C/EBP� and STAT-1 Are Required for TLR8 TranscriptionalActivity*□S

Received for publication, April 14, 2010, and in revised form, August 3, 2010 Published, JBC Papers in Press, September 9, 2010, DOI 10.1074/jbc.M110.133884

Claudia Zannetti‡§1, Francois Bonnay¶, Fumihiko Takeshita�, Peggy Parroche‡§, Christine Menetrier-Caux**,Massimo Tommasino‡, and Uzma A. Hasan‡§

From the ‡Infection and Cancer Biology Group, International Agency for Research on Cancer, Lyon 69008, France, §Infection,Immunity, and Vaccination, INSERM U851, Faculty of Medicine, University of Claude Bernard, Pierre Benite 69310, France, ¶UPR9022, CNRS, Institute of Molecular and Cellular Biology, Strasbourg 67084, France, the �Department of Molecular BiodefenseResearch, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan, and **Cytokines and Cancer,INSERM U590, Centre Leon Berard, Lyon 69008, France

Toll-like receptor 8 (TLR8), which is expressed primarily inmyeloid cells, plays a central role in initiating immuneresponses to viral single-stranded RNA. Despite the great inter-est in the field of TLR8 research, very little is known in terms ofTLR8 biology and its transcriptional regulation. Here, wedescribe the isolation of the hTLR8 promoter and the character-ization of the molecular mechanisms involved in its regulation.Reporter gene analysis and ChIP assays demonstrated that thehTLR8 regulation of the basal transcription is regulated viathree C/EBP cis-acting elements that required C/EBP� andC/EBP� activity. In addition,we observed thatR848 stimulationincreasesTLR8 transcriptional activity via an enhanced bindingof C/EBP�, and not C/EBP�, to its responsive sites withinthe TLR8 promoter. Moreover, we showed that IFN-� alsoincreased TLR8 transcription activity via the binding of STAT1transcription factor to IFN-� activated sequence elements onthe TLR8 promoter and enhanced TLR8 functionality. Theseresults shed new light on the mechanisms involved duringTLR8-mediated innate immune response.

Key players of the innate immune response are pattern-rec-ognition receptors that sense microorganisms and activateantimicrobial defenses (1, 2). Pattern-recognition receptorsalso comprise Toll-like receptors (TLRs),2 a family of trans-membrane proteins that recognize conserved microbial struc-tures named pathogen-associated molecular patterns. TLR7,-8, and -9 are grouped together phylogenetically and are locatedin intracellular endolysosomal compartments to recognize viralinfection in the form of foreign nucleic acids (3). Extensiveresearch has been performed in the field of TLR7 and -9 withthe assumption that TLR8 mobilizes in a similar manner (4);however, direct studies on TLR8 are in fact very limited.Restrictions in research are due to the existence of a nonfunc-tional TLR8 in mice, thus making in vivo studies difficult (5).The hTLR8 is predominantly expressed in lung and peripheralblood leukocytes and lies in close proximity to TLR7 on chro-

mosome X (6). These receptors present overlapping specificityin sensing ssRNA viruses including HIV, sendai virus, coxsackiB virus, and parechovirus 1 (7–9) leading to a possible redun-dancy among these receptors. TLR8 is abundant in myeloidcells and induces more effectively TNF-�, IL-12, and IL-6,whereas TLR7 and TLR9 and not TLR8 (10, 11), are stronglydetected in plasmacytoid dendritic cells and produce type I IFNin response to their respective ligands (5). Many studies havedemonstrated that the mRNA levels of different TLRs changeafter activation of immune response (12, 13). These changes inTLR expression could have positive or negative consequencesin regulation of an immune response. So far, the mechanismsresponsible for the changes in TLR expression are poorlydefined, and only few studies focused on the characterization ofTLR transcriptional regulation (12–16).C/EBP, CREB, Ets, and NF-�B transcription factors have

been identified as important regulators of the transcriptionactivity of the TLR9 promoter (13), whereas IRF-1 has beenshown to play a role in inducing the TLR7 mRNA level aftertype I IFN stimulation (12). In terms of transcriptional regula-tion of TLR8, nothing is currently known. In this report, wedescribe the molecular mechanisms required for basic andinducible expression of the human TLR8 gene.

EXPERIMENTAL PROCEDURES

Stimuli Used—LPS (InvivoGen), R848 (InvivoGen), IFN-�(catalog no. I3265, Sigma Aldrich), IL-6 (Peprotech), TNF-�(210-TA, R&D Systems), and uridine (Sigma Aldrich).Cell Culture—THP-1 were kindly provided by Jurge

Tschopp (University of Lausanne, Lausanne, Switzerland)and cultured as described previously (17). Peripheral bloodmononuclear cells and monocyte-derived dendritic cells(moDCs) were generated as described previously (18).Monocytes were purified from peripheral blood mononu-clear cells using CD14-positive selection (Miltenyi) andcultured as described previously (18). Huh-7 cells (kindlyprovided by Alex Weber, Deutsches Krebsforschungszen-trum) and HEK293 were grown in the same conditions asdescribed previously (19).Cloning of the hTLR8Gene 5�-Flanking Region and Construc-

tion of hTLR8 Gene Promoter-dependent Luciferase ExpressionVectors—Supplemental Fig. 1 shows the sequence of the clonedTLR8 promoter. The 5�-flanking region of the hTLR8 gene was

* This work was founded by “Lyon BioPole” and INSERM.□S The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. 1–11.1 To whom correspondence should be addressed. E-mail: claudia.zannetti@

inserm.fr.2 The abbreviations used are: TLR, Toll-like receptor; ssRNA, single-stranded

RNA; qPCR, quantitative PCR; GAS, IFN-� activated sequence.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 45, pp. 34773–34780, November 5, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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PCR-amplified from human genomic DNA with the ExpandHigh FidelityPLUS PCR System (Roche Applied Science) usingthe primers as listed in supplemental Fig. 11 and cloned into theNheI-HindIII site of the pGL3 basic vector (Promega).Expression Vector Constructs—Expression vectors for Spi1

(PU.1), SpiB, SpiC, CREB1, C/EBP�, C/EBP�, C/EBP�,C/EBP�, C/EBP�, c-Jun, c-Fos, p65, p50, c-Rel, IRF-3, and IRF-3constitutively active have been described previously (13).Expression vectors for IRF-1, IRF-5, and IRF-7 have been kindlyprovided by Kate Fitzgerald (UMass Medical School).Transient Transfection and Luciferase Assay—Transient

transfections were performed in duplicate using FuGENE 6(Roche Applied Science) as described previously (19).Antibodies—Anti-C/EBP� (M-17), anti-C/EBP� (C-19), and

anti-Stat1 p84/p91 (E-23) antibodies were supplied by SantaCruz Biotechnology. Antiphospho-Stat1 Tyr701 and anti-PARPantibodies were supplied by Cell Signaling Technology. Anti-IgG control was supplied byDiagenode. Peroxidase-conjugatedanti-rabbit or anti-mouse secondary antibodies were fromPromega.Immunoblot Analysis—To obtain cytoplasmic and nuclear

extracts 7 � 106 cells were harvested, lysed as described previ-ously (41). Twenty �g of protein extracts (determined by theBradford assay, Bio-Rad) were used for immunoblotting. Pro-teins were detected using ECL (Amersham Biosciences).Electrophoretic Mobility Shift Assay—EMSA and supershift

assays were performed using the C/EBP (1) EMSA kit andStat-1 EMSA kit (Panomics, Fremont, CA). For each bindingreactions, 5 �g of nuclear extracts (see under “ExperimentalProcedures”) were used.Chromatin Immunoprecipitation Assay—ChIP assay was

performed using the Shearing Optimization kit and the One-Day ChIP kit (Diagenode, Philadelphia, PA). For THP-1 cells,the sonication cycle lasted 15 s with 5 s on and 2 s off at 20% ofamplitude and was repeated six times. Immunoprecipitationwas performed overnight on a rotating wheel at 4 °C. 2.5 �l/re-action ofDNA solutionwas used for qPCR.The primers used toamplify TLR8 promoter regions are listed in supplementalFig. 11.ELISA—THP-1 were seeded at 1 � 105 cells/96 wells, pre-

treated for 3 h in triplicate with IFN-� or left untreated, washedtwice with PBS, and then stimulated with R848 and uridine.Supernatantswere harvested 24 h after treatment for analysis ofIL-8 or IL-6 (R&D Systems).RT-qPCR—Total RNA was extracted from cells using the

RNeasymini kit (Qiagen,Hilden,Germany). cDNAwas synthe-sized with the First Strand cDNA synthesis kit (Fermentas).Mx3000P real-time PCR system (Strategene, La Jolla, CA) wasused to perform qPCRwithMesa Green qPCRMasterMix Plus(Eurogentec), TLR8, TLR7, and �2-microglobulin-specificprimers (supplemental Fig. 11). Each reaction was run induplicate.Statistical Analysis—GraphPad (version 5) was used to cal-

culate unpaired and paired p values: ***, extremely significant,p � 0.001; **, very significant, p � 0.001–0.01; *, significant, ,p � 0.01–0.05; and ns, p � 0.05.

RESULTS

Identification of the TLR8 Regulatory Region—The 16.5-kbTLR8 gene is located on chromosome X and is composed ofthree exons (20). Fig. 1A diagrammatically depicts the existenceof two splice variants of TLR8 (isoform I and isoform II) with asole transcriptional start site (6).Wedetermined the expressionof the two variants in cells known to express functional TLR8,i.e. monocytes (using the THP-1 cell line), peripheral bloodmononuclear cells, and MoDCs. The presence of the TLR8transcript levels were determined by RT-PCR using specificprimers that would detect for the two spliced forms. Weobserved, however, that three bandswere amplified in the threecell types tested (Fig. 1B). Sequencing of the amplified productsled to the identification of isoform I (356 bp) and isoform II (220bp) in the cell types tested (as verified using BLAST search).The isoform IIwas highly expressed in bothTHP-1 andprimarycells. Becauseboth isoformscontain the same transcriptional startsite, we hypothesized that the upstream region of exon I is respon-sible for the transcriptional regulation of TLR8 gene. To consoli-date this hypothesis, a comparative analysis of TLR8 genomicsequences derived from different mammals (dog, human andmouse) showed that the exon I upstream sequences were indeedhighly conserved using FamilyRelations II software.To determine whether this region exerts transcriptional reg-

ulatory functions, the genomic area spanning�3496 bp to�47bp of the transcriptional start site was cloned into the luciferasereporter plasmid pGL3 and analyzed by transient transfection

FIGURE 1. The human TLR8 gene, the regulatory region, and splice vari-ants. A, physical map of the human TLR8 gene. Exons 1–3 are indicated. Openand dark gray boxes represent the untranslated and coding regions, respec-tively. Black boxes mark the position of high homology sequences amongmammals. The �3496-bp regulatory region is indicated above the TLR8 genestructure. Isoform I has an extended 19-amino acid (aa) N terminus encodedby exon 2. Isoform II uses an initiator methionine in exon 1 and lacks exon 2.The rest of the protein (1040 aa) is encoded by exon 3 shared by both splicedvariants. B, determination of the TLR8 isoforms variants in THP-1, moDCs, andperipheral blood mononuclear cells. RT-PCR was performed using primerslisted in supplemental Fig. 11. An asterisk indicates a nonspecific band. C, the�3496 TLR8 promoter is constitutively active in THP-1 cells. THP-1 cells weretransfected with the �3496 TLR8 promoter cloned in front of the lucifer-ase (Luc) reporter gene. Cells were harvested as described previously (19).The data are the mean of three independent experiments.

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in THP-1 cells. The luciferase construct displayed a 10-foldincrease in activity in comparison with the empty vector (Fig.1C). We determined whether another 1.6-kb genomic frag-ment, which also is conserved among mammals and is locatedupstream exon II could display transcriptional activity whencloned in front of the luciferase reporter gene. Transient trans-fection experiments in THP-1 cells showed that this fragmentwas inactive (supplemental Fig. S2). The activity of the �3496TLR8 promoter was myeloid cell-specific, as we did not observeluciferase expression in nonmyeloid cells such as HEK293 orHuh-7 cell lines (supplemental Fig. S3i and S3ii). Together, theseresults indicate that the regulatory regions responsible for thebasal TLR8 transcription are comprised in a 3.5-kb-long regionlocated upstream of exon I and their activity is myeloidcell-specific.

C/EBP� and C/EBP� Regulate Basal Transcription Activityof the TLR8 Promoter—Using MatInspector software (Geno-matix), we performed a computational sequence analysis of theTLR8 promoter region and found no canonical TATA box.Weidentified the presence of several putative responsive cis-elements such as CCAAT/enhancer binding proteins(C/EBP), STAT, IRF, Ets, and NF-�B family members (Fig.2A). To determine their effect on TLR8 trans-activity, weco-transfected the �3496 TLR8 promoter with the con-structs expressing different transcription factors includingCRE-associating proteins (CREB1), Ets proteins (SpiB, SpiC,Spi1(PU.1)), C/EBPs (C/EBP�, C/EBP�, C/EBP�, C/EBP�,and C/EBP�), interferon-regulated factors (IRF1, IRF3, IRF5,and IRF7), NF-�B proteins (p65, p50, and c-Rel) and AP-1proteins (c-Fos and c-Jun).

FIGURE 2. The role of C/EBP� and C/EBP� in driving basal TLR8 expression. A, identification of the putative responsive elements on the TLR8 promoter. Motifscanning was performed (Genomatix), and putative transcription factor binding sites identified are indicated. B and C, characterization of the trans-activity ofknown transcription factors on the TLR8 promoter. THP-1 cells were co-transfected with the expression vectors for a panel of different transcription factors withthe �3496 TLR8 promoter, and luciferase (Luc) activity was measured post-48 h. Values represent the mean of three independent experiments. D, THP-1 displaya basal C/EBP� DNA binding activity. A biotin-labeled C/EBP commercial probe was used in an EMSA using THP-1 nuclear extracts. For competition analysis, anexcess of the unlabeled probe was used. For supershift assays, antibodies against C/EBP family members are indicated above each line. E, ChIP assay deter-mining the in vivo binding of C/EBP� and C/EBP� to the TLR8 promoter in THP-1 cells. Chromatin was immunoprecipitated with the indicated antibodies. Arabbit IgG isotype control was used as negative control. The sites A, B, C, D, and E on the TLR8 promoter were amplified by qPCR from DNA-transcription factorcomplexes, and the values obtained are referred as relative to the initial amount of DNA (input). F, deletion analysis of the TLR8 promoter. Progressively deletedTLR8 promoter fragments were transfected into THP-1. After 48 h, cells were processed as described previously (19). Values represent the mean of threeindependent experiments.

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Overexpression of C/EBP� strongly activated the �3496TLR8 promoter (Fig. 2B), whereas a slight increase in luciferaseactivity (1.8-fold) was observed upon transfection of C/EBP�.By contrast, the other transcription factors did not have anysignificant effect on the TLR8 promoter when expressed alone.We observed synergistic enhancements on the �3496 TLR8promoter when co-expressing p50 with p65 or c-Rel proteins,suggesting a positive role played by NF-�B heterodimers (Fig.2C). Conversely, the co-expression of additional transcriptionfactors with C/EBP� did not further increase TLR8 promoteractivity versus C/EBP� alone (Fig. 2C). To corroborate the roleof C/EBP� in the basal transcription of theTLR8, we performedan EMSAusing nuclear extracts from the untreated THP-1 andC/EBP consensus probe. Two retarded bands were observed(Fig. 2D). Only the lower band was supershifted by an antibodyagainst C/EBP� but not by a C/EBP� antibody (Fig. 2D).We next determined whether C/EBP� and/or C/EBP� were

able to bind to the TLR8 promoter. We identified five putativeC/EBP responsive sites referred to as A, B, C, D, and E (Fig. 2A).We performed ChIP assays using THP-1 chromatin extracts.As shown in Fig. 2E, we observed a binding of both C/EBP� andC/EBP� at sites C (unpaired t test was applied compared withIgG control p � 0.006 and p � 0.0007 respectively), D, and E,which implies that these transcription factors play an impor-tant role in TLR8 basal transcription. No binding was observedat sites A and B (nonsignificant p values obtained for bothC/EBP� and C/EBP� compared with IgG alone). Accordingly,in a deletion assay using 5�-flanking region luciferase genehybrid constructs of the TLR8 gene promoter (Fig. 2F), thehighest level of luciferase activity was observed with the �849/�47 construct, which excludedC/EBP sites A and B (p� 0.006,Student unpaired t test). These data support the crucial roleplayed by sites C, D, and E to promote basal TLR8 expression.Additional deletions made from �849 to �396 and then from�396 to �172 regions, which excluded site C, then D and E,respectively, led to a further reduction of promoter basalactivity.TLR8 Transcription Regulation in Response to R848 and Proin-

flammatory Cytokines—Independent reports have showed previ-ously that certain T helper 1 cell-induced cytokines, includingIFN-�, IL-6, TNF-�, and TLR agonists such as R848 and LPSup-regulated TLR8 expression in myeloid cells (21, 22). Usingourmodel, we investigated the effect of the described cytokineson TLR8 transcription. As shown in Fig. 3A, IFN-� stronglyincreased TLR8 mRNA by qPCR at 3 h. An increase in TLR8mRNA also was observed after R848 treatment at 8 h. IL-6,TNF-�, and LPS also induced further TLR8 mRNA expressionbut only at later time points. These data indicate that IFN-� andR848 may directly up-regulate TLR8 transcription, whereasIL-6, TNF-�, and LPS may induce secondary response genesthat increase TLR8 mRNA levels. Accordingly, when we testedthe effects of these cytokines on the �3496 TLR8 promoterin transient transfection experiments, we observed that IL-6and TNF-� treatments induced marginal promoter activitycompared with nonstimulated cells (supplemental Fig. 4A).IFN-� significantly increased luciferase activity at 6 h (Fig. 3B,paired Student t test, p � 0.006) as well as at earlier time points(supplemental Fig. 4B). The induction of the promoter was also

observed after 16 h with R848 (paired Student t test, p� 0.006).These data are coherentwith the increase of TLR8mRNAaswenoted that R848 showed delayed kinetics in comparison toIFN-� stimulation (Fig. 3A). To corroborate our data in a morephysiological setting, we used moDC and human primarymonocytes to examine TLR8 levels. Similarly to THP-1 cells, aninduction of endogenous TLR8 gene was observed after treat-ments with IFN-� for 3 h, and at later times with R848 (Fig. 3,Cand D) or uridine (Fig. 3D). These results indicate that IFN-�-and TLR8-mediated signaling pathways are involved in theinduction ofTLR8 gene. Because TLR7 andTLR8 present over-

FIGURE 3. Transcriptional activation of TLR8 in myeloid cells. A, qPCR anal-ysis of the TLR8 expression in THP-1. Cells were serum-starved for 16 h andthen stimulated with R848 (10 �M), IL-6 (50 ng/ml), IFN-� (1000 units/ml), LPS(50 ng/ml), and TNF-� (10 ng/ml). The expression level of TLR8 was assessedby qPCR, normalized to the �2-microglobulin expression and expressed rel-ative to untreated cells. B, TLR8 promoter activity in R848- and IFN-�-treatedcells. THP-1 cells were stimulated as indicated with either R848 or IFN-� andharvested in total 48 h post transfection. Cells were then processed asdescribed previously for luciferase (Luc) activity (19). C and D, TLR8 geneexpression in moDC and primary monocytes. Cells were treated as indicated,and qPCR analysis of the TLR8 expression was performed in moDC (C) andprimary monocytes (D) as described above. Values represent the mean ofthree independent experiments. RLU, relative light units.

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lapping functions in sensing ssRNA motifs, we exploredwhether the two genes share similarities in terms of geneexpression upon TLR8 alone (uridine) or TLR7/8 (R848) stim-ulation. We observed that TLR7 expression is induced by R848at 3 h and by uridine at later times in THP-1 (supplemental Fig.5A). No significant induction of TLR7 was detected in moDC.(supplemental Fig. 5B). These data highlight the difference ingene expression among the two genes.R848 Enhances C/EBP� butNot C/EBP�-mediated Induction

of TLR8 Transcription—We have observed that C/EBP� andC/EBP� play an essential role in TLR8 basal transcription inmonocytes. Moreover, previous studies have shown thatCEBP� was induced by proinflammatory signals, includingTNF-� and IL-6, and that murine TLR4 stimulation leads to itsactivation in macrophages (23–25). We therefore determinedwhether activation of TLR8 signaling by R848 could lead toC/EBP� induction. For this purpose, the levels of the activatedCEBP� were analyzed by immunoblotting from untreatedTHP-1 cells or treated with R848, LPS, IFN-�, and cytokinesproduced by TLR8 activity such as IL-6 and TNF-�. �ineticswere performed at early time points indicative of a direct ligandeffect.Weobserved that nontreated cells expressedC/EBP� at abasal level (Fig. 4A). R848-TLR8 signaling triggered significantnuclear accumulation of C/EBP� as did other proinflammatorystimuli starting at 8 h for IL-6, IFN-�, and LPS and at 3.5 h withTNF-�. Notably, nontreated cells versus the R848-mediatedaccumulation of CEBP� levels tightly correlated with the R848-mediated induction of TLR8 promoter activity (Fig. 3A). Fur-thermore, we confirmed that human TLR4 also inducesC/EBP� (24).

To verify whether C/EBP� plays a role in the R848-mediatedinduction of TLR8 expression through the interaction with thecis-acting promoter elements, a ChIP assay was performed onC/EBP consensus sites A, B, C, D, and E in THP-1 stimulatedwith R848 for 8 h or left untreated (Fig. 4B). The qPCR analysisshowed that in response to R848, THP-1 displayed an increasedbinding of C/EBP� to sites C, D, and E, supporting the hypoth-esis that they are important for the R848-mediated TLR8 up-regulation (unpaired Student t test compared with C/EBP�binding in nonstimulated cells; p � 0.008, p � 0.006, and p �0.009, respectively). By contrast, C/EBP� appeared not to beinvolved in this signaling pathway. In fact, C/EBP� phosphoryl-ated levels were not affected by TLR8 activation (supplementalFig. 6), and no significant increase of binding of C/EBP� to sitesA, B, C, D, and E was observed after 8 h of R848 treatment inTHP-1 in comparison to untreated cells (supplemental Fig. 7).Therefore, in response to R848, C/EBP�, but not C/EBP�, isrecruited to the TLR8 promoter to further transactivate itsexpression, which suggests an auto-regulatory mechanism.IFN-� Triggers TLR8 Promoter Transactivation through

STAT-1 Binding Sites—Wehad observed previously that IFN-�increases the transcription of the TLR8 promoter (see Fig. 3, AandB). However, we did not note an increase inTLR8 promoteractivity when we overexpressed IRF family members (Fig. 2B).Therefore, we looked at the status of phosphorylation ofSTAT1 in THP-1 stimulated with IFN-�, IL-6, TNF-�, R848,and LPS versus untreated cells. We saw that phosphorylationand nuclear translocation of STAT-1 occurs at both 3.5 and 8 hafter treatment with IFN-� and R848 (Fig. 5A). We next per-formed gel shift experiments using nuclear extracts fromTHP-1 cells treated or not with IFN-� and R848 for 3.5 h and aGAS probe. IFN-�-stimulated cells displayed an enhancedDNA binding activity versus untreated cells (supplemental Fig.8i). R848 had no effect on DNA binding activity. Incubationwith STAT-1 antibody resulted in a decrease of IFN-�-inducedretarded band (supplemental Fig. 8ii).We performed a computational analysis and identified four

potential GAS cis-elements on the TLR8 promoter. These sitesare referred to as GAS1, GAS2, GAS3, and GAS4 (Fig. 2A). Tounderstand whether these sites are required for IFN-�-medi-ated enhanced TLR8 transcription, a transient transfectionassay was performed employing 5� deletion mutants progres-sively lacking promoter regionswithGAS-responsive elements.These data showed that IFN-�-mediated TLR8 transactivationis affected partially when deleting the promoter region contain-ing GAS2 and GAS3 elements (�1803 compared with �3496unpaired Student t test, p� 0.05; Fig. 5B). A further decrease ofpromoter activity was observed when deleting the region con-taining the GAS4 site (�396 compared with �3496 unpairedStudent t test, p � 0.05; Fig. 5B). To further demonstrate the invivo interaction of STAT1 with the GAS sites on TLR8 pro-moter, a ChIP assay was performed using STAT-1 antibody. Asshown in Fig. 5C, after 3.5 h of IFN-� treatment, chromatinextracts from THP-1 cells contained STAT-1, which bound toGAS-1, -2, -3, and -4 sites. No binding was observed inuntreated cells (Fig. 5C). These findings support a model inwhich TLR8 expression is induced upon IFN-� treatment. Fur-thermore, priming THP-1 cells with IFN-� for 3 h enhanced

FIGURE 4. C/EBP� nuclear accumulation and binding to TLR8 promoterspecific sites is induced upon R848 treatment in THP-1. A, determinationof C/EBP� nuclear levels in THP-1 by immunoblotting. PARP immunodetec-tion was performed as a loading control. B, ChIP determining the in vivo bind-ing of C/EBP� to the TLR8 promoter upon R848 treatment. Sheared chromatinfrom cells that have been stimulated R848 for 8 h was immunoprecipitatedwith C/EBP� or IgG control antibodies. Sites A, B, C, D, and E on TLR8 promoterwere amplified by qPCR, and specific binding was calculated relative to theinitial amount of DNA (input). NT, not treated.

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IL-8 and IL-6 production upon R848 or uridine treatment (Fig.5D). Together, these results show that IFN-� affectsTLR8 tran-scriptional level through STAT-1 transcription factor andenhances TLR8-mediated functionality.

DISCUSSION

To date, it remains difficult to consolidate a specific role forTLR8 due to its overlapping function with TLR7. However, adirect role of TLR8 in innate immunity is emerging. Recent datareported HIV ssRNA sequences that are specific for TLR8 (26)and epidemiological studies showed the association of a func-tionalTLR8 variant with HIV progression disease (27) and withpulmonary tuberculosis susceptibility (28). TLR8 activity is animportant source of TNF-� in the sinovial membrane culturesin rheumatoid arthritis (29). Based on these findings, it is thusimportant to understand how this receptor is regulated prior toand during an immune response.Here, we have characterized the mechanisms involved in

basicTLR8 expression and identified cellular pathways respon-sible forTLR8 transcriptional activation upon exposure toR848

and/or IFN-�. We found that members of the C/EBP family, �and �, have a role in driving TLR8 basal expression. Althoughwe did not observe binding of CEBP/� when performing theEMSA, ChIP experiments showed that both C/EBPs bind thethree C/EBP sites (C, D, and E) within theTLR8 promoter. Thisapparent discrepancy is most likely due to the fact that EMSAwas performed using a oligonucleotide containing a consensusC/EBP-binding sequence, whereas ChIP assays examinedC/EBP binding to the cis-elements present on the endogenousTLR8 promoter (Fig. 2, D and E). Several reports described thepotential overlapping or synergistic functions of C/EBP� andC/EBP� because both transcription factors can bind the samecis-elements as homodimer or heterodimer complexes (30, 31).Experiments in the mouse model showed that C/EBP� and

C/EBP� are induced via TLR-MyD88-dependent signalingpathways, which lead to the induction of proinflammatorycytokines (31). Here, we show for the first time that in humancells TLR8 and TLR4 stimulation led to a nuclear accumulationof C/EBP� but not C/EBP�. This event correlated with anincrease in TLR8 expression. The effect of TLR8 and/or TLR4

FIGURE 5. Identification of the GAS elements within the TLR8 promoter responsive to IFN-� treatment. A, determination of STAT-1 nuclear translocationand phosphorylation level by immunoblotting. Twenty �g of nuclear and cytoplasmic extracts from cells treated with IFN-� at the indicated time points wereanalyzed for STAT-1 and pSTAT-1 by immunoblotting. �-Actin immunodetection was performed as a loading control. B, deletion analysis of the IFN-�-mediatedTLR8 promoter transactivation. Progressively deleted TLR8 promoter fragments were transfected into THP-1. Forty-four h after transfection, cells were treatedwith IFN-� for 4 h or left untreated. Cells were harvested as described previously (19). C, ChIP assay determining the in vivo binding of STAT-1 to the TLR8promoter upon IFN-� treatment. The sheared chromatin from cells stimulated with IFN-� for 4 h or left untreated was immunoprecipitated with STAT-1 or IgGcontrol antibodies. The GAS- 1, -2, -3, and -4 sites on the TLR8 promoter were amplified by qPCR, and specific binding was calculated relative to the initial inputof DNA. D, enhanced cytokine production in IFN-� pretreated THP-1 cells following TLR8 triggering. Cell culture supernatants were harvested from THP-1treated for 24 h with R848 (i and iii) or uridine (ii and iv) with or without a 3-h pretreatment with IFN-�. IL-6 and IL-8 production was determined by ELISA. Resultsare representative of three independent experiments. NT, not treated.

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stimulation on C/EBP� appears not to be direct since we andothers (31) observed induction at eight hours post TLR8 andTLR4 stimulation. Many cytokines and chemokines are pro-duceddownstreamofTLR signaling that lead to sequential acti-vation of pathways that eventually accumulate on C/EBP� genetranscriptional activation. Accordingly, the C/EBP� gene hasbeen shown to be positively regulated by multiple cytokines,including IL-6, growth factors and various transcription factors(25, 32), and a regulatory network involving initially c-Rel (24).The molecular events occurring after induction of C/EBP� ontheTLR8 promoter are still unknown. C/EBP� is known to bindand recruit the histone acetylase CBP, which leads to chroma-tine opening (33) and nucleosome remodeling at the C/EBP-responsive site of the IL-12 p40 promoter (34). We can specu-late the same mechanism for the TLR8 promoter. Furtherinvestigations in this direction will help to clarify this issue interms of TLR8 transcription. We cannot exclude that othertranscription factors are likely to be involved in the TLR8 tran-scription, which among them is the NF-�B family. This familycomprises five different members p65 (RelA), RelB, c-Rel,NF-�B1 (p50), and NF-�B2 (p52), which can form homo- orheterodimers and play a crucial role in the transcriptional reg-ulation of many immunoregulated genes (35). Accordingly, weobserved that NF-�B heterodimers p65/p50 and c-Rel/p50transactivate the TLR8 promoter. Litvak and colleagues (24)showed that NF-�B activity is required for the transcriptionalregulation of C/EBP�. Thus, we cannot exclude that NF-�Bmay have a role in terms of regulating first C/EBP� or simulta-neously to activate theTLR8 promoter. Further studiesmust beperformed to better characterize the role of NF-�B in TLR8promoter transactivation.We also describe the mechanisms through which IFN-�

induces the TLR8 transcript. IFN-�-mediated TLR8 inductionat the promoter and mRNA level occurs at early times aftertreatment, (starting from 2 h) and increases significantlypost-12 h stimulation. We also observed that type I IFN tran-siently induces TLR8 at 2 h, yet this phenomenon was less evi-dent than that induced by IFN-�, as TLR8 levels returned tonormal post-6 h stimulation (supplemental Fig. 9). The rapidTLR8 up-regulation upon IFN-� treatment argues for a directmechanism in terms ofTLR8 transcription. The IFN-regulatoryfactor 1 (IRF1) is promptly induced after IFN-� treatment (36)and is one of the major transcriptional activators of IFN-�inducible genes. However, in our study, IRF1 did not affectTLR8 promoter activity in THP-1 cells, suggesting the involve-ment of another transcription factor. Sequence analysis of theTLR8 promoter identified four GAS sites. ChIP analysisrevealed the binding of STAT-1 at the four GAS sites on theTLR8 promoter. The IFN-�-mediatedTLR8 induction has beenobserved in primary cells such as eosinophils (21) and freshlypurified monocytes (supplemental Fig. 10).Interestingly, a 3-h pretreatment with IFN-� enhanced sen-

sitivity of THP-1 cells to R848 and uridine (which is specific toTLR8) versus unprimed cells, supporting the possibility thatIFN-� could enhance TLR8 functionality. We also observedthat TLR8 mRNA increased post-3 h treatment with IFN-�. Inmonocytes, TLR8 has been shown to respond to ssRNA andinduce IL-12 p70, which favors the differentiation of T helper 1

cells and the activation of natural killer cells (37, 38). Anincrease of IL-12 produced bymonocytes in response to ssRNAis observed in presence of natural killer cells and appears todepend on IFN-� production (39). Thus, we can hypothesizethat the increased IL-12 induction by TLR8 may be due to theIFN-�-mediated enhanced transcription and protein expres-sion of TLR8. Although we have extensively examined TLR8transcriptional regulation, protein expression is a technicalobstacle.We tested a totality of four commercial available anti-bodies byWestern blotting or by FACS analysis and found thatfor Western blotting, none of them worked in detecting eitherexogenous or endogenous TLR8 protein. For FACS, we wereable to recognize only exogenously expressed TLR8 (data notshown).TLR8 expression ismainly restricted tomyeloid cells, (mono-

cytes, macrophages, and moDCs) (4), which argues a cellularspecific TLR8 transcription regulation. Coherently, we did notdetect any TLR8 transcriptional activity in non-myeloid cellssuch as HUH-7 or HEK cell lines. Moreover, TLR8 promotersequence appeared to share common features with other mye-loid-specific promoters (16, 40) such as the lack of a TATAbox,the presence of C/EBP-responsive elements, and purine-richsequence motifs that are recognized by the Ets family. Regard-ing the role of Ets family members in TLR8 transcriptional reg-ulation, we observed that overexpression of SpiC, SpiB, or Spi1(PU.1) did not affectTLR8promoter activity.However, the pos-sibility that othermembers of the Ets family are involved in thisevent cannot be excluded.In summary, our data describe and characterize the molecu-

lar events that trigger TLR8 transcriptional activation uponR848 stimulation and IFN-� treatment and extend our under-standing of tissue-specific TLR8 gene expression.

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C/EBP� and STAT-1 Are Required for Transcriptional Activity

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Ménétrier-Caux, Massimo Tommasino and Uzma A. HasanClaudia Zannetti, François Bonnay, Fumihiko Takeshita, Peggy Parroche, Christine

Transcriptional ActivityTLR8 and STAT-1 Are Required for δC/EBP

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