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Originally published online as doi:10.1189/jlb.0705385 on October 5, 2006

Published online before print October 5, 2006
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(Journal of Leukocyte Biology. 2007;81:221-228.)
© 2007 by Society for Leukocyte Biology

TLR7/8 agonists impair monocyte-derived dendritic cell differentiation and maturation

Eric Assier*, Viviana Marin-Esteban*, Alain Haziot*,{dagger}, Enrico Maggi{ddagger}, Dominique Charron*,{dagger} and Nuala Mooney*,{dagger},1

* INSERM U662, Paris, France;
{dagger} Université Paris 7, Institut Universitaire d’Hématologie, Centre Hayem, Hôpital Saint-Louis, Paris, France; and
{ddagger} Department of Internal Medicine, Immunoallergology and Respiratory Disease Unit, University of Florence, Florence, Italy

1Correspondence: INSERM U662, Institut Universitaire d’Hématologie, Centre Hayem, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. E-mail: nuala.mooney{at}paris7.jussieu.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pathogen recognition by TLR activates the innate immune response and is typically followed by the development of an adaptive immune response initiated by antigen presentation. Dendritic cells (DC) are the most efficient APC and express diverse TLRs, including TLR7 and -8, which have been recently identified as targets for ssRNA recognition during viral infection. We have studied the effect of TLR7/8 agonists on DC differentiation and maturation from human monocytes. The synthetic agonist Resiquimod (R-848) or the physiological agonist ssRNA impaired monocyte differentiation to DC phenotypically and functionally. Induced expression of the nonclassical MHC molecules of the CD1 family in DC was inhibited at the protein and mRNA levels, and antigen acquisition was inhibited. Proinflammatory cytokine (including IL-6, IL-8, TNF-{alpha}, IL-1ß) and IL-10 production were induced during DC differentiation. Cross-talk between TLR4 and TLR7/8 was revealed as immature DC, which had been differentiated in the presence of R-848 were insensitive to LPS-mediated maturation and cytokine production but still induced allostimulation. These data lead us to suggest that ongoing viral activation of TLR7/8 could alter the adaptive immune response by modifying DC differentiation and by down-regulating DC responsiveness to a subsequent bacterial TLR4-mediated signal.

Key Words: human • viral • antigen presentation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) have been identified as the most efficient APC, as they have the unique ability to activate naïve T lymphocytes [1 ]. Characteristics of mature myeloid DC include high surface expression of antigen-presenting and costimulatory molecules, modified protease expression, and stabilized expression of peptide-associated MHC Class II molecules at the cell surface, all of which contribute to the efficacy of antigen presentation [2 3 4 5 ].

Resting immature DC sensing microbial products in peripheral tissues capture antigens and migrate to germinal centers, where they present antigens as mature DC. Replenishment of tissues with DC is partly accomplished through differentiation of blood monocytes [6 , 7 ]. In the absence of pathogenic signals, immature DC are produced continuously and are widely distributed within tissues where they can sample self-antigen continuously, move to lymphoid organs, and present self-antigen, thereby maintaining self-tolerance.

In contrast, pathogen recognition by pattern recognition receptors (PRR) causes maturation of immature DC and their migration to lymphoid organs. Tissues are then repopulated with monocytes, which differentiate into DC in the presence of diverse pathogens such as microbial products [8 ]. Although the induction of maturation of DC by pathogenic stimuli has been examined extensively, the effect of pathogenic stimuli in the course of DC differentiation remains unclear. Differentiation of monocytes to DC occurs in vivo and can be reproduced readily in vitro in the presence of GM-CSF and IL-4, and maturation can be induced by diverse proinflammatory stimuli [4 , 6 , 7 , 9 , 10 ]. Typically, monocyte differentiation to immature DC is characterized by loss of CD14 expression, enhanced MHC Class II molecule, CD80 and CD86 expression, and de novo expression of the nonclassical MHC molecule CD1a.

Monocytes and immature DC express PRR, including members of the TLR family, which recognize diverse microbial pathogens and microbial ligands [11 ]. LPS provides a potent, maturing stimulus for DC via activation of TLR4 and has been particularly well-documented in vitro and in vivo [4 , 12 ].

The implication of the TLRs in the antiviral immune response has been examined more recently. Activation of TLR3 by dsRNA led to the production of Type I IFNs (IFN-{alpha}/ß), which exert antiviral and immunostimulatory activities [13 ]. In humans, TLR7 and TLR8 have been identified as receptors for synthetic compounds belonging to the imidazoquinoline family, which are used clinically for treatment of herpes virus infection [14 ]. Synthetic agonists specific for TLR7 or TLR8 are currently under development [15 ]. Resiquimod (R-848) is a synthetic imidazoquinoline-like molecule, which activates NF-{kappa}B via TLR7 and TLR8 and is therefore considered as a TLR7/8 agonist. Thus, TLR7/8 were predicted to recognize nucleic, acid-like structures. This has been confirmed recently by the identification of TLR7/8 as receptors for guanosine- or uridine-rich ssRNA from viruses [16 17 18 ].

This study examines the outcome of the continued presence of TLR7/8 ligands during DC differentiation from monocytes such as occurs during viral infection. We have therefore determined the effect of the TLR7/8 synthetic agonist R-848 and the physiological ligand ssRNA on DC differentiation from monocytes and on DC maturation in response to a TLR4 ligand.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cytokine and reagents
Human recombinant (hr)GM-CSF was obtained from Novartis-Schering Plough (Switzerland; Leucomax, 4.44x106 IU). hrIL-4 was from PromoCell (France). Stock solutions of GM-CSF were prepared in the manufacturer’s buffer and then diluted in RPMI-1640 serum-free medium; aliquots were kept at –80°C. Stock solutions of IL-4 were prepared by dissolving the drug in water, and aliquots were frozen at 20°C. R-848 was obtained from InvivoGen (Cayla SAS, Toulouse, France). A stock solution was prepared by dissolving the drug in 20% ethanol and was kept at 4°C. ssRNA (RNA 40), synthesized in the phosphothioate-protected form, correspond to the sequence of the U5 region of HIV-1 RNA. ssRNA were used complexed to cationic lipids [1,2-dioleoyl-3- trimethylammonium propane (DOTAP)] to facilitate its uptake by monocytes. DOTAP was obtained from Roche Diagnostics (Nutley, NJ). LPS was obtained from Sigma Chemical Co. (St Quentin Fallavier, France; 0111:B4) and re-extracted twice with phenol.

Monocyte and DC cultures
Highly purified monocytes were obtained from PBMC of healthy volunteers by positive selection of CD14-positive cells with MACS technology (Miltenyi Biotec, Germany). Monocytes were cultivated for 7 days in medium (RPMI 1640 containing 10% FCS, 2 mM glutamine, 100 IU/ml penicillin, streptomycin, supplemented with 800 IU/ml GM-CSF and 1000 IU/ml IL-4). R-848 (2 µg/ml) and ssRNA (0.5 or 1 µg/ml complexes with DOTAP solution at a ratio of 5 µl/µg ssRNA) were added on Day 0. Half of the culture medium was replaced with fresh medium on Days 2 and 5; R-848 and ssRNA were replenished at each change of medium. Maturation of DC was performed by incubating cells for a further 2 days in fresh medium supplemented with LPS (0.5 µg/ml) or with R-848 (2 µg/ml).

Flow cytometry
Cell-surface staining was performed using the following anti-human mAb: anti-CD1aFITC (NA1/34) and control IgG1PECy5 were obtained from Dako (Denmark). Anti-CD1bFITC (WM25) was obtained from Cymbus Biotechnology (UK). Anti-CD1cFITC (AD5-8E7) was obtained from Miltenyi Biotec. Anti-HLA-DRPECy5 (IM2659), anti-CD14FITC (RM052), and control IgG1FITC were obtained from Immunotech (Marseille, France). Anti-CD80FITC (557 226), anti-CD83FITC (556 910), anti-CD86FITC (555 657), anti-CD11cPE (555 392), and anti-CXCR1FITC (555 939) were obtained from Becton Dickinson and PharMingen (BD BioSciences, Le Pont-du-Claix, France). Anti-CCR7FITC antibodies (FAB197F) were from R&D Systems Europe (Lille, France).

Nonadherent cells were collected and washed in PBS. Nonspecific binding to Fc{gamma}R was blocked by preincubation of cell suspensions in PBS containing 100 µg/ml nonimmune human IgG, 5% FCS, and 0.02% sodium azide, and staining was performed in the same conditions. After washing, cells were analyzed on a FACScan flow cytometer (BD BioSciences). Events (20,000) were acquired for each sample, and dead cells were excluded by their light-scatter properties.

Endocytosis assay
The ability of DC to capture antigen was determined by measuring uptake of BSA-FITC (Sigma Chemical Co.). Cells (5x105 per sample) were preincubated in RPMI-1640 complete medium for 15 min at 4°C or 37°C, before addition of BSA-FITC (50 µg/ml) for 30 min. After incubation, cells were washed twice with cold PBS and fixed with PBS 2.5% paraformaldehyde. Uptake of BSA-FITC was determined by flow cytometry. Events (20,000) were analyzed. Particulate antigen uptake was also determined by the same protocol using Dextran-FITC (50 µg/ml) in the place of BSA-FITC.

Immunoblotting of CD1 isoforms
Cell lysates (20 µg) from monocytes and immature DC obtained after 5 days culture in the presence or absence of 2 µg/ml R-848 were separated in a 10% SDS-PAGE gel and transferred to Hybond polyvinylidene difluoride membrane (Amersham, UK). Membranes were blocked overnight at 4°C with 5% BSA in PBS 0.1% Tween 20 and then probed with a goat polyclonal serum anti-CD1 (C19, Santa Cruz Biotechnologies, CA) at a dilution of 1/500. Membranes were washed in PBS 0.1% Tween 20 before incubating with HRP-conjugated rabbit antigoat (Amersham) at a dilution of 1/2000 for 1 h at room temperature. The blot was washed and developed using ECL chemiluminescence according to the manufacturer’s instructions.

Oligonucleotides and RT-PCR
Total RNA was extracted using RNeasy mini kit (Qiagen SA, France) from 2 x 106 monocytes and immature DC obtained after 7 days culture in the presence or absence of 2 µg/ml R-848 or ssRNA (0.5–1 µg/ml) complexes with DOTAP liposome solution. First-strand DNA was prepared using an Omniscript RT kit (Qiagen SA). PCR amplification was performed with 2.5 IU TaqDNA polymerase (Promega, France) in a final volume of 50 µl. Semiquantitative PCR was performed with oligonucleotide primer sets designed, as possible, to cross intron-exon boundaries to allow differentiation of PCR products from genomic DNA contaminants. Specific primers used were as follows: for CD1a, 5'-AGACGGGCTCAAGGAGCCTC-3' and 5'-TCCAGTTCCTTCCACTCCTC-3'; for CD14, 5'-TTATCGACCATGGAGCGCGC-3' and 5'-ACCAGTAGCTGAGCAGGAAC-3'; for GAPDH, 5'-GTCGTATTGGGCGCCTGGTCA-3' and 5'-AGGGGCCATCCACAGTCTTCT-3'. Reaction mixtures were heated at 94°C for 5 min before PCR amplification (45 s at 94°C, 45 s at 60°C, and 45 s at 72°C). Thirty cycles were performed for CD1a and CD14 and 21 cycles for GADPH. Final extension was obtained at 72°C for 15 min. GADPH product yields were quantified by densitometry to standardize RT products used for PCR amplifications of CD1a and CD14.

Statistical analysis
Statistical analysis was performed using the Statview5 software. The significance of the data was examined using the Student’s t-test; P values and standard deviations are indicated.

Analysis of cytokine production
Cytokines secreted in the course of monocyte differentiation to DC (with or without TLR7/8 ligands) were evaluated using the human inflammation cytometric bead array kit (BD Biosciences) in culture supernatants. IL-1ß, IL-6, IL-8, IL-10, IL-12p70, and TNF-{alpha} were captured simultaneously by a mix of six beads coated with specific antibodies. Briefly, supernatants of monocytes and DC were collected and stored at –80°C until analysis. When assayed, the supernatants were mixed with the capture beads and PE-conjugated detection antibodies and then incubated together for 3 h at room temperature to form sandwich complexes. After the incubation, the beads were washed and resuspended in PBS for analysis in the FL3 channel of a flow cytometer (FACSCalibur, BD Biosciences).

Allogenic MLR assay
The capacity of DC differentiated in the presence or the absence of R-848 and then activated with LPS to allostimulate T cells was evaluated by MLR assays. Allogenic PBMC (5x104 cells/200 µl/well) were seeded in 96-well U-bottom tissue-culture plates. The indicated numbers of irradiated DC were added and cultured for 6 days in RPMI 1640 containing 10% human serum, 2 mM glutamine, 100 IU/ml penicillin, and streptomycin. Fifteen hours before harvest, 1 µCi [3H]thymidine was added to each well. Cells were harvested, and radioactivity was measured in a ß-liquid scintillation analyzer (MicroBeta Trilux, Perkin Elmer, Wellesley, MA). Results are expressed as the means cpm of triplicate cultures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DC differentiation from monocytes is altered by TLR7/8 agonists
To determine the effect of TLR7/8 activation in the course of DC differentiation, human monocytes isolated from peripheral blood of healthy donors were cultured for 7 days in the presence of GM-CSF and IL-4, with or without the TLR7/8 agonists R-848 or ssRNA, and the phenotype of differentiated cells was analyzed.

The first row of Figure 1A (d0) shows the typical phenotype of the monocytes, and the second row (d7) illustrates the typical phenotype of the immature DC. The most striking modification induced in the presence of R-848 was the lack of induction of the CD1a molecule belonging to the nonclassical MHC family of CD1 molecules composed of CD1a, CD1b, and CD1c (Fig. 1A) . Moreover, the typical DC expression of the IL-8 receptor CXCR1 was not observed in the presence of either TLR7/8 agonist (Fig. 1B) . These differences contrasted with the expression profiles of other typical DC cell surface molecules including HLA-DR, CD80, and CD86, which were unaffected by R-848 or ssRNA treatment. The maturation marker CD83 was not or was minimally induced in the presence of R-848 or ssRNA (Fig. 1A and 1B) . CD11c expression was also unaltered by TLR7/8 activation during DC differentiation, and no morphological modifications of nonadherent cells were observed by light microscopy (data not shown). The phenotypes shown in Figure 1 were assessed on nonadherent cells. The phenotypic profile of nonadherent and adherent cells was compared in two experiments, and no difference was observed (data not shown).


Figure 1
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  		Figure 1. Alteration of monocyte-derived DC phenotype by differentiation in the presence of TLR7/8 agonists. (A) The phenotype of freshly isolated CD14+ cells (d0), cells that were differentiated in the presence of GM-CSF and IL-4 for 7 days (d7), and cells that were differentiated in the presence of R-848 (2 µg/ml) in addition to GM-CSF and IL-4 for 7 days (d7 R-848). Shaded histograms reveal surface labeling with antibodies specific for the indicated surface marker, and open histograms show isotype control Ig staining. Representative histograms of more than nine independent experiments are shown. The mean fluorescence intensity (MFI) is indicated. (B) One of four experiments in which CD14+ cells were differentiated with GM-CSF and IL-4 for 7 days supplemented with ssRNA at the indicated concentrations or R-848 (as above). The phenotype of the monocyte-derived DC, differentiated without addition of a TLR7/8 stimulus, is shown for comparison (d7).

 
The MFI of CD1a expression was compared in DC from nine individuals, which were differentiated with or without R-848 (2 µg/ml) for 7 days; the MFI fell from 268.3 ± 115.8 to 21.9 ± 10.3 (P<0.0003, n=9 donors).

Figure 1B shows the phenotype of monocyte-derived immature DC, which had been differentiated in the presence of ssRNA or R-848. The decreased expression of CD1a and maintained expression of CD14 in the majority of donors (albeit to different degrees with different donors) is similar to that observed with R-848. In addition, the lack of induction of CXCR1 in the presence of ssRNA or R-848 is shown.

The key function of immature DC is antigen uptake, and this function was tested first by examining uptake of FITC-labeled BSA in R-848- or ssRNA-treated cells. Immature DC clearly internalized BSA-FITC, as described previously (Medium), and the capacity for internalization was down-regulated by differentiation in the presence of ssRNA or R-848. The MFI of internalized BSA-FITC fluorescence of DC compared with R-848-treated DC was decreased from 81.2 ± 24.9 to 32.7 ± 8.5 (P<0.0465, n=6 donors, data not shown).

The same approach was taken to determine whether TLR7/8 activation altered particulate antigen uptake using Dextran-FITC as a model antigen. In two independent experiments with DC from different donors, R-848 (2 µg/ml) and ssRNA (0.5 or 1 µg/ml) decreased Dextran-FITC uptake by 45% in both experiments (data not shown). TLR7/8 activation therefore decreases the capacity of DC for antigen internalization.

Regulation of CD1 expression in DC by TLR7/8 agonists
The CD1 family of nonclassical MHC molecules is responsible for presentation of lipid and glycolipid antigens, and up-regulation of members of the CD1 family in the course of DC differentiation has been widely documented. We therefore examined the time course of expression of the different CD1 isoforms in R-848-treated cells.

Figure 2A shows the time course of induced surface expression of CD1a, -b, and -c during differentiation of monocytes to DC. Induced expression of CD1a and -b was visible from Day 2 of DC differentiation and was increased clearly by Day 5 (*, significant increases/decreases compared with expression at Day 0; P<0.005, n>3 donors). The increase in CD1c expression was much less marked even at Days 5 and 7. CD14 was expressed strongly on the starting monocyte population and was down-regulated by Day 2 of DC differentiation from monocytes in the presence of GM-CSF and IL-4.


Figure 2
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  		Figure 2. Induction of DC CD1 expression is inhibited by TLR7/8 agonists. (A) The time course of induced expression of CD1 isoforms and the down-regulation of CD14 during differentiation of CD14+ cells in the absence (A) or presence (B) of R-848. CD1a and -b were highly up-regulated by Day 5, and CD14 expression is decreased dramatically by Day 2 (A). Immature DC, differentiated in the presence of R-848, fail to up-regulate CD1a or CD1b, and expression remains unaltered even on Day 7 (B). Mean MFIs and SD from at least three separate experiments are shown. The differences in CD1a and CD1b expression levels in the absence or presence of R-848 were statistically significant on Days 5 and 7 (n=3, P<0.02).

 
Figure 2B shows the expression profile of CD1 isoforms on R-848-treated cells. The induced expression of CD1a and CD1b was abrogated, and this was not a result of a delay in expression, as even at Day 7, CD1a and -b expression had failed to increase significantly. The differences in CD1a and CD1b expression levels in the absence or presence of R-848 were statistically significant on Days 5 and 7 (*,{dagger}n=3, P<0.02).

We next determined whether the decrease concerned only surface CD1 or whether the total cellular protein was affected. An immunoblot was carried out with a pan-CD1 antibody on lysates containing equal amounts of protein from monocytes, DC, or R-848-treated cells. Figure 3A shows that the total CD1 protein expression was down-regulated when DC differentiation took place in the presence of R-848.


Figure 3
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  		Figure 3. (A) The immunoblot reveals that CD1 total protein was not detected in cell lysates from monocytes, whereas it is clearly expressed in monocyte-derived DC. The total protein levels of CD1 are reduced drastically by comparison with cells that had been differentiated in the presence of R-848. (B) The decrease in CD1a protein is paralleled by a clear decrease in mRNA levels in cells differentiated in the presence of ssRNA or R-848. In contrast, CD14 mRNA was expressed in immature DC (iDCs), and the level of expression was increased in the presence of ssRNA or R-848. One of three experiments using cells differentiated in the presence of a TLR7/8 stimulus (from different donors) is shown.

 
The mRNA level of CD1a was next examined in TLR7/8-stimulated cells. CD1a mRNA was increased in immature DC compared with monocytes (not shown). Figure 3B shows that the mRNA level of CD1a was decreased in DC, which had been differentiated in the presence of TLR7/8 agonists. Liposomes alone did not modify mRNA levels, whereas addition of ssRNA (at 0.5–1 µg/ml) during monocyte differentiation to DC decreased CD1a expression and was similar to the decrease observed with R-848. CD14 mRNA levels are shown for comparison. GADPH mRNA levels were quantified and used to confirm equal loading.

Cytokine secretion by DC differentiated in the presence of TLR7/8 agonists
Monocyte-derived cytokines contribute to the immune response. Activation of TLR4 in monocytes has been shown to induce cytokine production via activation of MAPK p38 [19 ]. We therefore determined the time course of cytokine production throughout the 7 days of DC differentiation from monocytes (Fig. 4 ). First, all of the cytokines were already detected after 2 days of monocyte culture with GM-CSF and IL-4 in the presence of R-848. Cytokine secretion was maintained (TNF-{alpha}, IL-6, IL-1ß, IL-10, or IL-12) or increased (IL-8) by Day 5 and remained elevated in comparison with non-R-848-treated cells on Day 7. As half of the medium was replaced by fresh medium on Days 2 and 5, the actual amount of the different cytokines secreted is underestimated. Moreover, the maintenance (see IL-6) or increase in cytokine secretion (see IL-8) indicates that the decreased secretions of some cytokines (see TNF) are not simply a result of a decrease in cell viability. The difference between the amount of cytokine produced in the presence or absence of R-848 was significant (*n=3 donors, P<0.05).


Figure 4
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  		Figure 4. Time course of cytokine secretion induced by R-848 during DC differentiation; the time course of cytokine secretion during 7 days of CD14+ cell differentiation in the presence or absence of R-848. Half of the medium was replaced by fresh medium on Days 2, 5, and 7. All of the cytokines were detected on Day 2 in R-848-treated cells, and the levels secreted remained high on Day 5. Significant differences in the levels of cytokines secreted with and without R-848 are indicated (*P≤0.005).

 
Cytokines were also measured in the supernatants of DC, which had been differentiated for 7 days in the presence of ssRNA (1 µg/ml), and IL-6 and IL-8 secretion were detected (data not shown).

TLR7/8 activation in the course of DC differentiation impairs maturation induced via TLR4
We next analyzed how DC, differentiated in the presence or absence of TLR7/8 agonists, responded to a maturation signal. One well-characterized maturation signal is generated by activation of TLR4 by its agonist LPS. First, we analyzed the phenotype of DC differentiated in the presence of GM-CSF and IL-4 only (Fig. 5A , Medium). Maturation in the presence of R-848 or LPS led to increased expression of HLA-DR, CD80, CD83, and CD86, although the increase was greater with LPS. CD1a expression was decreased by maturation with LPS or R-848.


Figure 5
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  		Figure 5. Dual role of TLR7/8 agonist in maturation: Maturation of conventional DC (A) and inhibition of maturation of DC derived from monocytes stimulated with TLR7/8 agonists (B). (A) The phenotype of monocyte-derived DC (d7) differentiated with GM-CSF and IL-4 for 7 days followed by maturation with R-848 or LPS for 24 h. Increased expression of HLA-DR, CD80, CD83, and CD86 is consistent with maturation. (B) The phenotype of DC given a maturation signal with LPS as above but after exposure to ssRNA or R-848 during the 7 days of differentiation from monocytes. There was no evidence of CD80, CD83, or CD86 up-regulation as shown in A. Closed histograms show specific antibody binding, and open histograms show isotype control Ig staining. Representative histograms of three independent experiments are shown in A and B.

 
However, when DC were differentiated in the presence of ssRNA or R-848, the increased expression of CD83, CD86 was abrogated as was CD1a expression (Fig. 5B) . In addition, the induced expression of CCR7 was no longer detected (Fig. 5B) .

The results shown in Figure 5 are representative of three independent experiments. Of a number of surface markers tested, increased CD83, CD86, and CCR7 expression was impaired. ssRNA (1 µg/ml) or R-848 prevented maturation via TLR4 (Fig. 5B , Row 1, LPS alone).

We next examined whether cytokine secretion function of mature DC was altered by the presence of R-848 during differentiation. Figure 6A shows that monocyte-derived DC, matured by addition of LPS or of R-848, secreted a qualitatively and quantitatively similar profile of proinflammatory cytokines including TNF-{alpha}, IL-6, and IL-8 in addition to IL-12 and a low level of IL-10. *, The level of cytokine secretion compared with DC, which were not given a maturation signal, is indicated (*n=3, P<0.05).


Figure 6
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  		Figure 6. TLR7/8 stimulation of differentiating DC prevents cytokine secretion induced by a TLR4-mediated maturation signal. (A) The cytokine secretion profile of monocyte-derived cells, which after 7 days of differentiation, were given a 24-h maturation signal by addition of LPS or of R-848 (as in Fig. 5 ). Up-regulation of TNF-{alpha} was particularly clear, and IL-12 secretion was also increased. IL-6 and IL-8 secretion were strongly induced. Means and SD from three independent experiments are shown. (B) In contrast, when monocyte-derived cells, which had been differentiated in the presence of R-848, were subsequently given a maturation signal with LPS or with R-848, there was no evidence for increased cytokine secretion. Only IL-8 was detected and was increased scarcely beyond the background level. Significant differences in cytokine secretion after pretreatment with R-848 compared with no treatment during differentiation are indicated (P≤0.005, n>3).

 
Figure 6B shows that differentiation in the presence of R-848 abrogated cytokine production in response to a maturation signal, as LPS stimulation failed to induce secretion of TNF-{alpha}, IL-6, IL-10, IL-12, or IL-1ß, and only a low level of IL-8 was detected. The difference in the level of cytokine production by cells, which had been pretreated with R-848 compared with nontreated cells, was significant where indicated ({dagger}P≤0.05, n=3).

Antigen-presentation capacity
Finally, the antigen-presenting capacity of DC derived from R-848-treated monocytes was examined by carrying out an allostimulation or MLR assay (Fig. 7 ). As expected, immature DC induced T lymphocyte proliferation, which was amplified when mature DC from the same donor were used.


Figure 7
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  		Figure 7. T lymphocyte proliferation induced by DC is unaltered by TLR7/8 activation during DC differentiation. Antigen presentation was tested by T cell proliferation assays using monocyte-derived DC, which had been differentiated in the presence or absence of R-848. The proliferation of allogenic T cells was lowest in the presence of immature DC, produced by 7 days of monocyte differentiation in the presence of GM-CSF and IL-4 (•), and was consistently higher in the presence of DC, which had received a maturation stimulus (LPS) for 2 days further ({blacksquare}). Cells, which had been differentiated in the presence of R-848, induced more proliferation than DC differentiated without R-848 ({circ}). However, after addition of a maturation signal (LPS), proliferation induced by cells differentiated in the presence or absence of R-848 was comparable ({square} vs. {blacksquare}).

 
When R-848-treated, immature cells were used, they consistently led to a slightly (but nonsignificantly different) higher level of T lymphocyte proliferation compared with immature DC. This could be a result of the slight increase in the expression of costimulatory molecules.

DC, which had been given a maturation stimulus by addition of LPS, induced overall the highest level of T lymphocyte proliferation regardless of pretreatment with R-848 (Fig. 7) . Therefore, despite the profound inhibition of the TLR4-mediated cytokine secretion in DC, which had been differentiated in the presence of Resiquimod, the capacity to induce T cell proliferation of the mature DC was not at all impaired.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Monocyte-derived DC are a physiologically relevant APC population, and previous studies have examined the effect of TLR4 activation on the differentiation and maturation of this population in vitro. The importance of TLR4 activation in the antimicrobial immune response has been demonstrated clearly. In contrast, few studies have examined the outcome of the continued presence of TLR7/8 ligands on the monocyte-derived DC population, such as occurs in the course of viral infection [20 21 22 ]. A natural ligand for TLR7 and -8, ssRNA has been described recently, whereas synthetic imidazoquinoline compounds and guanosine analogs with antiviral activity have been recognized as agonists for these TLRs for some time [14 , 18, 23 ]. Plasmacytoid DC recognize ssRNA via TLR7 and produce high levels of IFN-{alpha}, and it has been demonstrated recently that nonplasmacytoid DC share this ability to recognize ssRNA via TLR7 and thereby, contribute to the IL-6 and IL-12 response [17 ].

This study determined the outcome of the ongoing presence of TLR7/8 ligands on differentiation of monocyte-derived DC and on their subsequent maturation by addition of a TLR4 ligand. Although neither the morphology of the cells nor their allogeneic T cell stimulation capacity was disturbed, phenotypic and functional differences were observed including inhibition of expression of DC surface molecules such as CXCR1 and members of the CD1 family. CD1 is structurally similar to MHC Class I and Class II molecules and is responsible for presentation of nonpeptide molecules of lipid or glycolipid origin. CD1a, -b, and -c are expressed in vivo on dermal DC and Langerhans cells. We have particularly examined CD1a and observed that the decreased expression is mediated at a transcriptional level. The regulation of CD1 expression via TLRs is not restricted to TLR7/8, as TLR4 activation by LPS during DC differentiation also led to decreased expression of CD1a via a mechanism involving p38 MAPK. Previous studies have reported that LPS induced alterations in differentiation of monocyte-derived DC [19 , 24 25 26 ].

It is interesting that a recent study reported that topical application of a Resiquimod-containing cream led to a decrease in the number of CD1a-expressing cells, despite an increase in HLA-DR-expressing cells [27 ]. CD1a-expressing DC enrichment within human tumors has been associated with survival, leading to the suggestion that CD1a could be important in presenting tumor-derived glycolipid antigens [28 ]. Alteration of induced up-regulation of CD1 expression on DC has been documented in the presence of infective agents such as Mycobacterium bovis or Mycobacterium tuberculosis [29 ]. IL-10 secretion may be partly responsible, as an IL-10-mediated reduction of GM-CSF-induced CD1a and MHC Class II expression on monocytes has been reported, although other studies failed to block CD1a down-regulation in the presence of neutralizing IL-10 antibodies [30 ].

In addition to the loss of induced expression of CD1 on DC, addition of TLR7/8 ligands led to loss of induced CCR7 expression via TLR4. CCR7 is implicated particularly in DC migration to lymph nodes. TLR7/8 stimulation also led to loss of antigen-internalization capacity. Such losses could therefore alter key DC functions [31 ]. TLR activation of immature murine DC has been reported to lead to an early increase in antigen internalization, which is of short duration (30–45 feet). We tested internalization after 7 days of exposure to TLR7/8 agonists, and therefore, we cannot exclude that antigen uptake is increased initially after TLR7/8 activation before a later reduction [32 ].

In terms of the cytokine secretion profile of DC, two distinct effects of TLR7/8 agonists were observed: in immature DC, cytokine production associated with the inflammatory response in addition to IL-10 was induced during differentiation, whereas LPS-induced production of cytokines by mature DC was abrogated by the continuous presence of TLR7/8 ligands during DC differentiation. DC regulate polarization of T lymphocyte cytokine production toward Type 1 or Type 2 responses. Disruption of the cytokine secretion profile could therefore alter the outcome of the immune response to viral or microbial pathogens [1 ]. Taken together, these data suggest that TLR7/8 activation allows DC, differentiated in the presence of GM-CSF and IL-4, to develop cross-tolerance to TLR4, although DC differentiation driven by other cytokine combinations could be unaffected by TLR7/8 signaling. We have conducted experiments on monocyte differentiation in the presence of polyinosinic:polycytidylic acid, a ligand of TLR3, which does not signal through the MyD88 adaptor but exclusively via TLR-IL-1 receptor domain-containing adaptor-inducing IFN-ß. We have observed only partial inhibition of CD1 expression on immature DC, followed by a complete response to LPS during maturation in terms of up-regulation of costimulatory molecules and cytokine secretion (data not shown). This could suggest implication of a MyD88-dependent pathway, common to TLR4 and TLR7/8.

Taken together, the data support the notion that the continued presence of TLR7/8 ligands during replenishment of monocyte-derived DC populations could alter the adaptive immune response at two levels: first, by impairing DC differentiation, and second, by abrogation of the DC response to a subsequent TLR-activating pathogen, impairing DC maturation and function.


   ACKNOWLEDGEMENTS
 
This work was supported by EU Grant QLK3-CT-2002-02026. We are grateful to Dr. Ryad Tamouza, who provided CD1a primers for the RT-PCR studies, and Dr. Claire Rabian, who gave assistance with the MLR assays. We thank I. Sloma, M. Abdul, and T. Vasselon for helpful discussions and O. Leclercq for technical assistance.

Received July 11, 2005; accepted August 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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