-A2216 at relatively high concentrations

($0.3 mM) was more efficacious than CpG ODN-

B1826. Conversely, CpGODN-B1826 induced IFN-ab

expression only at lower ODN concentrations (0.01–0.3

mM), and IFN-ab declined to undetectable levels at

higher CpG-B ODN concentrations ($1.0 mM), which

also inhibited induction of IFN-ab by CpG-A ODN.

Data points represent means of duplicate samples from

a representative experiment. B–D, FLT3L-induced DCs

from wild-type C57BL/6 mice (closed bars) and

MyD882/2 mice (open bars) were incubated for 24 h

with medium alone, CpG ODN-A2336 (3 mM), CpG

ODN-B1668 (30 nM or 3 mM), non-CpG ODN-B2138

(30 nM), a combination of CpG ODN-A2336 and CpG

ODN-B1668 both at 3mM(A3/B3), or a combination of

CpG ODN-A2336 and non-CpG ODN-B2138 both at 3

mM(A3/non-B3). CpG-BODNat 3mMinhibitedCpG-

A induction of IFN-a and IFN-b but not CpG-A in-

duction of IL-12p40. E and F, Purified pDCs and mDCs

were incubated with ODNs as in B–D, revealing similar

CpG-B–induced inhibition of both IFN-a and IFN-b in

both cell types (although mDCs produce less IFN-ab

than pDCs). Data points inB–F represent mean6SD of

ELISA results pooled from three independent experi-

ments (each with duplicate samples). #p, 0.05 by one-

way ANOVA and Student-Newman-Keuls method for

comparison of IFN-a, IFN-b, or IL-12p40 expressionby

DCs with CpG ODN versus DCs with medium alone.

CpG-BODN at 3mMproduced significant inhibition of

IFN induction by 3 mMCpG-AODN. pp, 0.05.

3370 CpG-B ODNs INHIBIT TYPE I IFN IN DENDRITIC CELLS

inhibit induction of IFN-ab (Fig. 5C). Thus, CpG-B ODN inhibitedthe induction of IFN-ab by agonists of multiple receptors, includingTLRs and non-TLRs that signal through bothMyD88-dependent andMyD88-independent pathways. These findings were reproducedwith purified pDCs and mDCs and confirmed for both IFN-a andIFN-b (Fig. 5H, 5I). Two important conclusions derive from thesefindings. First, the inhibitory mechanism does not depend on com-petition of CpG-B ODN with other agonists for TLR9, because in-ductionof IFN-ab byagonists ofmanyother receptors is inhibitedbyCpG-B ODN. Second, the inhibitory mechanism may lie down-stream of initial signal transduction molecules (e.g., MyD88) thatdiffer between these receptors and likely involves downstream sig-naling steps that are common to all of these receptors.Comparison with earlier data (Fig. 1D) (10) suggests that CpG-B-

induced inhibition may be restricted to the induction of IFN-abwithout inhibition of other cytokines, so we also tested the effect ofCpG-BODNon induction of IL-12p40by strain 52-SeV, anagonist ofTLR7 (MyD88-dependent). Strain 52-SeV–induced IL-12p40 wasnot inhibited by CpG-B ODN (Fig. 5B), further indicating that CpG-B–induced inhibition is selective to the IFN-ab signaling pathway.Similarly, TNF-a protein expression induced by other TLR agonistswas not inhibited by CpG-B ODN (data not shown). Collectively,these data demonstrate a novel mechanism induced by CpG-B ODNthat selectively inhibits induction of IFN-ab (at a point of conver-

gence downstream of a variety of receptors) without inhibiting othercellular responses to activation of these receptors (Figs. 1D, 2, 5B, anddata on TNF-a that are not shown).

CpG-B ODN does not inhibit the IFN-induced positive feedbackloop and induction of second-wave IFN-ab in DCs

TLR signaling induces first-wave IFN-ab, which is restricted toIFN-b and IFN-a4, and the expression of these and other IFN-ab

FIGURE 2. CpG-A ODN and CpG-B ODN both induced DC maturation.

FLT3L-induced DCs were incubated for 3 h with or without 3 mM CpG

ODN-A2336, CpG ODN-B1668, or both ODN at 3 mM (A3/B3). Expres-

sion of mRNA for CD40, CD80, CD86, and CIITAwas determined by qRT-

PCR with normalization to GAPDH. A, Results for CD40, CD80, and

CD86, for which mRNA is increased by treatment with CpG ODN, are

expressed as fold-change relative to expression with medium alone. B,

Results for CIITA mRNA, which is decreased by treatment with CpG ODN,

are expressed as percent of control expression in medium alone. Data points

represent mean 6 SD for triplicate samples in a representative experiment.

Three independent experiments produced similar results. CpG ODN-A2336,

CpG ODN-B1668, and a combination of both ODNs all induced statistically

significant production of CD40, CD80 and CD86 mRNA (pp , 0.01 for

comparison with DCs with medium alone) and significant repression of

CIITA (#p , 0.05 for comparison with DCs with medium alone).

FIGURE 3. CpG-BODN inhibitsCpG-AODN-inducedexpressionof IFN-

a byhumanPBMCs.PBMCswere cultured for 48hwith orwithout 3mMCpG

ODN-A2336, 3mMCpGODN-B2006, orbothODNsat3mM(A3/B3). IFN-a

wasmeasuredbyELISA.Data represent five different donors (each assessed in

duplicate assays). Statistical analysis was done to compare the mean response

for the five different donors under each condition, and significance was as-

sessed with one-way ANOVA and Student-Newman-Keuls method. Results

reveal statistically significant (p, 0.05) induction of IFN-a by CpG-AODN

(comparison of PBMCs with CpG ODN-A2336 to PBMCs with medium

alone), higher induction of IFN-a by CpG ODN-A2336 than CpG ODN-

B2006 (p,0.05), and statistically significant (p,0.05) inhibitionofCpG-A–

induced IFN-a production by CpG-BODN (comparison of PBMCswith CpG

ODN-A2336 to A3/B3).

FIGURE 4. CpG-B ODN inhibits CpG-A ODN-induced IFN-a mRNA

expression. C57BL/6 FLT3L-induced DCs were incubated for 3 h with or

without 3 mMCpG ODN-A2336, CpG ODN-B1668, or both ODN at 3 mM

(A3/B3). Expression of mRNA for IFN-a and IFN-b was determined by

qRT-PCR with normalization to GAPDH and is expressed as fold-change

relative to expression with medium alone. Data represent mean and SD for

triplicate samples in a representative experiment. Three independent ex-

periments produced similar results. CpG ODN-A2336 induced statistically

significant production of IFN-a and IFN-b (#p, 0.01 for comparison with

DCs with medium alone), and CpG ODN-B1668 caused statistically sig-

nificant inhibition of CpGODN-A2336-induced IFN-a or IFN-b. pp, 0.01

for comparison with DCs with CpG ODN-A2336 alone.

The Journal of Immunology 3371

species can be substantially amplified by the autocrine/paracrineIFN-ab positive feedback loop, which induces second-wave IFN-ab(16, 18, 29). IFN-ab positive feedback involves signaling via theIFN-abR and consequent recruitment, phosphorylation, and heter-odimerization of STAT1 and STAT2, which associate with IRF9(IFN-stimulated gene factor 3 [ISGF3]-g) to form the STAT1/2:IRF9complex (ISGF3). ISGF3 translocates to the nucleus and inducestranscription of many genes, including the IFN-ab genes, inducinga second wave of IFN-ab (30). This mechanism is distinct from theTLR/MyD88 signaling pathway that induces the initial wave of IFN-ab. Therefore, in theory, the CpG-B–mediated inhibition of IFN-abexpression could involve inhibition of either first- or second-waveIFN-ab.We used two approaches to determine whether CpG-B–induced

inhibition of IFN-ab expression involves inhibition of the IFN-abpositive feedback loop to reduce second-wave IFN-ab. First, we in-cubated DCs for 24 h with 200 pg/ml rIFN-ab (rIFN-a or rIFN-b)with or without 3 mMCpG ODN-B1668; the cells were then washedand incubated for 24 h in chase medium without ODN or exogenousIFN to determine the subsequent release of endogenous IFN-a protein.CpG-B ODN did not inhibit IFN-ab–induced secretion of IFN-aprotein (Fig. 6A). As expected, CpG-B ODN did inhibit IFN-ab in-duced by CpG-A ODN; the inhibitory state was not reversed during

the 24 h chase, suggesting that CpG-B–induced inhibition of IFN-abmay be long-lived. Second, we determined that CpG-B ODN in-hibited IFN-ab induction by CpG-A ODN in IFN-abR2/2 DCs(Fig. 6B), indicating that the inhibitorymechanism is not dependent onIFN-abR, which is needed for the IFN-ab positive feedback loop.These data indicate that the IFN-ab positive feedback loop is not in-hibited byCpG-BODNand suggest that the inhibition occurs at earliersteps in the initial induction of first-wave IFN-ab by TLR signaling.

CpG ODNs induce IRF1, but CpG-B ODN does not inhibit IRF1induction or nuclear localization

IFN-ab gene induction is dependent on a number of regulatory andtranscription factors, particularly the IRFs, including IRF1 (15), IRF3(28, 31, 32), and IRF7 (14, 28). The dependence of IFN-ab gene in-duction on different IRFs varies with cell type and stimulus. Inductionof IFN-b by TLR9 signaling in mDCs and macrophages requiresIRF1 but not IRF7, whereas IRF7 functions in IFN-b induction byCpG-AODN in pDCs (15). These considerations led us to examine theeffects ofCpG-AandCpG-BODNsonIRFexpressionand localization(localization of IRFs to the nucleus results from their activation).The expression and localization of IRF1, IRF3, and IRF7 were

determinedbyWesternblot ofwhole cell lysates (Fig. 7A) andnuclearand cytoplasmic fractions (Fig. 7B, 7C). IRF7was induced byCpG-A

A B C

D E F

G H I

FIGURE 5. CpG-B ODN inhibits induction of

IFN-ab by multiple TLRs and other receptors, in-

cludingMyD88-dependent andMyD88-independent

receptors. DCswere incubated for 24 hwith receptor

agonists, including infectious strain 52-SeV at mul-

tiplicity of infection 2.5 (SeV-2.5), inactivated strain

52-SeV at 5 mg/ml (In-Sev-5), R837 (0.3 mg/ml),

polyinosinic:polycytidylic acid (50 mg/ml), dsB-

DNA(10mg/ml),LPS(10ng/ml),CpGODN-A2336

(3 mM), CpG ODN-B1668 (3 mM), or non-CpG

ODN-B2138 (3mM).Theagonistsother thanCpG-B

ODNwere added either alone or in combinationwith

3 mM CpG ODN-B1668 (B3) or non-CpG ODN-

B2138 (non-B2138, also at 3mM). ELISAwas used

to measure IFN-a (A, C, D, F–I) or IL-12p40 (B).

Expression of mRNA for IFN-a and IFN-b was de-

termined by qRT-PCR with normalization to

GAPDH and is expressed as fold-change compared

with expression with medium alone (E). FLT3L-

induced DCs were obtained from wild-type

C57BL/6 mice (A–G) or MyD882/2 mice (A, B).

Purified pDCs (H) or mDCs (I) were from wild-

type C57BL/6 mice. Data points represent

mean 6 SD of pooled data from three in-

dependent experiments, each of which included

duplicate ELISA or triplicate qRT-PCR assays for

each condition. All panels show statistically

significant agonist-induced production of IFN-a

(#p, 0.05 for comparisonwithDCswithmedium

alone) and statistically significant inhibition of

agonist-induced IFN-a or IFN-b by CpG ODN-

B1668 (pp , 0.05 for comparison of DCs with

agonist alone versus DCs with agonist plus CpG

ODN-B1668). Statistical analysis was done with

one-way ANOVA and Student-Newman-Keuls

method.

3372 CpG-B ODNs INHIBIT TYPE I IFN IN DENDRITIC CELLS

after∼7 h (slightly at 3.5 h),whereas IRF1was induced earlier at 3.5 h(slightly at 1 h) (Fig. 7A). IRF3 was constitutively expressed and wasnot upregulated by CpG stimulation. Subcellular fractionation wasused to separate nuclear and cytoplasmic fractions (Fig. 7B, 7C);effective separation was confirmed by detection of a-tubulin (cyto-plasmic marker) and Ku-70 (nuclear marker). IRF1 appeared in boththe cytoplasm and nuclear fraction of CpG-activated DCs at both 4 h(Fig. 7B) and 12 h (Fig. 7C),whereas IRF7was not detected at 4 h and

appeared only in the cytoplasm at 12 h (the possibility of low levels ofnuclear IRF7below the limit of detection in this assay or translocationof IRF7 after 12 h cannot be excluded). IRF3 was localized to thecytoplasmandnot detected in thenucleus at both4hand12h (Fig. 7B,7C). The addition of CpG-B to the stimulation condition (along withCpG-A) did not change the expression or localization of IRF1, IRF3,or IRF7. Our analysis subsequently focused on IRF1, because it wasinduced earlier than IRF7 within the time frame that CpG-inducedinhibition was observed. In contrast, IRF7 was induced later and wasnot present at early time points (e.g., 1–3 h) at which IFN-ab wasalready induced by CpG-A and CpG-B inhibition was already ap-parent. Although IRF7 is an established regulator of IFN-ab ex-pression, a role for IRF1 in IFN-ab induction also has beenestablished by prior studies (15). This does not preclude a role forIRF7 in later phases of IFN-ab induction or a different role for IRF7in other cell systems, but it indicates that IRF1 is more likely thanIRF7 to contribute to the mechanisms for inhibition of IFN-ab ex-pression that are rapidly induced by CpG-B ODN.

A

B

FIGURE 6. CpG-B ODN does not inhibit second-wave IFN-ab–induced

IFN-ab in DCs. A, C57BL/6 FLT3L-induced DCs were incubated for 24 h

without addition (medium) or with 3 mM CpG ODN-A2336, 3 mM CpG

ODN-B1668, 200 pg/ml rIFN-aA, 200 pg/ml rIFN-b, both CpG ODN-

A2336 and CpG ODN-B1668 at 3 mM (A3/B3), or 200 pg/ml rIFN-aA or

rIFN-bwith 3mMCpGODN-B1668 (rIFN-a/B3 or rIFN-b/B3). Cells were

washed and incubated for an additional 24 h in chase medium without ODN

or exogenous IFN to determine the subsequent release of endogenous IFN-a

(assessed by ELISA). As a negative control, “no cells” indicates wells in

which rIFN-aA or rIFN-b was added without cells to eliminate cellular

production of IFN-ab and serve as a control for carryover of exogenous

rIFN-ab. Each data point represents the mean 6 SD of pooled data from

three independent experiments (each containing duplicate ELISA samples

for each condition). Results show statistically significant rIFN-ab–induced

production of rIFN-ab, unaffected by coincubation with CpG-BODN. #p,0.05 by one-way ANOVA and Student-Newman-Keuls method for com-

parison of DCswith rIFN-ab or rIFN-ab plus CpG-BODNversus DCswith

medium alone. B, DCs from wild-type and IFN-abR2/2 mice were in-

cubated for 3 h with or without 3 mMCpG ODN-A2336, 3 mMCpG ODN-

B1668, or bothODNs at 3mM(A3/B3). Expression ofmRNA for IFN-bwas

determined by qRT-PCR with normalization to GAPDH and is expressed as

fold-change relative to expression with medium alone. Data represent mean

and SD for triplicate samples in a representative experiment. Two in-

dependent experiments produced similar results. CpGODN-A2336 induced

statistically significant production of IFN-b (p, 0.001 for comparison with

DCs of the same IFN-abR genotype with medium alone), and CpG ODN-

B1668 caused statistically significant inhibition of CpG ODN-A2336-in-

duced IFN-b. #p , 0.001 for comparison with DCs of the same IFN-abR

genotype with CpG ODN-A2336 alone.

A

B

C

FIGURE 7. IRF1 is rapidly induced by CpG-A and enters the nucleus.

A, C57BL/6 FLT3L-induced DCs (8–103 106) were incubated for various

periods with 3 mM CpG ODN-A2336 with or without 3 mM CpG ODN-

B1668. Cell lysates were prepared and analyzed by Western blot for IRF1,

IRF3, IRF7, and actin (loading control). DCs (5–8 3 106) were incubated

for 4 h (B) or 12 h (C) with or without 3 mM CpG ODN-A2336, 3 mM

CpG ODN-B1668, or both ODNs at 3 mΜ (A3/B3). Nuclear and cyto-

plasmic fractions were analyzed by SDS-PAGE and Western blot with anti-

IRF1, anti-IRF3, or anti-IRF7. Tubulin was used as a cytoplasmic marker,

and Ku-70 was used as a nuclear marker. Because IRF7 was not detected in

B, an IRF7-positive control was added in the rightmost lane of the gel; this

sample was derived from whole cell lysate of the CpG-A2336 24 h con-

dition analyzed in A. Data are representative of at least three independent

experiments.

The Journal of Immunology 3373

CpG-B ODN inhibits IRF1 binding and histone H4 acetylation atIFN-a4 and IFN-b promoter sequences

Although IRF1 expression and nuclear localizationwere not inhibitedby CpG-B ODN, these events do not establish effective induction ofIFN-ab by IRF1. Accordingly, we assessed the hypothesis that CpG-B ODN inhibits binding of IRF1 to IFN-a4 and IFN-b promoter se-quences, resulting in inhibition of IFN-ab promoter activation. ChIPassays used anti-IRF1 Ab to precipitate IRF1 along with promotersequences to which it was bound, and qPCR was performed usingprimers specific for IFN-a4 or IFN-b promoter sequences to de-termine association of IRF1with these promoters. Incubation of DCswith CpG-AODN for 3 h induced IRF1 binding to both IFN-a4 andIFN-b promoters (Fig. 8A, 8B). CpG-BODNwasmuch less effectiveat inducing IRF1 binding to these promoters. Furthermore, CpG-BODN inhibited induction of IRF1 binding by CpG-A ODN (A3/B3condition). As predicted by kinetic studies of IRF7 expression (Fig.7A), ChIP studies with anti-IRF7 Ab revealed no specific associationof IRF7 with the IFN-a4 and IFN-b promoters at 3 h; at this timepoint, IRF7 expression is not yet induced, yet the CpG-B inhibitoryeffect is already present. Thus, inhibition of IRF1 binding to IFN-a4and IFN-b promoters may contribute to CpG-B ODN inhibition ofIFN-ab induction.Changes in transcription factor binding can be associated with

altered local chromatin modification. Histone H4 acetylation was

previously reported to be associated with IFN-b transcriptional acti-vation (33). Accordingly, we assessed the effect of CpG ODNs onacetylation of histone H4 at the IFN-a4 and IFN-b promoters. ChIPassays using anti-acetyl histone H4 indicated that CpG-A ODN in-duced histone H4 acetylation at both IFN-a4 and IFN-b promoters(Fig. 8C, 8D), whereas CpG-B ODN did not. Furthermore, CpG-BODN inhibited CpG-A ODN-induced histone H4 acetylation whenboth ODNs were added together. This correlated with lower IRF1binding with CpG-B ODN and is consistent with decreased tran-scriptional activity at the IFN-a4 and IFN-b promoters.To demonstrate the promoter specificity of changes in histone H4

acetylation revealed by ChIP, we compared the results for the IFN-a4and IFN-b promoters to the results for CIITA promoter I, which israpidly repressed via histone deacetylation uponDCmaturation (34).ChIP with antiacetyl histone H4 and qPCR with CIITA promoter I-specific primers (35) showed a profound decrease in acetylation ofhistoneH4 at CIITA promoter I posttreatment with 3mMCpGODN-A2336, 3 mM CpG ODN B1668, or both ODNs at 3 mM (three in-dependent experiments, data not shown). These results are consistentwith the CIITA mRNA expression data shown in Fig. 2 and indicatethat theChIPassays reveal promoter-specific increasesor decreases inassociation of acetylated histone H4.

DiscussionPreviousstudies showed thatCpG-BODNs induce lowlevelsof IFN-abat low ODN concentrations but fail to induce IFN-ab at higher ODNconcentrations (9, 10). Previously, however, the mechanism for this bi-phasic response was unknown, and it was unclear whether CpG-BODNs simply lacked activity possessed by CpG-A ODNs that is re-quired for efficacious induction of IFN-ab or if higher concentrations ofCpG-B ODNs actively induce a mechanism that inhibits induction ofIFN-ab. Our studies reveal that CpG-B ODNs actively induce a mech-anism that inhibits expression of IFN-ab mRNA downstream of sig-naling by a wide variety of distinct receptors, including TLR9, TLR7,TLR3, TLR4, and a putative cytosolic receptor for dsB-DNA that hasbeen linked to RIG-I (19). Moreover, the IFN-ab response to SeV,potentially mediated by several membrane-bound and cytosolic re-ceptors, was also blunted by CpG-B ODNs. The inhibitory mechanismappears to be selective for induction of IFN-ab, becauseTNF-a and IL-12p40 expression are unaffected. In addition to FLT3L-induced DCs,which contain bothmDCs and pDCs, we observed the CpG-B–inducedinhibitory effect in purified mDCs and pDCs. These data suggest thatmDCs and pDCs, althoughmechanistically different in some aspects ofCpG ODN responses and signaling (28), are both affected by CpG-Binhibition. CpG-B–induced inhibition of IFN-ab is apparent at themRNA level as early as 3 h posttreatment with CpG-B ODN and issustained for at least 48hposttreatment.Weconclude thatCpG-BODNsinduce a novelmechanism that selectively inhibits the induction of IFN-ab in DCs by multiple pattern recognition receptors.Examination of different receptors and signaling pathways

provides some insight into the components of TLR9 and IFN-absignaling pathways that may be affected by CpG-B ODN. BecauseTNF-a and IL-12p40 expression are not affected by CpG-B ODN, theinhibition does not appear to occur at the level of TLR9 binding orinitial signal transduction molecule(s) (e.g., MyD88) that are commonto all signaling downstream of TLR9. Furthermore, because CpG-BODN inhibited IFN-ab induction through both MyD88-dependentand MyD88-independent receptors, the inhibitory mechanism mustinvolve components at or downstream of the convergence of thesediverse signaling pathways. Our finding that IFN-ab–induced second-wave IFN-ab production was not inhibited by CpG-B ODN indicatesthat the mechanism involves inhibition of first-wave IFN-b andIFN-a4, possibly by regulation of the activity of transcription factorsthat bind and regulate the IFN-a4 and IFN-b promoters.

B

D

A

C

FIGURE 8. CpG-BODNinhibits induction of IRF1binding andhistoneH4

acetylation at the IFN-a4 and IFN-b promoters. ChIP assessment of IRF1

binding to IFN-a4 (A) and IFN-b (B) promoter sequences. ChIP assessment of

histone H4 acetylation at the IFN-a4 (C) and IFN-b (D) promoters. For ChIP

analyses, DCs (8–103 106) were incubated for 3 hwith or without 3mMCpG

ODN-A2336, 3mMCpGODN-B1668, or bothODNs at 3mM(A3/B3). Cells

were fixed, nuclei were isolated, chromatin was sheared, and immunoprecip-

itation was performed with anti-IRF1 or anti–acetyl-histone H4. Precipitates

and input sheared chromatin were both analyzed by qPCR using primers for

IFN-a4 or IFN-b promoter sequences. Data are presented as percentage of

promoter sequence in the sheared chromatin that was immunoprecipitated.

CpG-A 2336 ODN (A3 condition) induced statistically significant IRF1

binding and histone H4 acetylation at both IFN-a4 and IFN-b promoters (p,0.05 for comparison with control treatment with no CpG ODN). The B3 and

A3/B3conditions produced statistically significant inhibition relative to theA3

condition for both IRF1binding (#p, 0.05 for comparisonwithDCswithCpG

ODN-A2336 alone) and histone H4 acetylation (pp , 0.01 for comparison

with DCs with CpG ODN-A2336 alone) for both IFN-a4 and IFN-b pro-

moters. B–E are representative of at least three independent experiments.

3374 CpG-B ODNs INHIBIT TYPE I IFN IN DENDRITIC CELLS

Waibler et al. (36) recently described a mechanism by which in-duction of IL-10 expression by mDCs inhibits IFN-ab expression bypDCs. Although such a mechanism could contribute to regulation ofIFN-ab, our findings indicate some differences from thismodel. First,we observed inhibition of IFN-ab induction by CpG-B ODN in pu-rified pDCs as well as mDCs; an mDC-mediated IL-10 paracrinemechanism is excluded in the purified pDC system. Similarly, we re-capitulated the same inhibitory effect in human PBMCs, even thoughhuman mDCs (and monocytes) do not express TLR9. In addition, ourmRNA data suggest a profound inhibitory effect as early as 3 h; al-thoughadditional studies are needed to assess possible contributions ofparacrine mechanisms, the mechanism must explain rapid regulationof IFN-ab expression, consistent with direct effects of TLR signalingor the involvement of rapidly induced paracrine mediators.Our results suggest that CpG-BODNs inhibit IFN-ab induction at

a stage that is after IRF activation and nuclear translocation and thataffects the induction of first-wave IFN-ab (IFN-a4 and IFN-b), notthe IFN-ab positive feedback loop. Although IRF7 is known tocontribute to IFN-ab regulation at some time points in pDCs, IRF1 ismore likely to contribute to the CpG-induced inhibitory mechanism,because IRF1 was induced and translocated into the nucleus rapidly,within 1–3 h, when the CpG-induced inhibition becomes apparent. Incontrast, IRF7was not induceduntil∼7 h, and IRF3was not regulatedby CpG-A or CpG-B ODN. Moreover, ChIP at 3 h postexposure toCpG-AODN showed association of IRF1with the IFN-a4 and IFN-bpromoters that was inhibited by CpG-B ODN. In contrast, ChIP re-vealed no specific association of IRF7 with these promoters at 3 hpoststimulation with CpG-A ODN. Consistent with the implied reg-ulation of first-wave IFN-ab in Fig. 6, the IRF1 and acetyl histoneH4ChIP results suggest that CpG-B ODN inhibits first-wave IFN-abmRNA by transcriptional regulation. Although IRF1 expression andnuclear localizationwere not inhibitedbyCpG-BODN,CpG-BODNinhibited binding of IRF1 to IFN-a4 and IFN-b promoter sequences,resulting in inhibition of promoter activation, inhibition of histoneH4acetylation, and decreased expression of IFN-ab mRNA.CpGODNsmodulate immune responses inmice and humans, but

the two species differ in some aspects of responses to CpGODNs. Inmice, mDCs, pDCs, B cells, and macrophages express TLR9, but inhumans, only pDCs and B cells express TLR9. There are also dif-ferences in the ODN sequences that are optimally recognized byhumanTLR9versusmouseTLR9.Nonetheless, theCpG-B–inducedinhibition of IFN-ab expression was demonstrated in humanPBMCs as well as mouse DCs. Thus, these data have relevance toinduction of IFN-ab in human cells and indicate the existence ofa mechanism for inhibiting expression of IFN-ab that might betargeted by future therapeutic agents developed for management ofautoimmune and other inflammatory pathological processes.

AcknowledgmentsWe thank Dr. Hung-ying Kao and Xiwen Cheng (Case Western Reserve

University), who provided valuable advice and assistance for ChIP assays.

We also thank Dr. Shizuo Akira (Osaka University) for generously provid-

ing MyD882/2 mice.

DisclosuresThe authors have no financial conflicts of interest.

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3376 CpG-B ODNs INHIBIT TYPE I IFN IN DENDRITIC CELLS