Sensitization to TLR7 Agonist in IFN-β-Preactivated Dendritic Cells

Generation of DC

DC were prepared as previously described (21). Briefly, PBMC were isolated from freshly collected buffy coats obtained from healthy voluntary blood donors (Blood Bank of University “La Sapienza,” Rome, Italy) by density gradient centrifugation using Lympholyte-H (Cedarlane Laboratories). Monocytes were purified by positive sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec). The recovered cells were > 99% CD14⁺ as determined by flow cytometry. DC were generated by culturing monocytes in 6-well tissue-culture plates (Costar) with 50 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D Systems). At day 5, the cells were 70–90% CD1a⁺ and 95% CD14⁻. At day 5, DC were starved from IL-4 and GM-CSF for 20 h before their stimulation.

Abs and other reagents

mAbs specific for CD1a, CD14, CD86, CD83, CD38, IgG1 (BD Pharmingen) were used as direct conjugates to FITC or PE. Sheep antiserum raised against human leukocyte IFN, a gift from G. Uzé (Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5124, Montpellier, France), was used at a 1/100 dilution (25). LPS from Escherichia coli 0111:B4 (Sigma-Aldrich) was used at a concentration of 1 μg/ml. IFN-β (Avonex; Biogen) was used at 200 pM. TLR7 (3M-001) and TLR8 (3M-002) ligands (3M Pharmaceutical) were used at 25 and 5 μM, respectively (13). These optimal doses of TLR7 and TLR8 ligands to induce DC maturation were evaluated by titrating different concentrations. Poly I:C (Amersham Biosciences) was used at 50 μg/ml. Pam₃CSK4 (Alexis) was used at 2 μg/ml. H3N2 influenza A (Flu) strain A/Beijing/353/89 virus was grown in 11-day-old embryonated chicken eggs and used at a concentration of 12.8 hemagglutination U/ml (gift of Dr. I. Julkunen, National Public Health Institute, Helsinki, Finland).

RNA isolation and real-time RT-PCR quantification

RNA was extracted with RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions that includes a DNase I treatment. Reverse transcriptions were primed with oligo(dT) and performed using the murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Quantitative PCR assays were done at least in triplicates using the Platinum TaqDNA Polymerase (Invitrogen Life Technologies) and the SYBR Green I (BioWhittaker Molecular Applications) on a LightCycler (Roche Diagnostics). All quantification data are presented as a ratio to the GAPDH level. The SEs (95% confidence limits) were calculated using the Student t test. Quantification standard curves were obtained using dilutions (4-log range) of the PCR products in 10 μg/ml sonicated salmon sperm DNA. The sequences of the primer pairs used for the quantification of GAPDH have been previously described (24). The primers used for mRNA quantification were: 2′-5′-oligoadenylate synthetase 2 (OAS2)_forward (for): 5′-AACTGCTTCCGACAATCAAC-3′; OAS2_reverse (rev): 5′-CCTCCTTCTCCCTCCAAAA-3′; SOCS1_for: 5′-AACTGCTTTTTCGCCCTTA-3′; SOCS1_rev: 5′-GCCACGTAGTGCTCCA-3′; Viperin_for: 5′-CTTTGTGCTGCCCCTTGAGGAA-3′; Viperin_rev: 5′-CTCTCCCGGATCAGGCTTCCA-3′; TLR7_for: 5′-TTACCTGGATGGAAACCAGCTACT-3′; TLR7_rev: 5′-TCAAGGCTGAGAAGCTGTAAGCTA-3′; CD38_for: 5′-GGCCTGGGTGATACATGGTGGA-3′; CD38_rev: 5′-ACAGCGACTGGCTCAGATCTCA-3′.

cDNA and antisense RNA (aRNA) synthesis

Five micrograms of total RNA were used for each analyzed condition (control, 4 h IFN-β, 24 h LPS, 24 h LPS plus anti-IFNs). As internal reference for microarray analysis, a pool of RNA from iDC collected from five healthy donors was used. First and second strand cDNA synthesis and the synthesis of aRNA were performed using the Amino-Allyl MessageAmp aRNA Amplification kit (Agilent). The quality of aRNA was tested by using Agilent Bioanalyzer 2100. The reaction allows the incorporation of amino-allyl-modified NTP that are subsequently labeled with the N-hydroxysuccinimide (NHS) ester-labeled Cy3/5 dye (Amersham Biosciences). The reference aRNA and the aRNAs from the different samples were labeled with Cy5 and Cy3, respectively. The labeled aRNAs (750 ng) were fragmented and prepared for the hybridization using the Agilent In Situ Hybridization kit Plus (Agilent).

Microarray analysis

Microarrays were purchased from Agilent. Agilent’s Human 1A Oligo Microarray (V2) contains 20,173 oligonucleotide probes (60-mer) spanning conserved exons across the transcripts of the targeted full-length genes. Microarrays were hybridized at 60°C in the Agilent Oligo Microarray Hybridization chamber (22 K format) for 17 h under constant rotation. Following hybridization, slides were washed once for 1 min in 6× standard saline phosphate/EDTA (SSPE)-0.005% N-lauroylsarcosine at room temperature, then washed for 1 min with 0.06× SSPE-0.005% N-lauroylsarcosine. Microarrays were stabilized and dried with the Stabilization and Drying Solution (Agilent). Using a ScanArray lite scanner and ScanArray Express software (Packard Biochip Technologies; PerkinElmer) microarrays were scanned at 532 and 633 nm. The results and images were quantified using Quantarray software 2.1 (Packard Biochip Technologies; PerkinElmer). Raw data and sample information were then entered into Genespring (Sylicon Genetics). The LOWESS normalization was performed because in experiments where two fluorescent dyes (red and green) are used, intensity-dependent variation in dye bias may introduce spurious variations in the collected data. LOWESS normalization merges two-color data, applying a smoothing adjustment that removes such variation. Moreover, the Student t test was applied to compare the different conditions.

Cell extracts

Nuclear cell extracts were prepared as previously described (26). Briefly, cell pellets (5 × 10⁶) were resuspended in 1 ml of buffer A (0.5% Nonidet P-40, 10 mM EDTA, 10 mM EGTA, 10 mM KCl, 10 mM HEPES (pH 7.9)) to which 1 mM DTT, 0.5 mM PMSF, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml antipain were freshly added) and incubated on ice for 10 min. Nuclei were sedimented by centrifuging the lysates at 1,200 × g for 10 min. The nuclear pellets were resuspended in 30–40 μl buffer C (1 mM EDTA, 1 mM EGTA, 0.4 M NaCl, 20 mM HEPES (pH 7.9), 5 mM MgCl₂, 25% glycerol, with fresh addition as above) and incubated for 10 min on ice with occasional mixing. The suspensions were clarified by centrifuging at 15,000 × g for 10 min. The supernatants were recovered as nuclear extracts and were rapidly frozen on crushed dry ice and stored at −80°C. Whole cell extracts were prepared as previously described (24). Briefly, cells (10⁷) were lysed in 30–50 μl of ice-cold whole cell extraction buffer (20 mM HEPES (pH 7.9), 50 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 10 mM EDTA, 2 mM EGTA, 10 μg/ml leupeptin, 100 mM NaF, 0.5 mM PMSF, 10 mM sodium orthovanadate, and sodium molybdate). The lysate was incubated for 30 min on a shaker at 4°C and insoluble debris was removed by centrifugation (13,000 × g at 4°C for 10 min) and the lysate was stored at −80°C.

Flow cytometry analysis

Approximately 1–2 × 10⁵ cells were aliquoted into tubes and washed once in PBS containing 2% FCS. The cells were incubated with mAbs at 4°C for 30 min. The DC were then washed and fixed with 2% paraformaldehyde before analysis on a FACScan using CellQuest software (BD Biosciences). The change in expression of the cell surface molecules was determined by subtracting the median fluorescent intensity (MFI) of the isotype control Ab from the MFI from the specific Ab.

Cytokine and chemokine determinations

Supernatants from DC cultures were harvested 24 h after treatments, and stored at −80°C. A CXCL-10-specific ELISA kit was obtained from R&D Systems. IL-6, IL-10, IL-12p70, and TNF-α were measured with the human inflammation cytometric bead assay (CBA; BD Biosciences). ELISA for IFN-α was obtained from PBL Biomedical Laboratories. All assays were conducted according to the manufacturers’ instructions.

Western blot analysis

Western blot was performed as previously described (24). Briefly, 30 μg of whole cell extracts were separated by 10% SDS-PAGE gel and blotted onto nitrocellulose membranes. Blots were incubated with the indicated Abs: IRF-7 Ab (Santa Cruz Biotechnology), TLR7 Ab (Novus Biologicals), or β-actin Ab (Sigma-Aldrich) and reacted with anti-rabbit HRP-coupled secondary Ab using an ECL system (Amersham Biosciences).

In silico analysis of human TLR7 promoter

The analysis of the human promoter of the TLR7 gene (accession NM_016562.3) was done by using the transcription factor binding site (TFBS) prediction program MatInspector (online at www.genomatix.de/matinspector.html). MatInspector uses informations of MatInd (that generates position weight matrices) to scan nucleotide sequences for matches to this pattern by calculating a matrix similarity score that reaches 1 only if the test sequence corresponds to the most conserved nucleotide at each position of the matrix. The analyzed sequence was a region spanning 1000 bp upstream the transcription starting site (TSS) (27).

EMSA

EMSA was conducted as previously described (26). Briefly, to measure the association of DNA-binding proteins with different DNA sequences, synthetic double-stranded oligonucleotides were end-labeled with [γ-³²P]ATP by T4 polynucleotide kinase. For the analysis of IRF-1 complexes, nuclear cell lysates (15 μg) were used in EMSA experiments. For supershift analysis, 1 μg of anti-IRF-1 Ab (Santa Cruz Biotechnology) was added to the reaction. The oligonucleotide used was as follows: IRF-1 site-TLR7 5′-CTATAAAAACGAAAGAAATTTGGT-3′. For competition analysis, a 100-fold molar excess of cold IRF-1 site-TLR7 and unrelated oligonucleotides was added to the binding reaction.

Plasmid and transfection assays

Rc-CMV empty vector and Rc-CMV IRF-1 have been previously described (28). All DNA plasmids were prepared using Endofree Plasmid Maxi kits (Qiagen). DC were transfected using Nucleofector technology according to the manufacturer’s instruction (Amaxa Nucleofector) as previously described (29). Twenty hours after transfection, total RNA was extracted and analyzed for TLR7 expression by real-time RT-PCR.

Statistical analysis

Statistical analysis was calculated using a two-tailed for paired data Student’s t test. A p value <0.05 was considered statistically significant.

Defining type I IFN-dependent response in monocyte-derived DC following TLR4 triggering

Before analyzing the IFN-β-related functional and phenotypical modifications occurring in DC following LPS treatment, initial studies were designed to test the efficacy of type I IFN neutralization in our experimental model. To this aim, we monitored IRF-7 expression by Western blotting because it is known that IRF-7 expression occurs following type I IFN release during most viral and bacterial infections (30, 31). Whole lysates were prepared from control iDC, or DC that had been treated for 24 h with IFN-β or LPS in presence or absence of anti-IFN Abs. These time points were judged to provide the best snapshot of IFN-mediated changes in LPS-matured DC and were used for all subsequent experiments. As expected, IRF-7 protein expression was strongly up-regulated after IFN-β and LPS treatments (Fig. 1⇓A). Moreover, the presence of anti-IFN Abs completely inhibited the LPS-induced IRF-7 expression, proving the requirement of IFN-β production for its induction and the ability of these neutralizing Abs to dramatically inhibit the autocrine/paracrine type I IFN-mediated intracellular signaling. No reduction of IRF-7 expression was observed by the addition of sheep serum to LPS-treated cultures (data not shown). Equal loading of all samples was verified by immunoblotting with an anti-β-actin Ab (Fig. 1⇓A, lower panel).

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FIGURE 1.

Type I IFN-dependent functional modifications in DC during LPS-induced maturation. A, iDC were left untreated (Ctr), stimulated for 24 h with IFN-β (200 pM), with LPS (1 μg/ml) in presence or absence of anti-IFN Abs. Whole cell extracts were prepared and 30 μg of lysates subjected to immunoblot analysis using anti-IRF-7 or anti-β-actin specific Abs. This is a representative experiment performed with DC obtained from other two donors. B, Supernatants were harvested from control cells (Ctr), LPS-treated DC with or without anti-type I IFN Abs or sheep antiserum (serum) as control for 24 h. The level of CXCL-10 was measured by ELISA. The results represent the means ± SE of three different experiments; p < 0.05 LPS vs LPS + anti-IFN Abs. C, Surface expression of CD38 was evaluated by FACS in iDC (Ctr) or LPS-matured DC in presence or absence of anti-IFN Abs. A total of 5000 cells were tested per sample. A representative FACS profile is shown. The analysis was repeated three additional times, using DC from a total of four different blood donors. α, anti.

We next monitored the production of the IFN-inducible CX chemokine ligand 10 (CXCL10) by ELISA. Matured DC produce CXCL10 and this has been shown to be IFN-regulated (32, 33, 34). DC were matured with LPS, with or without anti-IFN Abs, for 24 h and supernatants were then harvested. CXCL10 release, observed following LPS treatment, was significantly decreased in DC matured with LPS in presence of anti-IFN Abs (Fig. 1⇑B). To further confirm that the activity of these neutralizing Abs was specific, DC were treated with LPS in presence of sheep control serum; in this condition, no decrease of CXCL10 production was observed (Fig. 1⇑B).

Furthermore, we investigated whether the profile of CD38, a surface molecule known to be induced also by type I IFN (35, 36) was affected by anti-IFN Abs. Surface expression of CD38 was examined using flow cytometry analysis and as expected a marked reduction in the LPS-induced increase in CD38 expression was observed (Fig. 1⇑C).

Analysis of IFN-β-induced gene expression in LPS-matured DC

To evaluate the extent to which IFN-β contributes to the LPS-induced gene expression profile, we next performed microarray analysis in DC following 4 h of treatment with IFN-β or 24 h of treatment with LPS in the presence or absence of anti-IFN Abs. The RNA was collected from four different healthy donors and similar gene expression profiles were found between donors. aRNAs were hybridized to Agilent’s Human 1A Oligo Microarray (version 2) that contains 22 K oligonucleotide probes and the data were analyzed with Gene Spring Software. Because one of the principal goals of our study was to elucidate a specific list of genes regulated by type I IFN production during LPS treatment, we began our analysis by profiling the changes in gene expression that occur in DC in response to 4 h IFN-β treatment compared with that observed after 24 h stimulation with LPS. A total of 218 genes were found to be modulated by IFN-β and 315 genes by LPS. We focused our attention on the genes that were up-regulated by the two different treatments and which were down-modulated in the presence of anti-IFN Abs. The expression profile of 23 genes followed this criteria and 5 of these were validated by real-time RT-PCR (see below). A hierarchical clustering analysis of this set of genes is shown in Fig. 2⇓A. Some of these genes are part of the so-called “IFN signature,” a set of genes known to be regulated by type I IFN signaling (37), such as the OAS 1 and 2 (38), suppressor of cytokine signaling (SOCS) 1 (39, 40), protein kinase R (PKR) (41) and Viperin (42). Others were ISGs with unknown function but which have been shown to be induced via IFN production including IFN-induced protein 35 and IFN-induced protein with tetratricopeptide repeats 5 (37). We validated the results obtained by microarray analysis by quantifying the mRNA level of OAS2, SOCS1, and Viperin by real-time RT-PCR (Fig. 2⇓B). Moreover, the CD38 profile of expression, observed by FACS analysis (see Fig. 1⇑C), was confirmed also at the mRNA level by microarray (Fig. 2⇓A) and by real-time RT-PCR (Fig. 2⇓B).

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FIGURE 2.

Specific transcriptional profile induced by type I IFN release along LPS-induced DC maturation. Total RNA was isolated from DC either following 4 h IFN-β or 24 h LPS treatment in presence or absence of anti-IFN Abs. A, Hierarchical clustering of gene expression data by four different healthy donors is shown. Each row represents a separate gene and each column a different condition, as indicated. The normalized expression index for every transcript sequence (rows) in each sample (columns) is indicated by a color code. Red, yellow and green squares indicate that expression of the gene is greater than, equal to or less than the mean level of expression across the different donors. The scale extends from fluorescent ratios of 0.25 to 5.0. B, mRNA expression of OAS2, SOCS1, Viperin, CD38, and GAPDH was analyzed by real-time RT-PCR. Data were then normalized with GAPDH level of expression. Results are means ± SE of triplicate values. This is a representative experiment of four different analyzed donors. α, anti.

Interestingly, IFN-β and LPS treatments also induced the expression of a subset of genes which have not previously been reported to be IFN-inducible in DC. Importantly, two of these were PRRs including nucleotide-binding oligomerization domain 2 (NOD2) and TLR7 (Fig. 2⇑A). Whereas NOD2 (also called CARD15) plays a central role in the immune response to intracellular bacteria by recognizing muramyl dipeptide (MPD), a derivative of peptidoglycan (43), TLR7 recognizes ssRNA and is activated by infections with ssRNA viruses (9, 10). The TLR7 gene has been reported to be induced by type I IFN in monocytes/macrophages or in B cells (44, 45), but its inducibility by type I IFN has never been characterized in the context of DC maturation.

The expression of defensin-β1 (DEFB1) was also found to be IFN regulated. DEFB1 is an antimicrobial peptide implicated in the resistance of epithelial surfaces to microbial colonization and it has already been described that IFN-γ treatment or TLR3 and 4 triggering can drive its expression (46, 47).

Type I IFN-dependent regulation of TLR7 expression in LPS-treated DC

We focused our attention on the modulation of TLR7 expression to understand its regulation in mDC. Indeed, in humans the expression of TLR7, together with TLR9, is known to be restricted only to the endosomes of the pDC but not of the mDC, whereas TLR8 expression is constitutively expressed in mDC, albeit at low levels (5, 13). We confirmed the results obtained by microarray analysis measuring TLR7 mRNA level by real-time RT-PCR either in iDC or IFN-β- and LPS-treated DC in presence or absence of anti-IFN Abs (Fig. 3⇓A). Whole cell lysates were prepared from DC following 24 h of treatment with IFN-β or with LPS in the presence or absence of anti-IFN Abs. TLR7 protein expression was quantified by Western blotting (Fig. 3⇓B). Consistent with mRNA expression data, TLR7 protein levels were also induced following stimulation with LPS and were inhibited when anti-IFN Abs were added to the cultures, further proving a clear dependence on IFN production and signaling in the regulation of TLR7 by LPS. Equal loading of all samples was verified by immunoblotting with an anti-β-actin Ab (Fig. 3⇓B, lower panel).

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FIGURE 3.

TLR7 expression by LPS-induced IFN-β production. A, TLR7 gene expression was quantified by real-time RT-PCR in total RNA extracted from iDC (Ctr) or DC treated with IFN-β, LPS, or LPS + anti-IFN Abs as described in the legend of Fig. 2⇑. B, Whole cell lysates collected from cells treated for 24 h as described in (A) were analyzed by immunoblotting using a specific anti-TLR7 Ab. This analysis was repeated two additional times using DC from a total of three different blood donors. α, anti.

We next examined whether the activation of other TLRs expressed in DC could modulate TLR7 gene expression as we had observed following LPS stimulation. DC were stimulated for 24 h with synthetic bacterial lipopeptide (Pam3cysk4) and with synthetic dsRNA (poly I:C) which engage TLR2 and 3, respectively. RNAs were prepared and TLR7 mRNA levels were analyzed by quantitative PCR (Fig. 4⇓A). Poly I:C and LPS treatments both strongly induced TLR7 expression, in contrast to Pam3Cysk4 which did not stimulate TLR7 expression consistent with a failure of TLR2 ligands to induce type I IFNs (48).

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FIGURE 4.

Transcriptional regulation of the TLR7 gene. A, Total RNA was extracted from nonstimulated (Ctr), Pam3cysk4 (2 μg/ml), poly I:C (pI:C) (50 μg/ml), and LPS (1 μg/ml) treated DC upon 24 h treatment. Real-time RT-PCR was performed to measure TLR7 mRNA level. One representative of three different experiments is shown. B, Schematic map of ∼600 bp of the human TLR7 promoter. TATAAA is the consensus sequence for the TATA box. C, DC were treated with IFN-β or LPS with or without anti-IFN Abs for 4 h. Nuclear extracts (15 μg) were prepared and analyzed by EMSA using a specific radiolabeled oligonucleotide corresponding to the IRF-1 binding site present within TLR7 promoter. Supershift assay (SS) was performed using an anti-IRF-1 Ab, where indicated. This experiment was repeated additional two times. D, Nuclear extracts from LPS-treated DC (4 h) were used for competition studies where 100× excess of cold oligonucleotides containing the putative IRF-1 consensus sequence within the TLR-7 promoter and a sequence unrelated (unr) to IRF-1 were added to the binding reaction. E, Total RNA was also extracted from DC transfected with vectors expressing IRF-1 or with an empty vector (EV). TLR7 expression was evaluated by quantitative PCR. This is a representative experiment performed additional two times. α, anti.

This differential activation of the TLR7 gene prompted us to examine its promoter region for TFBS. Using the TFBS prediction program MatInspector (27), a region spanning 1000 bp upstream and 500 bp downstream of the TSS was analyzed. Interestingly, TFBS for IRF-1, STAT1, and NF-κB were detected within 600 bp of the TSS (Fig. 4⇑B). To probe the involvement of each of these factors to the regulation of TLR7 gene induction, oligonucleotides corresponding to the binding sites for IRF-1, STAT1, and NF-κB from the TLR7 promoter were designed. Nuclear extracts were prepared following stimulation of DC with IFN-β and LPS in the presence or absence of anti-IFN Abs and analyzed by EMSA for the specific binding of each factor to the respective site. No STAT1 binding and only a slight signal in the case of NF-κB were detected (data not shown). Conversely, a clear induction of IRF-1 DNA-binding activity was observed in LPS- and IFN-β-treated cells and this binding activity was completely lost when anti-IFN Abs were added (Fig. 4⇑C). The identity of the complexes was confirmed by supershift experiments using Abs raised against IRF-1. Moreover, complex formation was also inhibited by the addition of an excess of unlabeled oligonucleotide containing the putative IRF-1 consensus sequences within TLR7 promoter when nuclear extracts from IFN-β-stimulated (data not shown) or LPS-treated DC were used (Fig. 4⇑D).

We also examined the effect of IRF-1 on TLR7 mRNA expression in DC transiently transfected using nucleofection with a vector expressing IRF-1 or with an empty vector. As shown in Fig. 4⇑E, the expression of TLR7 was clearly increased by the presence of IRF-1 compared with DC transfected with the empty vector. All together, these results indicate a key role for IRF-1 in the regulation of TLR7 along LPS-induced DC maturation.

Maturation of IFN-β-pretreated DC by a specific TLR7 agonist

Although TLR7 and TLR8 are phylogenetically and structurally related, their selective activation leads to completely different immune responses. It has been shown that the small molecule imidazoquinoline analogues 3M-001 and 3M-002 are selective ligands for TLR7 and TLR8, respectively (13). 3M-001 directly activates pDC and, to a lesser extent, monocytes; conversely, 3M-002 activates monocytes and mDC. Because our findings support a model whereby TLR7 gene and protein expression are induced following type I IFN release, we examined whether a 24 h pretreatment of DC with IFN-β would enable these cells to respond to TLR7 ligands. Thus, IFN-β-pretreated DC were stimulated for 24 h with TLR7 agonist and expression of CD83, CD86, and CD38 surface markers were evaluated by flow cytometry analysis to assess the activation status of the cells (Table I⇓). We observed that a prestimulation of DC with IFN-β provoked the maturation of the 3M-001-treated DC, phenomenon not observed in the absence of IFN-β treatment. As expected, TLR8 triggering alone induced an up-regulation of the considered markers and IFN-β by itself caused an increase of CD38 and CD86.

Furthermore, the secretion of the proinflammatory cytokines, such as TNF-α and IL-6, and of the regulatory cytokines, IL-12p70, IL-10, and IFN-α was analyzed. Supernatants were harvested from DC treated for 24 h with the TLR7 agonist 3M-001 with or without 24 h IFN-β pretreatment. 3M-002-stimulated DC were used as our internal control and a strong production of all the analyzed cytokines was observed upon TLR8 triggering with the exception of the poor release of IFN-α (Fig. 5⇓). Conversely, in the pretreated DC, a clear induction of IL-12, IL-6, TNF-α, and IL-10 production was detected following TLR7 activation compared with DC treated with 3M-001 alone. Only a scant production of IFN-α was observed upon 3M-001 stimulation in DC pretreated with IFN-β compared with the levels which accumulated in the culture medium following FLU virus infection.

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FIGURE 5.

Analysis of cytokine production in IFN-β pretreated DC following TLR7 triggering (A–E). Cell culture supernatants were harvested from DC treated for 24 h with the TLR7 agonist 3M-001 with or without 24 h pretreatment with IFN-β, or stimulated either with 3M-002 or IFN-β alone. The level of IL-12 (A), TNF-α (B), IL-6 (C), or IL-10 (D) were measured by CBA. IFN-α production (E) was evaluated by ELISA. Results are means ± SE from four different experiments; p < 0.05 3M-001 vs IFN-β + 3M-001.