Glucocorticoids inhibit dendritic cell maturation induced by Toll-like ...

Article

Glucocorticoids inhibit dendritic cell maturation induced by Toll-like receptor 7 and Toll-like receptor 8 Alexandre Larange´, Diane Antonios, Marc Pallardy, and Saadia Kerdine-Ro¨mer1 University Paris-Sud, INSERM UMR-S 749 and INSERM UMR-S 996, and INSERM, Faculte´ de Pharmacie, 92296 Chaˆtenay-Malabry, France RECEIVED NOVEMBER 15, 2010; REVISED SEPTEMBER 12, 2011; ACCEPTED SEPTEMBER 14, 2011. DOI: 10.1189/jlb.1110615

ABSTRACT GCs are widely prescribed to treat inflammatory disorders and autoimmune and allergic diseases. Their antiinflammatory and immunosuppressive effects may be related, in part, to their ability to control the maturation and functions of DCs. Here, we report that GCs inhibit the maturation of human CD34-DCs induced by the TLR7 agonist imiquimod and the TLR8 agonist 3M-002. GCs down-regulate the expression of CD86, CD40, CD83, CCR7, and HLA-DR on DCs and inhibit IL-6 and IL-12p40 production by DCs following TLR7 and TLR8 stimulation. This inhibitory effect is abolished by RU486, suggesting a role for GR transcriptional activity. Our results also show that GCs do not affect TLR-mediated DNA-binding activity of NF-␬Bp65. We observe that GCs control the activation of JNK induced by TLR agonists, without affecting its upstream MKK4. However, p38MAPK activation is not affected by GCs. Concomitantly to JNK inhibition, we observe the induction of the DUSP MKP-1 but not of other DUSPs by GCs. However, although silencing of MKP-1 in DCs reverses GC-mediated JNK inhibition, no significant effect on GC-induced inhibition of DC maturation was evidenced. Our results show that GCs alter DC maturation in response to TLR7 or TLR8 through a mechanism involving GR transcriptional activity. J. Leukoc. Biol. 91: 105–117; 2012.

Introduction GCs are among the most potent immunosuppressive and antiinflammatory drugs currently available to treat inflammatory

Abbreviations: CD34-DC⫽DC derived from CD34 progenitors, CD40L⫽CD40 ligand, cMFI⫽corrected mean fluorescence intensity, Dex⫽dexamethasone, DUSP⫽dual-specificity MAPK, Flt3L⫽fetal liver tyrosine kinase 3 ligand, GC⫽glucocorticoid, GR⫽glucocorticoid receptor, HPC⫽hematopoietic progenitor cell, IRAK⫽IL-1R-associated protein kinase, mDC⫽myeloid DC, MFI⫽mean fluorescence intensity, MKK⫽MAPK kinase, Mo-DC⫽monocyte-derived DC, NP-40⫽Nonidet P-40, pDC⫽plasmacytoid DC, rdm⫽random, siRNA⫽small interfering RNA, TAK⫽TGF-␤-activated protein kinase 1 The online version of this paper, found at www.jleukbio.org, includes supplemental information.

0741-5400/12/0091-105 © Society for Leukocyte Biology

disorders and autoimmune and allergic diseases [1]. For several years, the immunosuppressive effects of GCs have been ascribed to their capacity to inhibit T cell-mediated immunity. Indeed, GCs are known to inhibit the production by T cells of cytokines such as IL-2 [2] and IFN-␥ [3], to inhibit IL-2-driven T cell proliferation [4], and to induce T cell apoptosis [5]. However, the pharmacological effects of GCs are not only a result of the inhibition of T cell functions, and it is now clearly accepted that GCs also affect the function of APCs. Indeed, GCs down-regulate the production of IL-1 [6], IL-6 [7], TNF-␣ [8], IL-12-p40 [9], and MIP-1␣ [10] by monocytes and macrophages. GCs also alter the maturation of DCs induced by LPS [11, 12], TLR2 and TLR3 agonists [12], CD40L [13], or proinflammatory cytokines, such as IL-1␤ and TNF-␣ [14]. Consequently, DCs treated by GCs are less efficient for inducing T cell proliferation, as a result of the inhibition of costimulatory molecule expression (CD86, CD80), CD83, and cytokine production (IL-12) [11, 13, 15]. TLRs are highly conserved receptors that recognize a variety of pathogen-breakdown products, including peptidoglycan and lipopeptides (TLR2), dsRNA (TLR3), LPS (TLR4), flagellin (TLR5), ssRNA (TLR7/8), and CpG DNA (TLR9) [16]. Among the TLR family, TLR3, TLR7, TLR8, and TLR9 are not expressed on the cell surface but are retained in endosomal compartments. TLR7 and TLR8 are similar in sequence and together with TLR9, form an evolutionarily related subgroup within the TLR superfamily [17]. TLR7 and TLR8 mediate the recognition of ssRNA by DCs and play a major role in the immune response during viral infection [18, 19]. Recently, synthetic compounds of the imidazoquinoline family have been identified as TLR7 and/or TLR8 agonists [20]. Among these agonists, the lead compound imiquimod, a TLR7 agonist, exerts strong antiviral and antitumoral activities and is commercialized for the treatment of external genital warts caused by human papillomavirus [21]. TLR7 is expressed in pDCs [22, 23], but its expression in CD11c⫹ mDCs is still controversial [24, 25]. In most studies, TLR7 expression was not detected in 1. Correspondence: Faculte´ de Pharmacie Paris Sud, 92296 Chaˆtenay-Malabry, France. E-mail: [email protected]

Volume 91, January 2012

Journal of Leukocyte Biology 105

Mo-DCs. However, a recent study showed that Mo-DCs and LC-like DCs derived from CD34⫹ HPCs (CD34-LCs) expressed low levels of TLR7 [26], suggesting that TLR7 expression may depend on the culture protocol used. In response to TLR7 stimulation, pDCs produce large amount of IFN-␣ [27], whereas mDCs produce IL-12 [24]. TLR7 activation also leads to DC maturation and up-regulation of CD40, CD80, CD86 [24], and CCR7 [22]. TLR8 is expressed in mDCs, monocytes, Mo-DCs, and CD34-LCs [16, 26, 28]. TLR8 activation in mDCs leads to IL-12p70 and TNF-␣ production [24, 27, 29] and to the up-regulation of CD40, CD80, and CD86 [24, 30]. We have shown recently that TLR7 or TLR8 induces the maturation of CD34-DC [31]. Signaling pathways activated following TLR engagement can be different depending on the recruitment or not of MyD88, which when recruited by most TLRs except TLR3, forms a complex with members of the IRAK family (IRAK1 and IRAK4) and TRAF6, which then activates TAK1, which in turn, activates the IKK complex, leading to I␬B degradation and NF-␬B activation. Simultaneously, TAK1 phosphorylates members of the MKK family, such as MKK4, MKK3, or MKK6, which subsequently, activates JNK and p38MAPK [32]. All of the TLR agonists converge to the MAPKs and the NF-␬B signaling pathways to exert, in fine, their biological effects [17]. Current literature about the control of DC maturation by GCs following TLR stimulation has focused mainly on TLR4induced maturation and on the regulation of TLR2, TLR3, and TLR4 expressions. In our study, we have investigated the effects of GCs on DC phenotype and the signaling pathways induced by TLR7, TLR8, or TLR4 agonists in CD34-DCs. Our results showed that GCs inhibited DC maturation, reduced JNK activation without affecting the activation of its upstream kinase MKK4, but had no significant effect on p38MAPK phosphorylation. NF-␬B activity was not altered by GCs. Concomitantly with JNK inhibition, we observed GC-induced expression of the DUSP MKP-1, known to regulate MAPK phosphorylation [33]. Inhibition of MKP-1 with a pharmacological inhibitor or the silencing of MKP-1 with a specific siRNA abolished GC-mediated inhibition of JNK phosphorylation, suggesting a role for MKP-1 induced by GCs. However, silencing of MKP-1 had no effect on GC-mediated inhibition of DC maturation. Together, our results showed that GCs can inhibit DC maturation induced by TLR7 or TLR8 agonists but that this process does not involve the MKP-1-mediated inhibition of JNK activation.

MATERIALS AND METHODS

Generation of DCs from human cord blood CD34ⴙ HPCs Umbilical cord blood samples were obtained after full-termed delivery from women who were clearly informed about the aim of the study and gave their informed consent. Mononuclear cells were prepared by density centrifugation using medium for lymphocyte isolation (Eurobio, Les Ulis, France). Cells bearing the CD34⫹ antigen were isolated from the mononuclear fraction through magnetic positive selection using MiniMACS separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany) and antiCD34⫹ antibodies coated on magnetic beads (Direct CD34 progenitor cell

106 Journal of Leukocyte Biology


isolation kits, Miltenyi Biotec, France). After purification, the isolated cells were 80 –95% CD34⫹ cells. Cultures were established in the presence of GM-CSF 200 U/ml (Leucomax 400, a kind gift from Novartis, Rueil-Malmaison, France), human rTNF-␣ 50 U/ml (kindly provided by Dr. Schmidt, Mainz, Germany), and Flt3L 50 ng/ml (PeproTech, Tebu, Le Perray-en-Yvelines, France) in RPMI 1640 containing Glutamax I, supplemented with 10% heat-inactivated FCS, 1 mM sodium pyruvate, and 1% penicillin and streptomycin antibiotic solution (all from Gibco Invitrogen, Paisley, UK). Cells were then incubated at 3 ⫻ 105 cells/ml at 37°C in a 5% CO2, 95% air atmosphere for 7 days.

Chemical treatment of immature DCs On Day 7, DCs were washed three times and then incubated at 106 cells/ ml. Cells were then treated with Dex (10⫺7 M; Sigma-Aldrich, St. Louis, MO, USA) for 2 h before treatment with LPS (25 ng/ml, from Escherichia coli O55:B5; Sigma-Aldrich), imiquimod (40 ␮M; Sigma-Aldrich), or 3M-002 (25 ␮M, 3M Pharmaceuticals, St. Paul, MN, USA; Supplemental Fig. 1) for different periods of time. In some experiments, DCs were pretreated for 30 min with RU486 (10⫺6 M; Sigma-Aldrich) or for 1 h with Ro-31-8220 (2.5 ␮M; Calbiochem, Darmstadt, Germany).

Flow cytometry analysis Cultured DCs were resuspended at 2.5 ⫻ 105 cells in 30 ␮l culture medium and incubated for 30 min at 4°C with specific mAb or appropriate isotypic controls. After three washes in cold PBS, supplemented with 0.5% BSA, cells were fixed with 1% paraformaldehyde in PBS. The following mAb were used: PE-Cy5-conjugated anti-CD86 (2331), Alexa Fluor 647-conjugated anti-CCR7 (3D12), PE-conjugated anti-CD83 (HB15a), allophycocyanin-conjugated anti-CD40 (5C3), and FITC-conjugated anti-HLA-DR (G46-6, all BD Biosciences, France). Appropriate isotype irrelevant antibodies were used at the same concentrations as controls. Cells were analyzed with a FACSCalibur cell analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) using the CellQuest software (Becton Dickinson). cMFI was calculated, corresponding to the ratio between the total MFI obtained using the specific antibody to the total MFI obtained using a control fluorescent antibody of the same isotype.

Oligonucleotide pulldown assay Following treatments, cells were lysed in NP-40 lysis hypertonic buffer. In brief, cell pellets were resuspended in buffer containing 0.2% NP-40, 20% glycerol, 20 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 0.125 ␮M okadaic acid, 62.5 mM EDTA, 40 ␮M EGTA, 0.5 mM PMSF, 1 ␮g/ml leupeptin, 1 ␮g/mL aprotinin, and 1 ␮g/ml pepstatin and incubated with very gentle agitation at 4°C for 30 min. Cellular debris was removed by centrifugation at 4°C at 15,000 rpm for 20 min. The following 5⬘-biotin-labeled oligonucleotides (MWG Biotech, Ebersberg, Germany) were hybridized: 5⬘-TTG AGG GGA CTT TCC CAG G-3⬘ and 5⬘-CCT GGG AAA GTC CCC TCA A-3⬘, according to the human NF-␬B promoter sequence. Mutated oligonucleotides were used as control for unspecific binding (5⬘-TTG AGG CGA CTT TCC CAG G-3⬘ and 5⬘-CCT GGG AAA GTC GCC TCA A-3⬘). Whole cell extracts (200 ␮g) were incubated at 4°C for 90 min with 2 ␮g doublestranded 5⬘-biotinylated oligonucleotide coupled to 30 ␮L streptavidin-agarose beads (Sigma-Aldrich). Complexes were washed in binding buffer and eluted by boiling in reducing sample buffer, and proteins were separated on an 8% SDS-PAGE gel, followed by Western blot analysis and probed for p65 level with a p65 mAb (sc-1191, Santa Cruz Biotechnology, Tebu, Le Perray-en-Yvelines, France).

Western blot analysis Cultured DCs (106 cells/ ml) were washed in cold PBS before lysis in 50 ␮l lysis buffer [20 mM Tris (pH 7.4), 137 mM NaCl, 2 mM EDTA (pH 7.4), 1% Triton, 25 mM ␤-glycerophosphate, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM PMSF, 10 ␮g/ml aprotinin, and 10 ␮g/ml

www.jleukbio.org

Larange´ et al. leupeptin]. The homogenates were centrifuged at 15,000 rpm for 20 min at 4°C. Equal amounts of denatured proteins were loaded onto 12.5% SDSPAGE gel and transferred on a PVDF membrane (Amersham Biosciences, Les Ulis, France). Membranes were then incubated with antibodies raised against the phosphorylated forms of p38MAPK, JNK1/2, ERK1/2, MKK3/6, and MKK4 (all from Cell Signaling Technology, Ozyme, St Quentin en Yvelines, France); I␬B-␣ (C21, Santa Cruz Biotechnology); or against MKP-1 (M18, Santa Cruz Biotechnology). Membranes were stripped and reprobed with antibodies against total p38 MAPK (p38 N20, Santa Cruz Biotechnology), total MKK3 (Cell Signaling Technology), or total JNK (Cell Signaling Technology) as loading controls. Immunoreactive bands were detected by chemiluminescence (ECL solution, Amersham Biosciences). Bands were quantified by densitometry using the ImageQuant software (for JNK quantification, p46 and p54 isoforms were quantified as one band).

RT-PCR analysis Total RNA was extracted after lysis of cells in TRIzol reagent (Gibco Invitrogen) by the guanidium thiocyanate method, as mentioned by the manufacturer. RNA was quantified by spectrophotometry. First-strand cDNA was synthesized from total RNA extracted in RNase-free conditions. The reaction was performed on 2 ␮g total RNA with an oligo dT primer (MWG Biotech) and 2 U AMV RT (Promega, San Luis Obispo, CA). PCR reaction was performed using 1 U Taq polymerase (QBiogen, Montre´al, Canada). Specific primers were used in PCR reaction mixture (forward and reverse primers, respectively): mkp-1: 5⬘-GCT GTG CAG CAA ACA GTC GA-3⬘ and 5⬘-CGA TTA GTC CTC ATA AGG TA-3⬘/dusp-2: 5⬘-AGG AGC TGA CTG TGG ACT GG-3⬘ and 5⬘-GCG TCT AGC TGA TTT CTG CC-3⬘/dusp-4: 5⬘-GCA GAA GTT GGG ACT GAG C-3⬘ and 5⬘-TGA AAC TGA CAC ATA AAC CAA ACC-3⬘/dusp-5: 5⬘-GAC TCT TGG GAT CAT CTA ACT CAG C-3⬘ and 5⬘-CTA ACC GGC AAG AGA ATT CCT TA-3⬘/ dusp-6: 5⬘-ACC GAC ACA GTG GTG CTC TA-3⬘ and 5⬘-GTT GGA CAG CGG ACT ACC AT-3⬘/dusp-8: 5⬘-AAG TGC ACG AAA GCT CGG-3⬘ and 5⬘-ACG TTT CTA AGC AAT ACG AGG C-3⬘/dusp-9: 5⬘-GCC CAC TCG TGT GGC AAG-3⬘ and 5⬘-AGC GTG GTC CCG CCA TAG-3⬘/dusp-10: 5⬘GGC ACT GTA ACC AGA ATC AAA TC-3⬘ and 5⬘-GGA ACT GAC CAT TAT ATG CCT TCA C-3⬘/dusp-16: 5⬘-ATC CCA TTT TAA ACA ATT CTT TGA-3⬘ and 5⬘-GCT GAA CCA CCA GGA ACC T-3⬘/dusp-26: 5⬘-CTC AAT GCC TCA CAC AGC CG-3⬘ and 5⬘-ATC TTC CCT CCT GGC TGG CT-3⬘/ tlr7: 5⬘-GAT GTC ACT CTG GAT GTT-3⬘ and 5⬘-GAT GTC TGG TAT GTG GTT-3⬘/tlr8: 5⬘-TTA CCC CAA ATA CCC TCT-3⬘ and 5⬘-AAA ATA GCA GTT CA GGC-5⬘/␤-actin: 5⬘-GGG TCA GAA GGA TTC CTA TG-3⬘ and 5⬘GGT CTC AAA CAT GAT CTG GG-3⬘. PCR products were visualized by addition of ethidium bromide on a 1% agarose gel. ␤-Actin was used to control and calibrate cDNA synthesis. The folds indicate the ratio of dusp gene to ␤-actin gene from treated cells to control cells. Real-time RT-PCR analysis was performed using SYBR Green technology on a LightCycler rapid thermal cycler (Roche Diagnostics, Meylan, France). Forward and reverse primers were each designed in a different exon of the target gene sequence, eliminating the possibility of amplifying genomic DNA. For each set of primers, a BLAST search revealed that sequence homology was obtained only for the target gene. To confirm the specificity of the amplification, the PCR product was subjected to a melting curve analysis and agarose gel electrophoresis. PCR amplification was performed in duplicate in a total reaction volume of 10 ␮l. The reaction mixture consisted of 1 ␮l diluted template, 2 ␮l FastStart DNA Master PLUS SYBR Green I kit, and 0.5 ␮M forward and reverse primers. After an 8-min activation of Taq polymerase, amplification was allowed to proceed for 30 – 45 cycles, each consisting of denaturation at 95°C for 10 s, annealing at 55°C (mkp-1, ␤-actin) or 58°C (gapdh) for 5 s, and extension at 72°C for 5 s or 9 s, depending on the target gene. Fivefold serial dilutions from soleus total RNA were analyzed for each target gene and allowed us to construct linear standard curves from which the concentrations of the test sample were calculated. Results were normalized to ␤-actin and gapdh transcription to compensate for variation in input RNA amounts and efficiency of reverse transcription.

www.jleukbio.org

Glucocorticoids affect TLR7/8-induced DC maturation

ELISAs for cytokine production The culture supernatants of DCs were removed 24 h after the different treatments and kept at – 80°C until cytokine measurement. IL-6 and IL12p40 productions were measured using Quantikine immunoassay ELISA kits (R&D Systems, Minneapolis, MN, USA) in 96-well microtiter plates according to the manufacturer’s instructions. Results were expressed as ng/million cells. The ELISA sensitivities were ⬍15 pg/ml for IL-12p40 and ⬍0.7 pg/ml for IL-6, according to the manufacturer.

Electroporation of CD34-DCs with siRNA This protocol was described previously for Mo-DCs [34] and adapted to CD34-DCs. At Day 6 of differentiation, DCs were washed once with serumfree medium and once with PBS. Cells were resuspended in serum-free medium at 4 ⫻ 107 cells/ml. Equivalent amounts of nonsilencing control siRNA (Qiagen, Courtabeuf, France) or MKP-1 siRNA (ID s4363, Applied Biosystems, Foster City, CA, USA) were transferred into a 4-mm cuvette (BioRad, Marnes-La-Coquette, France) and filled up to a final volume of 100 ␮L with serum-free medium. Cells (4⫻106) were added and pulsed in a GenePulser II (BioRad; 300V, 150 ␮F). Cells were then resuspended in complete medium at a concentration of 106/ml for 24 h.

Statistics analysis The comparison of two data groups was analyzed by Mann-Whitney's test. A P value of ⬍0.05 was considered statistically significant. Data were analyzed with Statistica version 7.1 software.

RESULTS

Dex inhibits CD34-DC maturation induced by TLR4, TLR7, and TRL8 agonists GCs are known to inhibit DC maturation induced by different signals, such as LPS, CD40L, or NiCl2 [11, 13–15, 35, 36]. However, there is no evidence to date that GCs are able to modulate DC maturation induced by TLR7 or TLR 8 triggering. In the first part of this work, we evaluated the effect of Dex on the phenotype of DCs activated by LPS, imiquimod, or 3M-002, which are known agonists of TLR4, TLR7, and TLR8, respectively. CD34-DCs were or not pretreated with Dex (10⫺7 M) for 2 h, and LPS (25 ng/ml), imiquimod (40 ␮M), or 3M002 (25 ␮M) was then added for 24 h. The concentrations of LPS, imiquimod, and 3M-002 used were the lowest noncytotoxic concentrations that induced DC maturation (data not shown). The three agonists induced DC maturation, characterized by the up-regulation of CCR7, CD83, CD86, CD40, and HLA-DR on the cell surface (Fig. 1A and B), and the secretion of IL-12p40 and IL-6 (Fig. 2). As we described already [31], imiquimod seemed to be a less-potent inducer of DC maturation compared with LPS or 3M-002 in this model. Treatment with Dex inhibited DC maturation induced by the TLR ligands. Indeed, Dex treatment reduced the up-regulation of CCR7, CD83, CD86, CD40, and HLA-DR induced by the three agonists (Fig. 1A and B). Dex inhibited, by ⬎40%, the expression of CD83 on DCs stimulated with imiquimod, and this inhibition was close to 50% in DCs treated with LPS or 3M-002. Dex down-regulated, by ⬎60%, the expression of CD86 at the surface of DCs treated with imiquimod or LPS; however, this effect was less pronounced in 3M-002-treated DCs (37%). IL-6 and IL-12p40 productions were also affected by Dex treatment (Fig. 2). In DCs treated with LPS or imiVolume 91, January 2012


Figure 1. Dex inhibits DC maturation induced by TLR agonists. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ ml), imiquimod (40 ␮M), or 3M002 (25 ␮M) for 24 h. Cell surface molecule expression was then analyzed by flow cytometry. (A) CCR7, CD83, CD86, CD40, and HLA-DR expression. Numbers represent cMFI, which is the ratio between the total MFI obtained using the specific antibody (solid lines) to the total MFI obtained using a control fluorescent antibody of the same isotype (broken lines). (B) Analysis of results shown in A. The percentage of positive cells according to control antibody and cMFI is shown for the expression of each surface molecule. Results are from one representative experiment out of four performed on different donors.

quimod, Dex inhibited, by ⬎70% and ⬎60%, the production of IL-12p40 and IL-6, respectively. Dex inhibitory effects were less pronounced in 3M-002-treated DCs (46% and 29% inhibition for IL-12p40 and IL-6, respectively). Additionally, tlr7 and tlr8 mRNA expressions were not affected by Dex treatment (data not shown).

I␬B-␣ degradation and p65 NF-␬B DNA binding activity induced by TLR4, TLR7, and TRL8 agonists are not affected by Dex treatment in CD34-DCs Activation of the NF-␬B pathway is an important process in DC maturation [37– 40]. NF-␬B is regulated by the inhibitory mol108 Journal of Leukocyte Biology


ecule I␬B-␣, which retains NF-␬B in the cytoplasm under normal conditions. Upon cellular stimulation, I␬B-␣ is degraded, allowing NF-␬B to translocate to the nucleus, where it binds to target gene promoters [41]. We used the degradation of I␬B-␣ as a readout for NF-␬B activation. DCs were treated or not with Dex (10⫺7 M) during 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) during 1 h. The three agonists induced I␬B-␣ degradation, which was not affected by Dex pretreatment (Fig. 3A). To further assess the effect of Dex treatment on NF-␬B, we evaluated the DNA binding activity of the p65 subunit of NF-␬B. Using an oligonucleotide pulldown assay, we observed that LPS, imiquimod,

www.jleukbio.org

Larange´ et al. Glucocorticoids affect TLR7/8-induced DC maturation

Figure 2. Dex inhibits cytokine production induced by TLR agonists in CD34-DC. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GMCSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 24 h. Supernatants were collected. IL-6 (upper) or IL-12p40 (lower) production was measured by ELISA. Results are the respective mean values of three individual experiments; *P ⬍ 0.05 by Mann-Whitney test.

and 3M-002 induced p65 DNA binding activity after 1 h of stimulation (Fig. 3B). When cells were pretreated with Dex, the p65 DNA binding activity induced by the agonists was not altered (Fig. 3C).

Dex treatment inhibits JNK activation induced by TLR4, TLR7, or TRL8 agonists in CD34-DCs MAPK signaling pathways are known to play a major role in the maturation of DCs and in cytokine production [42– 46]. Moreover, we have shown previously that p38MAPK and JNK are involved in DC maturation induced by TLR7 and TLR8 agonists [31]. To assess if the inhibitory effect of Dex on DC maturation was related to an inhibition of these pathways, we evaluated JNK and p38MAPK activities upon Dex treatment. DCs were treated or not with Dex (10⫺7 M) during 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) during 1 h. As shown in Fig. 4A, Dex pretreatment inhibited JNK phosphorylation induced by LPS or imiquimod and to a lesser extent, JNK phosphorylation induced by 3M-002. However, inhibition was statistically significant only for imiquimod and 3M-002 but not for LPS, as a result of experimental variability (Fig. 4B). On the other hand, p38MAPK phosphorylation induced by imiquimod or 3M-002 was not affected by Dex pretreatment (Fig. 4B).

www.jleukbio.org

TLR4, TLR7, or TRL8 agonist-mediated activation of MKK and MKK3/6 is not inhibited by Dex treatment in CD34-DCs JNK and p38MAPK are activated by upstream kinases, such as MKK4, MKK7, and MKK3/6, in several cell types [47]. DCs were treated with Dex and TLR agonists as described previously. All three agonists were able to induce MKK4 and MKK3/6 phosphorylation (Fig. 5A and B). Imiquimod was a less-potent inducer of MKK4 phosphorylation compared with LPS or 3M-002. MKK7 phosphorylation was not detectable for any agonist used (data not shown). Dex treatment did not significantly alter the phosphorylation of MKK3/6 and MKK4 in DCs activated with LPS, imiquimod, or 3M-002 (Fig. 5A and B).

RU486 prevents Dex inhibition of CD34-DC maturation GCs act by interacting with the GR, which then migrates to the cell nucleus. Activated GR can directly induce or repress gene transcription by binding to specific DNA sites. However, GR can also interact with AP-1 or NF-␬B, thereby inhibiting their transcriptional activity [1]. To evaluate whether the transcriptional activity of the GR was involved in Dex-mediated inhibition of DC maturation, the GR antagonist RU486 [48] was used. As shown in Table 1, Dex Volume 91, January 2012


Figure 3. Effect of Dex on I␬B-␣ degradation and p65 DNA binding activity induced by TLR agonists in CD34-DCs. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and treated or not with Dex (10⫺7 M) for 2 h and then stimulated or not (control cells) with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. (A) I␬B-␣ degradation was evaluated by Western blot. Membrane was then reprobed with an anti-p38 MAPK antibody for loading control. Blots are from representative results obtained in three independent experiments performed on different donors. Bands were quantified by densitometry using the ImageQuant software. Results were expressed as the ratio of the density value of I␬B-␣ protein to the density value of p38MAPK protein and annotated under each corresponding band. T0, Time 0. (B) Cells were stimulated or not (control cells) with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. p65 DNA binding activity was then evaluated using an oligonucleotide pulldown assay. (C) Cells were treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. p65 DNA binding activity was then evaluated using an oligonucleotide pulldown assay. Results are from one representative experiment out of three performed on different donors.

inhibition was nearly, totally abolished when RU486 (10⫺6 M) was added before Dex. These results suggested that GC-mediated inhibition of DC maturation involves GR-induced gene transcription.

ate cyclase 1), dusp4 (mkp-2), dusp8 (human VH5), and dusp10 (mkp-5) mRNAs were expressed in immature, untreated DCs. Among these phosphatases, Dex up-regulated the mRNA of mkp-1, whereas others did not appear to undergo significant variations (Fig. 6).

Dual-specifity phosphatase mRNA expression in GCtreated CD34-DCs

Dex induces MKP-1 expression in CD34-DCs

MAPK activities are known to be regulated by reversible phosphorylation. In mammalian cells, inactivation of MAPKs is primilarly mediated by the DUSP family of proteins. In fact, DUSPs are able to dephosphorylate the threonine and tyrosine residues on MAPKs [49]. In line with our results obtained with RU486, we hypothesized that the inhibitory effect of Dex on JNK phosphorylation in CD34-DC, cotreated with Dex and TLR7/8 agonist, could be a result of the up-regulation of one or several of these phosphatases. DCs were treated with Dex (10⫺7 M) during 1.5 h, and mRNA expression of a large panel of DUSPs was then measured by semiquantitative RT-PCR. Our results showed that dusp1 (mkp-1), dusp2 (human pituitary adenyl-

We then focused our interest on the effect of Dex on mkp-1 expression in DCs. As a result of the rapid effect of Dex on JNK phosphorylation, we evaluated, using real-time RT-PCR, the early kinetic induction of mkp-1 mRNA in immature DCs, treated or not with Dex (10⫺7 M), which induced mkp-1 mRNA expression in DCs as early as 30 min and reached a maximum at 45 min (sevenfold increase). The expression of mkp-1 mRNA was maintained for 2 h after Dex addition (Fig. 7A). We then evaluated whether the MKP-1 protein was expressed at the time that we observed the inhibition of JNK phosphorylation by Dex. Although mkp-1 mRNA was present in



www.jleukbio.org


Figure 4. Effect of Dex on p38MAPK and JNK phosphorylation induced by TLR agonists in CD34-DCs. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. (A) Cells were then lysed, and the level of phosphorylation of p38MAPK (P-p38) and JNK (P-JNK) was evaluated by Western blot using antibody against the phosphorylated forms of these proteins. Membrane was then reprobed with an anti-p38MAPK antibody for loading control. Blots show representative results obtained in three independent experiments performed on different donors. (B) Bands were quantified by densitometry using the ImageQuant software. Results are expressed as the ratio of density value of the phosphorylated form to the density value of equivalent, nonphosphorylated p38MAPK. Results are the respective mean values of three individual experiments; *P ⬍ 0.05 by Mann-Whitney's test.

untreated DCs and was up-regulated in Dex-treated DCs, the MKP-1 protein was not detectable in cells treated with Dex alone. Interestingly, MKP-1 expression was detectable in DCs treated with LPS and 3M-002 but not with imiquimod. When Dex was added prior to the three agonists, MKP-1 expression was strongly up-regulated upon addition of LPS or 3M-002 and to a lesser extent, with imiquimod (Fig. 7B).

MKP-1 impairment abolishes Dex-mediated inhibition of JNK activation in CD34-DCs To assess the involvement of MKP-1 in the control of JNK phosphorylation by GCs, we first used the MKP-1 inhibitor Ro-

www.jleukbio.org

31-8220 [50]. DCs were pretreated with Ro-31-8220 (2.5 ␮M) during 1 h, treated or not with Dex (10⫺7 M) during 2 h, and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) during 1 h. Ro-31-8220 was used at a concentration that inhibits MKP-1 activity, measured by the increase of MAPK phosphorylation. We observed that Ro-31-8220 abolished Dex-mediated inhibition of JNK in DCs stimulated with the TLR agonists (Fig. 8A and B). To confirm the involvement of MKP-1 in the control of JNK inhibition by GCs, we inhibited MKP-1 expression in DC by using specific siRNA. CD34DCs were electroporated with nonsilencing control siRNA or mkp-1 siRNA. Twenty-four hours after electroporation, DCs Volume 91, January 2012


Figure 5. Effect of Dex on MKK4 and MKK3/6 phosphorylation induced by TLR agonists in human DCs. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. Cells were then lysed, and the level of phosphorylation of MKK4 (P-MKK4; A) and MKK3/6 (P-MKK3/6; B) was evaluated by Western blot using antibody against the phosphorylated forms of these proteins. Membrane was then reprobed with an anti-MKK3 antibody for loading control. Blots show representative results obtained in three independent experiments performed on different donors. Bands were quantified by densitometry using the ImageQuant software. Results are expressed as the ratio of density value of phosphorylated form to the density value of equivalent, nonphosphorylated MKK3 and annotated under each corresponding band.

were treated or not with Dex (10⫺7 M) during 2 h and then stimulated with imiquimod (40 ␮M) or 3M-002 (25 ␮M) during 1 h. As shown in Fig. 8C, mkp-1 siRNA was efficient to silence MKP-1 expression in CD34-DCs. We observed that in the absence of MKP-1, Dex-mediated inhibition of JNK was abolished (Fig. 8C). This result shows that GC-mediated inhibition of JNK activation involves MKP-1.

MKP-1 silencing does not affect GC-mediated inhibition of DC maturation To assess the involvement of MKP-1 in the control of DC maturation by GCs, we silenced MKP-1 expression in DCs by using specific siRNA. At Day 6, DCs were electroporated with nonsilencing control siRNA or mkp-1 siRNA. Twenty-four hours after electroporation, DCs were treated or not with Dex (10⫺7 M) during 2 h and then stimulated with imiquimod (40 ␮M) or 3M-002 (25 ␮M) during 24 h. We observed that in the absence of MKP-1, Dex-mediated inhibition of DC maturation induced by imiquimod was slightly affected and that Dex-mediated inhi-

bition of DC maturation induced by 3M-002 was not affected (Table 2). This result showed that MKP-1 did not play a central role in GC-mediated inhibition of DC maturation.

DISCUSSION In this work, we showed that Dex inhibited the expression of CD83, CCR7, CD86, CD40, and HLA-DR in response to TLR7 and TLR8 agonists. The secretions of IL-12p40 and IL-6, in response to TLR7 and TLR4 agonists in CD34-DCs, were inhibited by Dex. Moreover, GCs altered the MAPK signaling pathways, known to be involved in DC maturation. Dex reduced JNK activation induced by the three agonists without affecting MKK4 phosphorylation. Interestingly, p38MAPK activation was not affected by Dex in stimulated CD34-DCs, stimulated by imiquimod or by 3M-002. Alteration of DC function by GCs was dependent on GR-induced gene expression, and our results indicated that the phosphatase MKP-1 was involved in the control of JNK phosphorylation by GCs in human DCs,

TABLE 1. Effect of the GR Antagonist RU486 on Dex-Mediated Inhibition of DC Maturation Dex inhibition (%)

LPS Imiquimod 3M-002

Dex RU486 ⫹ Dex Dex RU486 ⫹ Dex Dex RU486 ⫹ Dex

CD83

CCR7

CD86

CD40

HLA-DR

48 13 41 0 60 1

40 7 25 0 38 0

66 13 68 12 65 10

18 0 36 0 36 0

14 6 39 4 32 0

DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM- CSF, TNF-␣, and Flt3L for 7 days. Cells were then washed and pretreated or not with the GR antagonist RU486 (10– 6 M) for 0.5 h, treated or not with Dex (10–7 M) for 2 h, and then stimulated with LPS (25 ng/ml), imiquimod (42 ␮M), or 3M-002 (25 ␮M) for 24 h. Cell surface expression of CD83, CCR7, CD86, CD40, or HLA-DR was analyzed by flow cytometry. Results were expressed in percentage of inhibition of the expression of these molecules induced by Dex treatment in the absence or in the presence of RU486. Results are from one representative experiment out of three performed on different donors.



www.jleukbio.org


Figure 6. DUSP expression in immature DCs, treated or not with Dex. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. DUSP mRNA expression was investigated in DCs stimulated with Dex (10⫺7 M) for 1.5 h in comparison with unstimulated DCs using semiquantitative RTPCR. Bands were quantified by densitometry using the ImageQuant software. The ratio of the density value of DUSP to density value of ␤-actin was calculated. These values were reported to those of untreated cells (fold). Results are representative of three independent experiments performed on different donors.

www.jleukbio.org

but MKP-1 did not play a significant role in the control of DC maturation by GCs. In our model, GCs control DC maturation induced by TLR7, TLR8, and TLR4 agonists, by inhibiting CD83, CD86, CD40, CCR7, and HLA-DR expression and secretion of IL-6 and IL-12p40. Several groups have shown that GCs inhibit DC maturation induced by LPS [11, 12, 51, 52]. Moreover, Rozkova et al. [12] recently found that Dex inhibited DC maturation, induced by TLR2 and TLR3 agonists and assessed by the inhibition of CD83, CD86, and CD80 expression and IL-6 and TNF-␣ secretion. MAPK [41, 45] and NF-␬B signaling pathways are central for DC maturation [17]. We have already shown that imiquimod and 3M-002 induced MAPK activation in CD34-DCs. Although p38MAPK participated in the up-regulation of maturation markers in response to TLR7 activation, this kinase exerted an inhibitory effect on CD40 expression and IL-12 production in TLR8-stimulated DCs [31]. In this report, we described that imiquimod and 3M-002 induce MKK3/6 and also MKK4 activation. Interestingly, the TLR8 agonist strongly induced MKK4 and JNK phosphorylation, whereas the TLR7 agonist activated these kinases only slightly. This observation seems to be relevant considering the difference of concentrations used between imiquimod and 3M-002, as we observed no difference on the level of activation of MKK3/6 between these two agonists measured using Western blot experiments. Concerning the effect of GCs on MAPK signaling pathways, we found that Dex reduced JNK phosphorylation, induced by imiquimod, 3M-002, and LPS. GCs did not affect MKK4 phosphorylation induced by the three agonists. This observation suggests that GCs altered JNK activation without affecting upstream kinases, indicating that GCs could directly target JNK activity. Similarly to our results, Sakai et al. [53] reported in HeLa cells that Dex inhibited JNK activity induced by IL-1␤ without affecting MKK4. GCs were also shown to inhibit JNK phosphorylation in monocytes [54] and macrophages [55] and also p38MAPK phosphorylation induced by LPS in HeLa cells [55] and macrophages [56]. Recently, it has also been found that GCs inhibit JNK and p38MAPK phosphorylation activated by the TLR3 or TLR9 agonist in macrophages in a TAK-dependant manner. In this latter case, GCs had no effect on JNK activation when TLR3 and TLR9 activation was combined [57]. In our model, imiquimod and 3M-002 activated the NF-␬B pathway, confirming what was already shown in the mouse cell line XS52 [58], in human monocytes [30], and in macrophages [59]. In our hands, Dex treatment did not modify TLR-induced p65 NF-␬B DNA binding activity or I␬B-␣ degradation. Guiducci et al. [60] have observed that GCs did not affect p65 NF-␬B phosphorylation or DNA binding activity in pDCs activated through TLR7 or TLR9. In macrophages, Battacharyya et al. [57] reported that GCs had no effect on I␬B-␣ degradation induced by the TLR3 agonist (polyinosinic:polycytidylic acid), whereas GCs inhibited I␬B-␣ degradation and also impaired translocation to the nucleus of phosphorylatedp65 induced by the TLR9 agonist (CpG). The biological actions of GCs are mediated through the GR, which regulates numerous genes. GR can bind directly to Volume 91, January 2012


Figure 7. MKP-1 expression in Dextreated DCs. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. (A) DCs were treated or not with Dex (10⫺7 M) for the indicated time. mkp-1 mRNA expression was then measured using real-time RT-PCR. Results were normalized to ␤-actin and gapdh, and obtained values were reported to T0. (B) DCs were treated or not with Dex (10⫺7 M) for 2 h and then stimulated with LPS (25 ng/ml), imiquimod (40 ␮M), or 3M-002 (25 ␮M) for 1 h. Cells were then lysed, and MKP-1 protein expression was evaluated by Western blot. Membrane was then reprobed with an anti-p38 MAPK antibody for loading control. Blots show representative results obtained in three independent experiments performed on different donors. Bands were quantified by densitometry using the ImageQuant software. Results are expressed as the ratio of density value of MKP-1 to the density value of p38MAPK (p38) and annotated under each corresponding band.

DNA, acting as a transcription factor. GR can also bind or interfere with other transcription factors, altering their activities [1]. To assess the involvement of gene transcription by GR in the inhibitory effect of GCs, we used the GR antagonist RU486, known to inhibit the transcriptional activity of GR [48]. RU486 abolished Dex-mediated inhibition of DC maturation, indicating that the inhibitory effect of Dex on TLR7 and TLR8 activation involved genes directly regulated by the GR.

DUSPs are known to dephosphorylate the threonine and tyrosine residues on MAPKs, leading to their inactivation [49]. We observed that Dex significantly up-regulated the mRNA expression of mkp-1. However, the MKP-1 protein was not detectable when cells were treated with Dex alone, whereas TLR4, TLR7, or TLR8 activation led to MKP-1 protein expression in CD34-DCs. The addition of GCs with the TLR agonists strongly up-regulated MKP-1 expression com-

Figure 8. Effect of impairment of MKP-1 on Dex-mediated inhibition of JNK phosphorylation. DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. (A and B) Cells were then washed and pretreated or not with the MKP-1 inhibitor Ro-31-8220 (2.5 ␮M) for 1 h, then treated or not with Dex (10⫺7 M) for 2 h, and then stimulated with imiquimod (40 ␮M) or 3M-002 (25 ␮M) for 1 h. (C) Cells were electroporated at Day 6 with nonsilencing siRNA (rdm) or specific mkp-1 siRNA. At Day 7, cells were washed, treated or not with Dex (10⫺7 M) for 2 h, and then stimulated with 3M-002 (25 ␮M) for 1 h. Cells were then lysed, and the MKP-1 expression was assessed by Western blot. Level of phosphorylation of JNK was evaluated by Western blot using antibody raised against the phosphorylated forms of these proteins. Membrane was then reprobed with an anti-p38 antibody for loading control. Blots show representative results obtained in three independent experiments performed on different donors.



www.jleukbio.org

Larange´ et al.

TABLE 2. Effect of MKP-1 Silencing on Dex-Mediated Inhibition of DC Maturation Dex inhibition (%)

Dex ⫹ imiquimod Dex ⫹ 3M-002

siRNA siRNA siRNA siRNA

rdm MKP-1 rdm MKP-1

CD83

CCR7

CD86

42 30 69 61

19 10 38 29

54 41 67 61

DCs were generated by culturing CD34⫹ cells from cord blood in the presence of GM-CSF, TNF-␣, and Flt3L for 7 days. Cells were electroporated at Day 6 with nonsilencing siRNA (rdm) or specific MKP-1 siRNA. At Day 7, cells were washed, treated or not with Dex (10–7 M) for 2 h, and then stimulated with imiquimod (40 ␮M) or 3M-002 (25 ␮M) for 24 h. Cell surface expression of CD83, CCR7, and CD86 was analyzed by flow cytometry. Results were expressed in percentage of inhibition of the expression of these molecules induced by Dex treatment in the presence of nonsilencing siRNA (rdm) or in the presence of mkp-1 siRNA. Results are from one representative experiment out of two performed on different donors.

pared with the agonists alone. Several studies have shown in different cell types that GCs can induce the expression of MKP-1 [61]. Moreover, it was demonstrated recently that MKP-1 contributes to the anti-inflammatory effects of Dex in vitro and in vivo [62]. In macrophages, TLR stimulation with LPS [63, 64], TLR2, TLR3, or TLR9 agonists [64] upregulated MKP-1 expression, which in turn, deactivated JNK and p38MAPK. Our results indicate that one or several sig-


naling pathways activated following TLR activation are needed for Dex-induced MKP-1 protein synthesis. Indeed, p38MAPK and its downstream kinase MAPKAPK 2 have been shown to play a critical role in the expression of MKP-1 at a post-transcriptional level in peptidoglycan-stimulated macrophages [65]. When CD34-DCs were treated with Ro-31-8220, a known inhibitor of MKP-1 [66], we observed that Dex-mediated inhibition of JNK phosphorylation was abolished. This result was confirmed using specific siRNA against MKP-1. Indeed, silencing of MKP-1 reversed Dex-mediated inhibition of JNK activation. However, Dex-mediated inhibition of DC maturation was not impaired when MKP-1 was silenced in 3M-002-treated DCs, and this inhibition was only reduced slightly in imiquimodtreated DCs. These results led us to the conclusion that MKP-1 does not play a pivotal role in GC-mediated inhibition of DC maturation. In conclusion, our work showed for the first time that GCs inhibited DC maturation induced by TLR7/8 agonists. We have also shown that GCs: reduced JNK activation without affecting the activation of its upstream kinase MKK4; had no major effect on p38MAPK activated by 3M-002 or imiquimod; and didn't alter NF-␬B activation. We also found that Dex-mediated JNK inhibition was mediated by MKP-1. However MKP-1 silencing did not affect GC-mediated inhibition of CD34-DC maturation. Together, our results showed that GCs can inhibit one aspect of DC maturation induced by TLR7 or TLR8 agonists, but this process does not involve the MKP-1-mediated inhibition of JNK activation (Fig. 9).

Figure 9. Schematic view of the effects of Dex on DC maturation induced by TLR7/8 agonists. In human CD34-DCs, the TLR7 agonist imiquimod and the TLR8, 3M-002, activated JNK, p38MAPK, and NF-␬B, which positively regulated DC maturation. Dex strongly reduced JNK activity in DCs, and this effect involved GC-induced MKP-1 expression. MKP-1 did not affect Dexmediated inhibition of DC maturation.

www.jleukbio.org



AUTHORSHIP A.L. designed the study, performed experiments, analyzed data, and wrote the manuscript. D.A. performed experiments. M.P. analyzed data and wrote the manuscript. S.K-R. designed the study, performed experiments, analyzed data, and wrote the manuscript.

ACKNOWLEDGMENTS This work was supported by the Institut National de la Sante´ et de la Recherche Me´dicale and the Acade´mie Nationale de Me´decine. We thank J. Bertoglio (INSERM UMR-S 749) for critical reading of this manuscript and 3M Pharmaceutical Division for the generous gift of TLR8 agonist 3M-002.

REFERENCES

1. Rhen, T., Cidlowski, J. A. (2005) Antiinflammatory action of glucocorticoids—new mechanisms for old drugs. N. Engl. J. Med. 353, 1711–1723. 2. Boumpas, D. T., Anastassiou, E. D., Older, S. A., Tsokos, G. C., Nelson, D. L., Balow, J. E. (1991) Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription. J. Clin. Invest. 87, 1739 –1747. 3. Kunicka, J. E., Talle, M. A., Denhardt, G. H., Brown, M., Prince, L. A., Goldstein, G. (1993) Immunosuppression by glucocorticoids: inhibition of production of multiple lymphokines by in vivo administration of dexamethasone. Cell. Immunol. 149, 39 – 49. 4. Vacca, A., Felli, M. P., Farina, A. R., Martinotti, S., Maroder, M., Screpanti, I., Meco, D., Petrangeli, E., Frati, L., Gulino, A. (1992) Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements. J. Exp. Med. 175, 637– 646. 5. Perrin-Wolff, M., Bertoglio, J., Bressac, B., Bohuon, C., Pallardy, M. (1995) Structure-activity relationships in glucocorticoid-induced apoptosis in T lymphocytes. Biochem. Pharmacol. 50, 103–110. 6. Snyder, D. S., Unanue, E. R. (1982) Corticosteroids inhibit murine macrophage Ia expression and interleukin 1 production. J. Immunol. 129, 1803–1805. 7. Waage, A., Slupphaug, G., Shalaby, R. (1990) Glucocorticoids inhibit the production of IL6 from monocytes, endothelial cells and fibroblasts. Eur. J. Immunol. 20, 2439 –2443. 8. Beutler, B., Krochin, N., Milsark, I. W., Luedke, C., Cerami, A. (1986) Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 232, 977–980. 9. Blotta, M. H., DeKruyff, R. H., Umetsu, D. T. (1997) Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4⫹ lymphocytes. J. Immunol. 158, 5589 – 5595. 10. Berkman, N., Jose, P. J., Williams, T. J., Schall, T. J., Barnes, P. J., Chung, K. F. (1995) Corticosteroid inhibition of macrophage inflammatory protein-1 ␣ in human monocytes and alveolar macrophages. Am. J. Physiol. 269, L443–L452. 11. Vieira, P. L., Kalinski, P., Wierenga, E. A., Kapsenberg, M. L., de Jong, E. C. (1998) Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential. J. Immunol. 161, 5245–5251. 12. Rozkova, D., Horvath, R., Bartunkova, J., Spisek, R. (2006) Glucocorticoids severely impair differentiation and antigen presenting function of dendritic cells despite upregulation of Toll-like receptors. Clin. Immunol. 120, 260 –271. 13. Rea, D., van Kooten, C., van Meijgaarden, K. E., Ottenhoff, T. H., Melief, C. J., Offringa, R. (2000) Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigenpresenting cells that secrete IL-10. Blood 95, 3162–3167. 14. De Jong, E. C., Vieira, P. L., Kalinski, P., Kapsenberg, M. L. (1999) Corticosteroids inhibit the production of inflammatory mediators in immature monocyte-derived DC and induce the development of tolerogenic DC3. J. Leukoc. Biol. 66, 201–204. 15. Matasic´, R., Dietz, A. B., Vuk-Pavlovic, S. (1999) Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells. J. Leukoc. Biol. 66, 909 –914. 16. Iwasaki, A., Medzhitov, R. (2004) Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987–995. 17. Kawai, T., Akira, S. (2006) TLR signaling. Cell Death Differ. 13, 816 – 825.



18. Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., Reis e Sousa, C. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529 –1531. 19. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G., Wagner, H., Bauer, S. (2004) Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303, 1526 –1529. 20. Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi, T., Tomizawa, H., Takeda, K., Akira, S. (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196 –200. 21. Gupta, A. K., Cherman, A. M., Tyring, S. K. (2004) Viral and nonviral uses of imiquimod: a review. J. Cutan. Med. Surg. 8, 338 –352. 22. Jarrossay, D., Napolitani, G., Colonna, M., Sallusto, F., Lanzavecchia, A. (2001) Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31, 3388 –3393. 23. Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R. W., Kastelein, R. A., Bazan, F., Liu, Y. J. (2001) Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194, 863– 869. 24. Ito, T., Amakawa, R., Kaisho, T., Hemmi, H., Tajima, K., Uehira, K., Ozaki, Y., Tomizawa, H., Akira, S., Fukuhara, S. (2002) Interferon-␣ and interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets. J. Exp. Med. 195, 1507–1512. 25. Krug, A., Towarowski, A., Britsch, S., Rothenfusser, S., Hornung, V., Bals, R., Giese, T., Engelmann, H., Endres, S., Krieg, A. M., Hartmann, G. (2001) Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31, 3026 –3037. 26. Renn, C. N., Sanchez, D. J., Ochoa, M. T., Legaspi, A. J., Oh, C. K., Liu, P. T., Krutzik, S. R., Sieling, P. A., Cheng, G., Modlin, R. L. (2006) TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response. J. Immunol. 177, 298 –305. 27. Gibson, S. J., Lindh, J. M., Riter, T. R., Gleason, R. M., Rogers, L. M., Fuller, A. E., Oesterich, J. L., Gorden, K. B., Qiu, X., McKane, S. W., Noelle, R. J., Miller, R. L., Kedl, R. M., Fitzgerald-Bocarsly, P., Tomai, M. A., Vasilakos, J. P. (2002) Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell. Immunol. 218, 74 – 86. 28. Osterlund, P., Veckman, V., Siren, J., Klucher, K. M., Hiscott, J., Matikainen, S., Julkunen, I. (2005) Gene expression and antiviral activity of ␣/␤ interferons and interleukin-29 in virus-infected human myeloid dendritic cells. J. Virol. 79, 9608 –9617. 29. Gorden, K. B., Gorski, K. S., Gibson, S. J., Kedl, R. M., Kieper, W. C., Qiu, X., Tomai, M. A., Alkan, S. S., Vasilakos, J. P. (2005) Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174, 1259 –1268. 30. Levy, O., Suter, E. E., Miller, R. L., Wessels, M. R. (2006) Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells. Blood 108, 1284 –1290. 31. Larange´, A., Antonios, D., Pallardy, M., Kerdine-Ro¨mer, S. (2009) TLR7 and TLR8 agonists trigger different signaling pathways for human dendritic cell maturation. J. Leukoc. Biol. 85, 673– 683. 32. Dunne, A., O'Neill, L. A. (2003) The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE 2003, re3. 33. Keyse, S. M. (2000) Protein phosphatases and the regulation of mitogenactivated protein kinase signaling. Curr. Opin. Cell Biol. 12, 186 –192. 34. Antonios, D., Rousseau. P., Larange´. A., Kerdine-Ro¨mer. S., Pallardy. M. (2010) Mechanisms of IL-12 synthesis by human dendritic cells treated with the chemical sensitizer NiSO4. J. Immunol. 185, 89 –98. 35. Singh, S., Aiba, S., Manome, H., Tagami, H. (1999) The effects of dexamethasone, cyclosporine, and vitamin D(3) on the activation of dendritic cells stimulated by haptens. Arch. Dermatol. Res. 291, 548 –554. 36. Vanderheyde, N., Verhasselt, V., Goldman, M., Willems, F. (1999) Inhibition of human dendritic cell functions by methylprednisolone. Transplantation 67, 1342–1347. 37. Kabashima, K., Honda, T., Nunokawa, Y., Miyachi, Y. (2004) A new NF-␬B inhibitor attenuates a TH1 type immune response in a murine model. FEBS Lett. 578, 36 – 40. 38. Laderach, D., Compagno, D., Danos, O., Vainchenker, W., Galy, A. (2003) RNA interference shows critical requirement for NF-␬ B p50 in the production of IL-12 by human dendritic cells. J. Immunol. 171, 1750 – 1757. 39. Lyakh, L. A., Koski, G. K., Telford, W., Gress, R. E., Cohen, P. A., Rice, N. R. (2000) Bacterial lipopolysaccharide, TNF-␣, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14⫹ monocytes through distinct pathways that activate NK-␬ B. J. Immunol. 165, 3647–3655. 40. Rescigno, M., Martino, M., Sutherland, C. L., Gold, M. R., Ricciardi-Castagnoli, P. (1998) Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188, 2175–2180.

www.jleukbio.org

Larange´ et al. 41. Doyle, S. L., O'Neill, L. A. (2006) Toll-like receptors: from the discovery of NF␬B to new insights into transcriptional regulations in innate immunity. Biochem. Pharmacol. 72, 1102–1113. 42. Arrighi, J. F., Rebsamen, M., Rousset, F., Kindler, V., Hauser, C. (2001) A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-␣, and contact sensitizers. J. Immunol. 166, 3837–3845. 43. Boisle`ve, F., Kerdine-Romer, S., Pallardy, M. (2005) Implication of the MAPK pathways in the maturation of human dendritic cells induced by nickel and TNF-␣. Toxicology 206, 233–244. 44. Boisle`ve, F., Kerdine-Romer, S., Rougier-Larzat, N., Pallardy, M. (2004) Nickel and DNCB induce CCR7 expression on human dendritic cells through different signalling pathways: role of TNF-␣ and MAPK. J. Invest. Dermatol. 123, 494 –502. 45. Nakahara, T., Uchi, H., Urabe, K., Chen, Q., Furue, M., Moroi, Y. (2004) Role of c-Jun N-terminal kinase on lipopolysaccharide induced maturation of human monocyte-derived dendritic cells. Int. Immunol. 16, 1701– 1709. 46. Osawa, Y., Iho, S., Takauji, R., Takatsuka, H., Yamamoto, S., Takahashi, T., Horiguchi, S., Urasaki, Y., Matsuki, T., Fujieda, S. (2006) Collaborative action of NF-␬B and p38 MAPK is involved in CpG DNA-induced IFN-␣ and chemokine production in human plasmacytoid dendritic cells. J. Immunol. 177, 4841– 4852. 47. Raman, M., Cobb, M. H. (2003) MAP kinase modules: many roads home. Curr. Biol. 13, R886 –R888. 48. Rajpert, E. J., Lemaigre, F. P., Eliard, P. H., Place, M., Lafontaine, D. A., Economidis, I. V., Belayew, A., Martial, J. A., Rousseau, G. G. (1987) Glucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions. J. Steroid Biochem. 26, 513–520. 49. Liu, Y., Shepherd, E. G., Nelin, L. D. (2007) MAPK phosphatases—regulating the immune response. Nat. Rev. Immunol. 7, 202–212. 50. Beltman, J., McCormick, F., Cook, S. J. (1996) The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J. Biol. Chem. 271, 27018 –27024. 51. Vizzardelli, C., Pavelka, N., Luchini, A., Zanoni, I., Bendickson, L., Pelizzola, M., Beretta, O., Foti, M., Granucci, F., Nilsen-Hamilton, M., Ricciardi-Castagnoli, P. (2006) Effects of dexamethazone on LPS-induced activationand migration of mouse dendritic cells revealed by a genomewide transcriptional analysis. Eur. J. Immunol. 36, 1504 –1515. 52. Matsue, H., Yang, C., Matsue, K., Edelbaum, D., Mummert, M., Takashima, A. (2002) Contrasting impacts of immunosuppressive agents (rapamycin, FK506, cyclosporin A, and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation. J. Immunol. 169, 3555–3564. 53. Sakai, A., Han, J., Cato, A. C., Akira, S., Li, J. D. (2004) Glucocorticoids synergize with IL-1␤ to induce TLR2 expression via MAP kinase phosphatase-1-dependent dual inhibition of MAPK JNK and p38 in epithelial cells. BMC Mol. Biol. 5, 2. 54. Ma, W., Gee, K., Lim, W., Chambers, K., Angel, J. B., Kozlowski, M., Kumar, A. (2004) Dexamethasone inhibits IL-12p40 production in lipopolysaccharide-stimulated human monocytic cells by down-regulating the activity of c-Jun N-terminal kinase, the activation protein-1, and NF-␬ B transcription factors. J. Immunol. 172, 318 –330.

www.jleukbio.org


55. Chen, P., Li, J., Barnes, J., Kokkonen, G. C., Lee, J. C., Liu, Y. (2002) Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J. Immunol. 169, 6408 – 6416. 56. Lasa, M., Abraham, S. M., Boucheron, C., Saklatvala, J., Clark, A. R. (2002) Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol. Cell. Biol. 22, 7802–7811. 57. Bhattacharyya, S., Ratajczak , C. K., Vogt , S. K., Kelley , C., Colonna , M., Schreiber , R. D., Muglia, L. J. (2010) TAK1 targeting by glucocorticoids determines JNK and I␬B regulation in Toll-like receptor-stimulated macrophages. Blood 115, 1921–1931. 58. Thatcher, T. H., Luzina, I., Fishelevich, R., Tomai, M. A., Miller, R. L., Gaspari, A. A. (2006) Topical imiquimod treatment prevents UV-light induced loss of contact hypersensitivity and immune tolerance. J. Invest. Dermatol. 126, 821– 831. 59. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., Akira, S. (2002) Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4. Nature 420, 324 –329. 60. Guiducci, C., Gong, M., Xu, Z., Gill, M., Chaussabel, D., Meeker, T., Chan, J. H., Wright, T., Punaro, M., Bolland, S., Soumelis, V., Banchereau, J., Coffman, R. L., Pascual, V., Barrat, F. J. (2010) TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus. Nature 465, 937–941. 61 . Clark, A. R. (2003) MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids? J. Endocrinol. 178, 5–12. 62. Abraham, S. M., Lawrence, T., Kleiman, A., Warden, P., Medghalchi, M., Tuckermann, J., Saklatvala, J., Clark, A. R. (2006) Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1. J. Exp. Med. 203, 1883–1889. 63. Sa´nchez-Tillo´, E., Comalada, M., Farrera, C., Valledor, A. F., Lloberas, J., Celada, A. (2006) Macrophage-colony-stimulating factor-induced proliferation and lipopolysaccharide-dependent activation of macrophages requires Raf-1 phosphorylation to induce mitogen kinase phosphatase-1 expression. J. Immunol. 176, 6594 – 6602. 64. Chi, H., Barry, S. P., Roth, R. J., Wu, J. J., Jones, E. A., Bennett, A. M., Flavell, R. A. (2006) Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc. Natl. Acad. Sci. USA 103, 2274 –2279. 65. Hu, J. H., Chen, T., Zhuang, Z. H., Kong, L., Yu, M. C., Liu, Y., Zang, J. W., Ge, B. X. (2007) Feedback control of MKP-1 expression by p38. Cell. Signal. 19, 393– 400. 66. Mikami, F., Lim, J. H., Ishinaga, H., Ha, U. H., Gu, H., Koga, T., Jono, H., Kai, H., Li, J. D. (2006) The transforming growth factor-␤-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacteria via a dual mechanism involving functional cooperation with NF-␬B and MAPK phosphatase 1-dependent negative cross-talk with p38 MAPK. J. Biol. Chem. 281, 22397–22408.

KEY WORDS: signal transduction 䡠 MKP-1 䡠 MAPKs 䡠 TLR signaling

