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The Journal of Immunology, 2007, 178, 7607 -7615
Copyright © 2007 by The American Association of Immunologists, Inc.
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IL-27 Synthesis Induced by TLR Ligation Critically Depends on IFN Regulatory Factor 31

Céline Molle*, Muriel Nguyen*, Véronique Flamand*, Joelle Renneson*, François Trottein{dagger}, Dominique De Wit*, Fabienne Willems*, Michel Goldman* and Stanislas Goriely2,*

* Institute for Medical Immunology, Université Libre de Bruxelles, Charleroi, Belgium; and {dagger} Institut National de la Recherche Médicale, Unité 547, Institut Pasteur de Lille, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3–/– upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-27, a member of the IL-12 family, is a heterodimeric cytokine composed of EBV-induced gene 3 (EBI3)3 and p28 subunits (1). EBI3 has been identified as an IL-12p40, whereas homolog p28 is structurally related to IL-12p35 and IL-6. IL-27 was first suggested to promote Th1 responses by inducing STAT-1-dependent expression of the transcription factor T-bet in naive CD4+ T cells, leading to up-regulation of IL-12Rbeta2 and IFN-{gamma} synthesis (2). However, both Th1- and Th2-type inflammatory responses were found to be amplified in IL-27R-deficient mice infected with parasites or bacteria, indicating a regulatory role for IL-27 on effector T cell activities (3, 4, 5). Indeed, IL-27 was recently shown to suppress pathogenic Th17 cell activities in experimental autoimmune encephalomyelitis and chronic Toxoplasma gondii infection (6, 7). Furthermore, Wirtz et al. (8) demonstrated that IL-27 neutralization protects mice against lethal septic peritonitis by enhancing the influx and oxidative burst capacity of neutrophils and macrophages. Altogether, IL-27 now emerges as a key regulatory cytokine which controls effector functions of several types of immune cells involved in inflammation and adaptive immune responses.

Like other members of the IL-12 family, IL-27 is rapidly produced by APCs upon exposure to TLR ligands (9). TLRs elicit distinct signaling cascades depending on the recruitment of various Toll/IL-1R (TIR)-containing adaptors (10). The myeloid differentiation factor 88 (MyD88), coupled to most TLRs, except TLR3 which drives activation of NF-{kappa}B, MAPKs, and IFN regulatory factor (IRF) 5 (11, 12). In contrast, recruitment of TIR domain-containing adaptor inducing IFN-beta (TRIF) associated with TLR3 and TLR4 results in activation and nuclear translocation of IRF3, a critical transcription factor for IFNbeta gene expression (13). As previously published in this journal, EBI3 expression was shown to be induced in dendritic cells (DCs) upon TLR stimulation via activation of the transcription factors NF-{kappa}B and PU-1 (14). In this study, we provide evidence that TLR-inducible expression of the p28 subunit critically involves activation of the TRIF/IRF3 pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

MyD88-deficient (11) and LPS2 (15) mice were obtained from Dr. S. Akira (Osaka University, Osaka, Japan) and B. Beutler (The Scripps Research Institute, La Jolla, CA), respectively, and were bred in the animal facility of the Transgenose Institute (Centre National de la Recherche Scientifique, Orleans, France). IRF3-deficient mice (16) were obtained from the Riken BioResource Center (Ibaraki, Japan) with the approval of T. Taniguchi (University of Tokyo, Tokyo, Japan). Mice were bred and maintained in specific pathogen-free conditions according to institutional guidelines.

Cells and reagents

Human monocyte-derived DCs (moDCs) and murine bone marrow-derived DCs (BMDCs) were generated as previously described (17). The RAW 264.7 murine macrophage cell line and HEK-293 human kidney cell line were obtained from LGC Promochem. Ultra-pure LPS from Escherichia coli (0111:B4) and resiquimod (R-848) were obtained from Cayla and Pharmatech, respectively. Polyinosine-polycytidylic acid (polyI:C) and S- [2.3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH (Pam3CSK4) trihydrochloride were purchased from GE Healthcare and EMC Microcollections, respectively. Recombinant murine IFN-beta was obtained from R&D Systems. For LPS challenge, each mouse was i.v. injected with 500 µg of LPS from E. coli (055:B5; Sigma-Aldrich) dissolved in PBS.

Virus and infection of moDCs

Human CMV (hCMV) strain TB40/E isolated from a bone marrow transplant recipient was provided by Dr. Z. Tabi (University of Wales College of Medicine, Cardiff, U.K.) and was propagated on human foreskin fibroblasts as previously described (18). Viral titers varied between 107 and 108 PFU/ml. To determine the proportion of infected cells, intracellular hCMV IE Ag was detected by flow cytometry (data not shown). At a multiplicity of infection (MOI) of 0.5, between 5 and 35% (mean 19.8%) of moDCs were infected by hCMV while 60–88% (mean 70.1%) of cells were found to be infected at a MOI of 10.

Plasmid constructs

A 1-kb fragment of the IL-27(p28) gene (nt –1061/+13) was amplified by PCR from human genomic DNA and subsequently cloned into the pCR-II-Blunt-TOPO vector (Invitrogen Life Technologies). The insert was subcloned into the pGL3-BASIC vector (Promega) as a HindIII-XhoI insert to generate the luciferase reporter plasmid. The plasmids p28lucMut A, Mut B, and Mut AB are derivatives of p28-lucWT in which the IFN-stimulated response element (ISRE) site was altered by the QuickChange Site-directed Mutagenesis Method (Stratagene). IRF3 5D expression vector was provided by R. Lin (McGill University, Montreal, Quebec, Canada).

Quantification of cytokine production in sera and culture supernatants

Murine IL-27p28, IL-12p70 were determined in cell-free supernatants or serum by specific ELISA (Quantikine; R&D Systems) with detection limits of 3.9 pg/ml. IL-12p40 levels in cell-free supernatants were determined using Duoset ELISA (R&D Systems) with a detection limit of 15 pg/ml. IL-6, IL-12p40, TNF-{alpha}, and GM-CSF levels in the sera were measured by Multiplex bead array (Luminex; BioSource International).

RNA purification and real-time RT-PCR

Total RNA was extracted using a MagnaPure LC RNA-High Performance Isolation kit (Roche Diagnostics). RT- and real-time PCR were then conducted using LightCycler-RNA Master Hybridization Probes (one-step procedure) on a Lightcycler apparatus (Roche Diagnostics). Primer sequences are described in Table I.


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  		Table I. Oligonucleotide sequences used for PCR

 
EMSAs

EMSAs were performed as previously described (17). For competition analysis, we used unlabeled ISRE from IFN-stimulated gene 15 (5'-GATCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3'), mutated ISRE from ISG15 (ISG15Mut; 5'-GATCCTCGGGAAAGGGAGGCCGAGGCTGAAGCC-3'), and NF-{kappa}B consensus (5'-AGTTGAGGGACTTTCCCAGGC-3'). For supershift assays, polyclonal Abs against IRF3 or IRF1 (Santa Cruz Biochemicals) were added to the binding-reaction mixture.

Transient transfection and luciferase assays

RAW 264.7 cells and HEK 293 cells were transfected using FuGENE-6 (Roche Diagnostics) as previously described (17). Promoter activities were analyzed using the Dual-Glo Luciferase Reporter Assay system (Promega). Promoter activities were then normalized to Renilla luciferase activities.

Chromatin immunoprecipitation (ChIP) assay

The ChIP experiments were performed using the ChIP-IT kit from Active Motif according to manufacturer’s instructions as previously described (17). Anti-IRF3 and anti-acetyl histone H4 Abs were obtained from Active Motif and Upstate (Biognost), respectively. Primer sequences are detailed in Table I.

Statistical analysis

The Student t test was used for statistical calculation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Both MyD88 and TRIF are involved in activation of EBI3 and p28 genes in LPS-stimulated mouse DCs

In a first series of experiments, we compared the ability of BMDCs to produce IL-27 in response to distinct TLR agonists. As shown in Fig. 1A, we found that TLR4 and TLR3 stimulation by LPS and polyI:C, respectively, induced high levels of IL-27. In contrast, IL-27 levels were low or undetectable in response to TLR2 and TLR7/8 engagement by Pam3CSK4 and R848, respectively. In comparison, important amounts of IL-12/23p40 were produced by BMDCs under the same experimental conditions, albeit at lower levels than in response to LPS (Fig. 1A, right panel). We then compared IL-27 protein synthesis by LPS-stimulated BMDCs obtained from wild-type, MyD88-deficient, and LPS2 mice, the latter carrying a mutation in the TRIF gene (15). IL-27 was not induced in LPS-stimulated TRIF-deficient LPS2 DCs, indicating that TRIF activity is critically required for TLR4-mediated IL-27 protein synthesis (Fig. 1B). In contrast, MyD88–/– DCs were stimulated to produce IL-27 upon LPS exposure although at lower levels than wild-type mice (Fig. 1B). We then quantified mRNA levels for EBI3 and p28 subunits under the same experimental conditions. In agreement with a previous report indicating the role of NF-{kappa}B in EBI3 gene activation (14), EBI3 mRNA levels upon LPS stimulation were found to be decreased in both MyD88- and TRIF-deficient mice as compared with wild-type mice (Fig. 1C). IL-27p28 mRNA levels were also decreased in MyD88-deficient mice as compared with wild-type mice (Fig. 1D). The most striking finding was the dramatic decrease of p28 mRNA induction in TRIF-deficient LPS2 DCs. Collectively, these data establish that IL27p28 gene activation and IL-27 protein synthesis critically require TRIF-dependent signals downstream of TLR4.


Figure 1
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  		FIGURE 1. TRIF signaling is required for IL-27p28 expression in TLR-stimulated mouse DCs. A, BMDCs from C57BL/6 wild-type (WT) mice were incubated in medium alone or stimulated with optimal concentrations of LPS (100 ng/ml), Pam3CSK4 (10 µg/ml), polyI:C (10 µg/ml), or R848 (10 µg/ml). After 24 h of stimulation, supernatants were collected and analyzed for IL-27 and IL-12/23p40 levels by ELISA. Results represent the mean ± SEM of 10 independent experiments. *, p < 0.05 and ***, p < 0.001 as compared with LPS stimulation. B, WT, MyD88–/–, or LPS2 (TRIF–/–) mice were stimulated with LPS (100 ng/ml). After 24 h of stimulation, supernatants were collected and analyzed for IL-27 levels by ELISA. Results represent the mean ± SEM of five independent experiments. ***, p < 0.001 as compared with WT mice; IL-27 levels were below 15 pg/ml in supernatants of DCs cultured in medium alone whatever the mouse strain. C and D, Total RNA was extracted and analyzed by real-time RT-PCR. Levels were normalized using beta-actin mRNA as reference. One representative experiment of three is shown.

 
Critical role of IRF3 in IL-27p28 synthesis

Because of the critical role of IRF3 in the TRIF-dependent signaling cascade (19), we investigated the possible involvement of IRF3 in the regulation of IL-27 synthesis. As shown in Fig. 2A, IL-27 protein synthesis by LPS-stimulated IRF3–/– DCs was profoundly depressed in comparison with DCs from wild-type mice, even after prolonged incubation. Furthermore, LPS-induced p28 mRNA levels were found to be strongly reduced in IRF3–/– DCs as compared with wild-type DCs whereas EBI3 mRNA levels were similar in IRF3–/– and wild-type DCs (Fig. 2B). Because IRF3 activation downstream of TLR4 leads to IFN-beta synthesis, we tested whether addition of rIFN-beta could restore IL-27 production in LPS-stimulated IRF3–/– BMDCs. Incubation with rIFN-beta induced detectable levels of IL-27 in both wild-type and IRF3–/– cells (Fig. 3). Addition of both LPS and rIFN-beta did not significantly increase IL-27 production in wild-type or IRF3–/– BMDCs as compared with single stimulus, whatever the dose of rIFN-beta used. Importantly, even in this condition, IL-27 production by IRF3–/– cells was strongly and statistically decreased in comparison to wild-type BMDCs. These results indicate that decreased IFN-beta production in LPS-stimulated IRF3–/– cells could contribute to impaired IL-27 production but does not directly account for this observation.


Figure 2
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  		FIGURE 2. Critical role of IRF3 in IL-27p28 gene activation in LPS-stimulated BMDCs. BMDCs from C57BL/6 wild-type (WT) or IRF3–/– mice were either incubated with medium alone or stimulated with LPS (100 ng/ml). A, Supernatants were collected and analyzed at different time points for IL-27 levels by ELISA. Results represent the mean ± SEM of triplicates in one representative experiment of three. B, Total RNA was extracted after 6 h and analyzed by real-time RT-PCR. Results are shown as mean ± SEM of five independent experiments. *, p < 0.05 as compared with WT mice.

 

Figure 3
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  		FIGURE 3. Addition of rIFN-beta does not circumvent deficient IL-27 production in LPS-stimulated IRF3–/– BMDCs. BMDCs from C57BL/6 wild-type (WT) or IRF3–/– mice were either incubated for 24 h with medium alone or stimulated with LPS (100 ng/ml) and/or the indicated concentration of rIFN-beta (units per milliliter). Results are expressed as mean ± SEM of at least four independent experiments. *, p < 0.05 and ***, p < 0.001 as compared with WT BMDCs.

 
To assess the contribution of IRF3 to IL-27 synthesis in vivo, wild-type and IRF3–/– mice were injected i.v. with LPS. We detected high IL-27 levels in the sera of wild-type animals 2 h after LPS challenge (Fig. 4). IL-27 levels were substantially and significantly decreased in IRF3–/– mice, indicating the critical role of IRF3 in IL-27 production induced by TLR4 ligation in vivo. Interestingly, production of other cytokines, including IL-6, IL-12/23p40, IL-12p70, and GM-CSF, was found to be comparable in both groups of mice 2 h after LPS injection. However, the inflammatory response was attenuated at later time points in IRF3–/– mice (Fig. 4). We also observed a nearly 2-fold decrease of TNF-{alpha} levels in the sera of these mice. Thus, in addition to its critical role in IFN-beta (20) and IL-27 induction, the TRIF/IRF3 pathway is also involved in the late amplification of the innate response in the course of endotoxemia. These results are in line with a previous report showing the role of IFN-beta in LPS-mediated activation of several proinflammatory cytokines (20) and also with the resistance of IRF3–/– mice to endotoxic shock (21).


Figure 4
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  		FIGURE 4. Critical role of IRF3 in IL-27 synthesis upon LPS challenge in vivo. Wild-type (WT) or IRF3–/– mice (six to nine mice per group) were injected i.v. with PBS or LPS (500 µg). After the indicated time, blood was collected and sera were assayed for IL-27 and IL-12p70 levels by ELISA and other cytokines by Multiplex bead array. Results are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 as compared with WT mice.

 
IRF3 physically interacts with a critical cis-acting element of the proximal IL-27p28 promoter

To determine how IRF3 might regulate IL-27p28 gene expression, we analyzed the p28 promoter region. The nucleotide sequences of the mouse and human proximal promoters (–200/+50) display >80% similarity. The sequence of the 5' flanking region of the human IL-27p28 gene is represented in Fig. 5A. Several putative cis elements were predicted by computer-assisted scanning. We identified a potential ISRE site at position –60 to –48 relative to the transcriptional initiation site. As shown in Fig. 5B, the region encompassing this site is highly conserved in human and murine promoters. We designed a double-stranded oligonucleotide (designated ISREp28wt, Fig. 5B) which was used as a probe in EMSAs for DNA-protein interaction with recombinant N-terminal IRF3. As shown in Fig. 5C, IRF3 strongly interacts with the ISREp28wt probe. Competition experiments with an excess of different double-stranded oligonucleotides indicated that this binding was specific: it was inhibited in presence of homologous unlabeled ISREp28wt or the ISRE element from the ISG15 promoter (ISG15 WT) whereas it was unaffected by mutated version of ISG15 (ISG15 Mut) or ISREp28MutA and MutAB oligonucleotides. The ISREp28MutB competitor slightly reduced IRF3 binding.


Figure 5
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  		FIGURE 5. Localization and characterization of an IRF3-binding site within the human IL-27p28 promoter. A, Sequence of the human IL-27p28 proximal promoter. Putative cis elements were predicted using the MatInspector software. The main transcription initiation site (TIS + 1) is shown (27 ). B, IRF3 consensus DNA-binding sequence is shown (38 ). The oligonucleotide used as probe in EMSA (human ISRE p28wt) encompasses the putative ISRE site (indicated by the underlined nucleotides) and is aligned with the corresponding sequence of the murine promoter. *, Nucleotides that differ from the consensus. C, Competition assays. Recombinant N-terminal IRF3 (2 ng) was incubated with radiolabeled ISREp28wt probe in the absence or presence of 12.5-, 25-, and 50-fold excess of the indicated unlabeled competitor. D, Competition and supershift assays. Wild-type (WT) and IRF3–/– BMDCs were incubated with medium alone, LPS, or polyI:C for 2 h. Nuclear extracts (5 µg) were incubated with radiolabeled ISREp28wt probe. Four inducible protein-DNA complexes (A–D) were observed in WT BMDCs conditions. For competition assay, nuclear extracts from LPS-stimulated WT BMDCs were incubated with radiolabeled ISREp28 WT probe in the absence or the presence of the indicated unlabeled competitor. *, The inducible and slow migrating complex in IRF3–/– BMDCs. NS, Nonspecific; SS, supershift.

 
To determine whether IRF3 interacts with the ISREp28 site upon activation of DCs, we performed EMSA experiments with nuclear extracts from wild-type or IRF3–/– DCs (Fig. 5D). Several complexes were induced after stimulation of wild-type DCs with LPS (lane 3) or polyI:C (a ligand of TLR3 which is coupled to TRIF, lane 4) (complexes A–D, Fig. 5D). They were found to be specific as their formation was inhibited in presence of homologous unlabeled ISREp28wt (lane 8) or ISG15 WT (lane 10) whereas it was affected neither by a heterologous NF-{kappa}B consensus competitor (lane 12) nor by ISG15Mut (lane 11) or ISREp28MutAB (lane 9). Upon activation, IRF3 associates with coactivators such as p300 or CBP and therefore is part of slow migrating complexes (22). We observed a supershift upon addition of anti-IRF3 Ab, which decreased formation of both complexes A and B (lane 5). As expected, no supershift was observed in conditions using nuclear extracts from IRF3–/– DCs (lane 16). Interestingly, another slow migrating complex was observed with nuclear extracts from polyI:C-activated IRF3–/– DCs (lane 15, marked by an asterisk (*)). It is possible that other IRF members participate to the formation of complex B in absence of IRF3. However, we were unable to inhibit the formation of this complex with anti-IRF-7 or anti-IRF-9/p48 Abs (data not shown). The lower m.w. complexes C and D were supershifted by addition of anti-IRF-1 Ab in both wild-type and IRF3–/– conditions (lanes 6 and 17). Overall, these experiments indicate that both recombinant and endogenous IRF3 physically interact in vitro with the ISRE site of the IL-27p28 promoter.

To examine the functional role of this ISRE site in the transcriptional activity of the IL-27p28 promoter, single nucleotide mutations (MutA and B), or a combination of the two (MutAB), were introduced by site-directed mutagenesis in the context of the –1061/+13 human IL-27p28 reporter plasmid. In RAW cells transiently transfected with the wild-type hIL-27p28 construct, we found that p28 promoter activity was induced in response to LPS or polyI:C (Fig. 6A). Both mutations reduced basal promoter activity and its inducibility upon LPS or polyI:C stimulation. Next, we cotransfected the IL-27p28 reporter plasmid with a plasmid expressing IRF3 5D. As this IRF3 mutant was found to be constitutively active in HEK293 cells (23), we used this cell line for these experiments. As shown in Fig. 6B, IRF3 5D plasmid significantly up-regulated IL-27p28 promoter activity in a dose-dependent fashion. This positive regulatory role of IRF3 was strongly affected by mutations A and B. We concluded from this set of experiments that the ISRE element is critically involved in IL-27p28 transcriptional activation induced by LPS or IRF3.


Figure 6
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  		FIGURE 6. Critical role of the ISREp28 site in IL-27p28 promoter activity. A, RAW 264.7 cells were transiently transfected with 1 µg of IL-27p28lucWT, IL-27p28lucMutA, or IL-27p28lucMutAB, and 100 ng of pRL-TK as an internal control plasmid. Cells were stimulated with either LPS (1 µg/ml) or polyI:C (20 µg/ml). Data are normalized against unstimulated wild-type promoter activity which is set at 1. Values represent the mean ± SEM of six independent experiments performed in triplicates. B, Ectopic expression of IRF3 5D up-regulates IL-27p28 promoter activity. HEK 293 cells were cotransfected with 400 ng of reporter plasmid, 100 ng of pRL-TK, and the indicated amounts of IRF3 5D plasmid. The total amount of DNA transfected was maintained constant by complementing with the empty vector (pCMV2). Results represent the mean ± SEM of triplicates. A representative experiment of three is shown. Data are normalized against unstimulated wild-type promoter activity which is set at 1; RLU, relative light units.

 
IL-27p28 gene activation in human DCs is preferentially induced by TRIF-coupled TLR ligands and involves IRF3 recruitment to the promoter region

We then checked whether our observations on mouse DCs could be generalized to human DCs. For this purpose, we stimulated human moDCs with various TLR ligands. We observed that the IL-27p28 gene was efficiently activated by LPS and polyI:C but not by optimal concentrations of TLR2 and TLR8 ligands (Fig. 7A). In contrast, EBI3 mRNA was up-regulated by all TLR ligands that we tested (Fig. 7A). This set of experiments indicates that the p28 subunit is preferentially expressed upon activation of TLRs that recruit TRIF, which is in line with the conclusions of our mouse experiments.


Figure 7
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  		FIGURE 7. IL-27p28 gene activation in human DCs is preferentially induced by TRIF-coupled TLR ligands and involves IRF3 recruitment to the promoter region. Human monocyte-derived DCs were incubated in medium alone or stimulated with optimal concentrations of LPS (1 µg/ml), Pam3CSK4 (10 µg/ml), polyI:C (10 µg/ml), or R848 (10 µg/ml). A, Total RNA was extracted after 8 h and analyzed by real-time RT-PCR. Levels were normalized using beta-actin mRNA as reference and compared with unstimulated conditions. Data are shown as mean ± SEM of three independent experiments on different donors. B and C, ChIP experiments. moDCs were either incubated with medium alone or stimulated with LPS (1 µg/ml). Three hours after stimulation, cells were treated with 1% formaldehyde to cross-link proteins bound to DNA. After sonication, chromatin samples were immunoprecipitated with anti-IRF3 rabbit polyclonal Abs or anti-acetyl-histone H4 rabbit polyclonal Abs. B, Purified DNA samples were subjected to PCR amplification (32 cycles) using primers specific for IL-27p28 promoter region. For each sample, 2% of the cross-link-released chromatin was saved and used as input control. Water indicates PCR without sample. C, Real-time PCR was performed on immunoprecipitated DNA samples to quantify IRF3 binding to the IL-27p28 promoter. Data are expressed as relative expression against the value obtained in the medium alone with IRF3 Ab condition.

 
To determine whether IRF3 physically interacts with the endogenous IL-27p28 promoter region upon activation of human DCs, we performed ChIP experiments on human DCs stimulated with LPS, using primers encompassing the ISRE site. As illustrated in Fig. 7B, IRF3 binding to the IL-27p28 promoter region was readily detectable in response to TLR4 engagement. This result was confirmed using real-time PCR (Fig. 7C). We also observed that IL-27p28 gene activation by LPS was accompanied by H4 histones hyperacetylation, suggestive of local chromatin remodeling (Fig. 7B). These results therefore establish that TLR4-induced signaling results in the recruitment of IRF3 to the promoter region of the endogenous IL-27p28 gene. Taken together, these data on human DCs are in line with those obtained in mice and confirm that LPS-induced IL-27 synthesis critically depends on the action of IRF3 on the IL-27p28 gene.

IL-27p28 gene is activated in human DCs upon infection with CMV

As IRF3 is commonly activated following viral infection, we analyzed the expression of both IL-27p28 and EBI3 mRNA in human moDCs infected with live CMV (hCMV). Infection with this virus was shown to induce antiviral genes through the direct activation of IRF3 (21, 24, 25). As shown in Fig. 8A, IL-27p28 mRNA levels observed following hCMV infection were in the range of those obtained upon LPS stimulation. However, the kinetics of IL-27p28 mRNA induction was delayed in comparison to LPS conditions. In sharp contrast, EBI3 mRNA induction in infected cells was very weak in comparison to LPS stimulation (Fig. 8B). These experiments therefore establish that the p28 subunit of IL-27 is preferentially expressed in response to signaling events induced by viral infection of human moDCs.


Figure 8
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  		FIGURE 8. IL-27p28 gene is activated upon infection with hCMV. moDCs (5 x 105/ml) were either infected with hCMVTB40/E at MOI of 0.5, and 10 PFU/cell or mock infected with 0.5 ml of MEM-5% FCS for 3 h at 37°C. After removal of the virus or medium, moDCs were washed and cultured in complete RPMI 1640 containing 10% FCS for the indicated time. Total RNA was extracted at different time points and analyzed by real-time RT-PCR. IL-27p28 (A) and EBI3 (B) mRNA levels were normalized using beta-actin mRNA as reference and compared with unstimulated conditions. Data are shown as mean ± SEM of four independent experiments on different donors. In parallel, the same cell preparations were stimulated with LPS (1 µg/ml) and processed in the same manner.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There is growing evidence that IL-27 plays a crucial regulatory role to limit inflammation upon exposure to microbes. In the course of chronic infections with intracellular pathogens such as T. gondii or Mycobacterium tuberculosis, IL-27p28 mRNA is highly induced, leading to suppression of excessive effector immune functions (3, 4). Inducible IL-27p28 expression was also observed in draining mesenteric lymph nodes following Trichuris muris infection, thereby down-regulating innate and effector Th2 type responses (5). During septic peritonitis, both EBI3 and p28 mRNA are rapidly induced, leading to reduction of neutrophil migration and activation (8). In this study, we show that IL-27 is differentially regulated by TLR ligands and that efficient IL-27 production involves TRIF-dependent activation of IRF3. Together with the effect of type I IFN, these data are reminiscent of previous findings on IL-12p35 (17, 26), suggesting an evolutionary conserved pathway leading to activation of these two related cytokines. While this article was being submitted, another report indicated the role of MyD88 in LPS-dependent expression of IL-27p28 gene activation in peritoneal macrophages (27). We obtained similar findings using MyD88–/– BMDCs. However, we observed a residual production of IL-27p28 protein in these cells suggesting that the relative importance of MyD88-dependent and -independent pathways in TLR4-triggered p28 expression could be cell-type dependent. The same report demonstrates that IFN-{gamma} signaling contributes to transcriptional activation of IL-27p28 through the recruitment of IRF1 to the promoter region. We confirmed this result and also observed the inducible binding of IRF1 to the ISREp28 site in response to TLR3 or TLR4 stimulation (Fig. 5D). Thus, multiple signaling pathways, including MyD88-dependent, TRIF/IRF3-dependent, type I and type II IFNs, contribute to p28 gene activation through the recruitment of IRF family members to the ISRE site.

IRF3–/– mice display increased ability to clear infection with Listeria monocytogenes (28). This protection was associated with decreased production of type I IFNs, which induce apoptosis of immune cells in infected wild-type animals. The present data suggest that decreased production of IL-27 in IRF3–/– mice could represent an additional mechanism contributing to this protection against L. monocytogenes.

IL-27 also plays an important role in the down-regulation of Th2-dependent lung inflammation during the development of allergic asthma (29) and Th17-dependent brain inflammation in the course of experimental autoimmune encephalomyelitis (29). Early exposure to certain microbes or microbial products including bacterial endotoxin appears to ensure some level of protection against the development of autoimmune or allergic diseases, supporting the "hygiene hypothesis" (30, 31). It will be important to determine whether expression of IL-27 participates to this phenomenon. Indeed, it appears that the same transcription factor, IRF3, is critically involved in the synthesis of two key cytokines regulating autoimmune demyelination, namely IFN-beta (32) and IL-27 (6).

In addition to its anti-inflammatory role, IL-27 was shown to favor early Th1-type responses during Leishmania major and bacillus Calmette-Guérin infection (33, 34). This suggests that IL-27 might act in synergy with other IRF3-dependent factors such as IL-12p70, type I IFNs, CXCL9, CXCL10, and CXCL11 to promote Th1 polarization in the early phase of immune responses. Furthermore, IL-27 was shown to increase CD8+ T cell-dependent cytotoxicity in murine carcinoma (35). IRF3 is activated upon exposure to virus (16) and we show herein that moDCs infected by hCMV express high levels of IL-27p28 mRNA. However, as induction of EBI3 mRNA is weak in the same experimental conditions, it remains to be determined whether bioactive IL-27 heterodimer is efficiently produced in the course of viral infections. A recent report indicates that IL-27 production triggered by human papilloma virus-like particles (which are known to signal through TLRs (36)) is a potent inhibitor of HIV replication in PBMC (37). These data suggest that apart from its immunoregulatory role in the course of infectious diseases and cancer, IL-27 could be directly involved in antiviral responses.

In sum, the present report establishes a critical role for the TRIF-IRF3 signaling pathway in TLR-mediated IL-27 synthesis. Beside their relevance to the control of IL-27 synthesis in the course of infections, we suggest that these findings could lead to novel therapies based on endogenous IL-27.


   Acknowledgments
 
We thank Drs. S. Akira (Osaka University, Osaka, Japan), B. Beutler (The Scripps Research Institute, La Jolla, CA), and T. Taniguchi (University of Tokyo, Tokyo, Japan) for the gift of MyD88–/–, LPS2, and IRF3–/– mice, respectively. Drs. B. Ryffel and V. Vasseur (Centre National de la Recherche Scientifique GEM2358, Orleans, France) are acknowledged for mice breeding (Transgenose Institute, Orleans, France). We also thank Dr. R. Lin (McGill University, Montreal, Canada) for providing IRF3 5D plasmid, purified IRF3 protein and Z. Tabi (Cardiff, U.K.) for providing hCMV strain TB40/E.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The Institute for Medical Immunology was sponsored by the government of the Walloon Region and GlaxoSmithKline Biologicals. This study was supported by the Fonds National de la Recherche Scientifique and an Interuniversity Attraction Pole of the Belgian Federal Science Policy. S.G. was a Postdoctoral Researcher of the Fonds National de la Recherche Scientifique. V.F. was a Research Associate of the Fonds National de la Recherche Scientifique and J.R. was supported by a grant from the Fonds National de la Recherche Scientifique-Telévie program. Back

2 Address correspondence and reprint requests to Dr. Stanislas Goriely, Institute for Medical Immunology, 8 rue Adrienne Bolland, B-6041 Charleroi-Gosselies, Belgium. E-mail address: stgoriel{at}ulb.ac.be Back

3 Abbreviations used in this paper: EIB3, EBV-induced gene 3; TIR, Toll/IL-1R; IRF, IFN regulatory factor; TRIF, TIR domain-containing adaptor inducing IFN-beta; DC, dendritic cell; moDC, monocyte-derived DC; BMDC, bone marrow-derived DC; hCMV, human CMV; MOI, multiplicity of infection; ISRE, IFN-stimulated response element; ChIP, chromatin immunoprecipitation; polyI:C, polyinosine-polycytidylic acid; Pam3CSK4, 2.3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser- Lys4-OH; MyD88, myeloid differentiation factor 88. Back

Received for publication January 3, 2007. Accepted for publication April 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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