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. Author manuscript; available in PMC: 2014 Jun 11.
Published in final edited form as: J Immunol. 2011 Jan 10;186(4):1951–1962. doi: 10.4049/jimmunol.1000918

Critical Role of IRF-8 in Negative Regulation of TLR3 Expression by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-2 Activity in Human Myeloid Dendritic Cells

Alessandra Fragale *, Emilia Stellacci *,†, Ramona Ilari *, Anna Lisa Remoli *, Angela Lanciotti *,‡, Edvige Perrotti *, Iart Shytaj *, Roberto Orsatti *, Harshani R Lawrence §, Nicholas J Lawrence §, Jerry Wu §, Michael Rehli , Keiko Ozato ||, Angela Battistini *
PMCID: PMC4053178  NIHMSID: NIHMS494682  PMID: 21220691

Abstract

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1– and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8–induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase–dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

Toll-like receptors are pattern recognition receptors crucially involved in sensing of infectious agents (1, 2). They are predominantly expressed by cells involved in immune functions as well as in tissues exposed to the external environment such as lungs and the gastrointestinal tract, where they recognize conserved microbial molecules called pathogen-associated molecular patterns. Upon recognition of viral pathogen-associated molecular patterns, TLRs trigger a cascade of events leading to the induction of proinflammatory cytokines and IFNs, which in turn orchestrate innate immunity, chemokines, and costimulatory molecules that promote T cell activation and specific immunity (1, 3). Among TLRs, TLR3 has been implicated in the recognition of dsRNA-derived from several viruses and of its synthetic analog polyinosinic-polycytidylic acid (poly-IC) (4). TLR3 expression has been shown to be cell type specific, with this receptor preferentially expressed in dendritic cells (DCs), where it is functionally localized in endosomal compartment, as well as fibroblasts, epithelial cells, and CNS-resident cells (5).

The establishment of the adaptive T cell-mediated response is ultimately dictated by DCs. TLR signaling induces DC maturation, a process characterized by enhanced expression of costimulatory molecules, MHC–peptide complexes, and increased secretion of cytokines that trigger activation and polarization of naive T cells. Human DCs are a heterogeneous family of leukocytes composed of functionally distinct subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Each DC subset is endowed with a different TLR repertoire, which has important implications in the way different types of immune responses are generated (6). Thus, in response to intracellular pathogens including viruses, TLR9 and TLR7 are expressed in endosomes of human pDCs, but not in human mDCs or IL-4/GM-CSF–differentiated monocyte-derived DCs (MDDCs). In contrast, TLR4 and TLR3 are expressed only in human mDCs and MDDCs but not in human pDCs. Selective TLR3 expression in mDCs suggests that TLR3 plays a key role in the antiviral response by induction of the adaptive immune response mediated by this subset of DCs (6). Furthermore, the ability of DCs to present peptides derived from exogenous Ags in a complex with MHC class I molecules, a process known as cross-priming, is largely dependent on TLR3 expression (7). Therefore, TLR3 also acts as a sensor for viruses that do not directly infect DCs (8).

TLR stimulation, however, presents a double-edged sword: it is indeed essential for triggering innate and adaptive immunity against pathogens, but the strength and the duration of activation of TLR signaling pathways must be tightly controlled, because TLR overactivation is known to contribute to pathogenesis of autoimmune, chronic inflammatory, and infectious diseases. The balance is maintained by multiple negative regulatory mechanisms that ensure very tight regulation. The expression of most negative regulators acting by degradation or destabilization of signal transduction factors can be induced by the engagement of TLRs themselves, thus providing a negative-feedback loop to terminate TLR activation (9). Although research in TLR biology has been mainly focused on identification of ligands and signaling pathways triggered through ligand–TLR interaction, less evidence is available concerning the transcriptional regulation of the TLR3 gene in human DCs, even though it represents a crucial aspect of their function (10). Indeed, TLR3 expression is upregulated during differentiation from monocytes to DCs as well as by viral infections and IFN-α, resulting in an enhanced response to viral RNA (11–14).

The IFN regulatory factor (IRF) family comprises nine mammalian members all sharing a well-conserved DNA-binding domain of 120 aa containing a unique helix–turn–helix DNA binding domain located at the N terminus, which is responsible for binding to the IRF element (IRF-E) for which the sequence is almost indistinguishable from the IFN-stimulated response element. This element was first identified in the promoters of genes that encode type I IFNs but is also found in the promoters of genes that are induced by type I IFN signaling as well as of many other genes involved in immunity and oncogenesis (15). Accordingly, besides regulating the IFN system, the IRF family participates in directing the development of innate and adaptive immunity, a role best illustrated by the activity of IRF-1, IRF-2, IRF-4, and IRF-8 (15). In particular, we and others (16–20) showed a clear correlation among IRF-1, IRF-2, and IRF-8 deficiency and severe defects in conventional DC maturation and function.

Recently, IRF-1 and IRF-2 have been implicated in the regulation of TLR3 expression (21). Promoter regions of the TLR3 gene in mice and humans, despite the overall diversity, bear similar IRF-E, which are responsible for both basal- and IFN-β–induced activation that constitutively binds IRF-2 and recruits IRF-1 upon stimulation. Accordingly, TLR3 expression is impaired in splenic DCs from IRF-1 knockout (KO) mice that were also unable to undergo full phenotypic activation upon in vitro culture in presence of maturation stimuli such as or poly-IC and Newcastle disease virus infection (20). Impaired TLR3 expression and responsiveness to poly-IC stimuli has also been observed in DCs from IRF-8 KO mice, thus also implicating IRF-8 in TLR3 regulation (16).

IRF-8 interacts with cis elements containing IRF-E/IFN-stimulated response element or composite Ets/IRF-E consensus sequences and either activates or represses transcription, depending upon the target gene, cellular context, and its phosphorylation status (22). In differentiating myeloid cells, IRF-8 activates transcription of genes such as gp91PHOX, p67PHOX, TLR4, IL-18, IL-1β, and IL-12p40 (22). Transcriptional activation involves binding of IRF-8 to the cis elements of these genes via DNA-bound PU.1 and/or another IRFs. This interaction requires phosphorylation of conserved tyrosine residues in the IRF domain of IRF-8 (23, 24). IRF-8 also represses transcription of the gene encoding 2′,5′-oligoade-nylate synthase (OASE) (25), ISG15 (26), and PTPN13 (27). Recently, non-IRF domain tyrosine residues necessary for switching from repressor to activator function of IRF-8 in differentiated myeloid cells have been identified (28). Nonphosphorylated IRF-8 directly bound DNA and represses transcription, whereas tyrosine-phosphorylated IRF-8 did not bind DNA (28, 29).

Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP2) is an ubiquitously expressed intracellular phosphatase that positively regulates the signaling pathways of cytokines and growth factors, whereas it negatively regulates the JAK/STAT signaling pathway initiated by IFN-α and IFN-γ (30). Of note, it has been reported that IRF-8 is a substrate for SHP2 and that SHP2 negatively regulates poly-IC–induced TLR3-activated signaling (31–33).

In this study, we addressed the role of IRF-1 and IRF-8 in the regulation of TLR3 expression in human MDDCs, both basal and ligand induced. Reporter assays showed that IRF-1 and IRF-2 stimulate TLR3 promoter activity, whereas IRF-8 totally abrogates such an effect. We found that IRF-1, IRF-2, and IRF-8 were expressed during MDDC differentiation and bound to the human (h)TLR3 promoter in vivo. IRF-8 negatively controlled TLR3 transcription upon SHP2 association that allowed dephosphorylated IRF-8 to strongly bind to the hTLR3 promoter competing with IRF-1 and dampening its activity.

Overall, our data highlight a new important mechanism for regulation of TLR3 expression and identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses.

Materials and Methods

Cells

Monocytes were isolated from PBMC obtained from healthy donor (HD) buffy coats by immunomagnetic selection using CD14 microbeads (MACS monocyte isolation kit; Miltenyi Biotec), according to the manufacturer’s instructions. This procedure yielded at least a 98% pure population of monocytes, as assessed by FACS analysis of lineage-specific surface markers (CD14, CD1a). To obtain MDDCs monocytes were cultured at 1.5–2 × 106 cells/ml in RPMI 1640 medium containing 10% FBS in the presence of GM-CSF (50 ng/ml) (PeproTech) and IL-4 (500 U/ml) (R&D Systems). Cytokines were added to the cultures every 2 d. On day 5, MDDCs were transfected with 20 μg/ml poly-IC–Fugene 6 complexes (Roche) to further activate intracellular TLR3, and the cells were collected after 24 or 72 h at the durations described. FACS analysis of CD80, CD86, and HLA-DR markers was then performed 24 and 72 h poststimulation. In IRF-8 phosphorylation experiments, MDDCs were pretreated with 50 μM SHP1/2 inhibitor NSC-87877 (Tocris Bioscience) (34) for 2 h and then transfected with poly-IC–Fugene 6 complexes. The human monocytic THP-1 cells were maintained in RPMI 1640, and the human lung epithelial A549 cells were grown in IMDM and human embryonic kidney (HEK) 293T cells in DMEM; all culture medium contained 10% FBS and supplements.

Transfections, luciferase assay, and nucleofection

HEK 293T cells were cotransfected with 1 μg each IRF-1, IRF-2, and IRF-8 (35) and ICSBPpcDNAamp and Y2-351F (12Y) ICSBPpcDNAamp (28)-expressing vectors (ratio 1:1:1) or together with 1 μg 588-bp genomic fragment of the human TLR3 gene promoter wild-type (WT) or the same carrying the mutated IRF-E (21) or the IL-12p40 promoter (36), all cloned in PGL3 vector, with 0.1 μg Renilla luciferase control vector (pRL-Act Renilla) by using Fugene 6. Twenty-four hours later, firefly and Renilla luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega). Transfections were performed in triplicate and repeated at least three times. Data were normalized to the activity of Renilla luciferase. A549 cells were pretreated with SHP2 inhibitors NSC-87877 or SPI-112 Me (37) for 2 h then cotransfected with IRF-1, IRF-8, or combination of expressing vectors with the calcium phosphate method and 6 h later transfected with 20 μg/ml poly-IC–Fugene 6 complexes. Cells were collected 24 h after. Transfections were performed in duplicate and repeated three times. Primary MDDCs were nucleofected with the Human Dendritic Cell Nucleofection Kit following the manufacturer’s instruction (Amaxa Biosystems). Briefly, 2 × 106 primary MDDC cells at day 5 of culture were electroporated using the U-O2 program of the Nucleofector with 2 × 20% polyethylene glycol/2.5 M NaCl-precipitated endotoxin-free (Qiagen) IRF-1 and IRF-8 or a combination and pmaxGFP-expressing vector DNAs. Transfection efficiency was monitored in all samples by FACS analysis of GFP fluorescence, and it ranged from 30–50% depending on the donor in three independent experiments. After 20 h, cells were collected, and RNA was extracted as described above. Transfections were performed in duplicate and repeated three times.

DNA affinity binding assay

For the DNA affinity binding assay, biotinylated oligonucleotides corresponding to the WT and mutant hTLR3 sequences were synthesized. The WT hTLR3 sequence was 5′-AGC TTTA CTT TCA CTT TCG AGA GTG C-3′; and the mutant hTLR3 sequences were: 5′-AGC TTT ACA CGC ACT TTC GAG AGT GC-3′ and 5′-TCG AAA TGT GCG TGA AAG CTC TCA CG-3′. DNA affinity binding assays were performed as described (38). Eluted material was separated onto 10% SDS-PAGE followed by immunoblotting with anti–IRF-1 (sc-497), anti–IRF-2 (sc-101069), anti–IRF-8 Abs (sc-6058), anti-p300 (sc-585), anti–CREB-binding protein (CBP) (sc-369), and anti-histone deacetylase 3 (HDAC3) (sc-8138; Santa Cruz Biotechnology).

Real-time RT-PCR

Total RNA was extracted from MDDCs or A549 cells using the Rneasy Mini kit (Qiagen). RNAs were DNase I digested (Roche) and reverse transcribed as previously described (38). Quantitative PCR (qPCR) was performed in duplicate by the real-time fluorescence detection method with the fluorescent DNA binding dye SYBR green (Power SYBR Green PCR master kit; Applied Biosystems) using an ABI PRISM 7000 sequence detection system (Applied Biosystems) according to the manufacturer’s protocol. Primers and conditions used for real-time PCR have been previously described, except the sequences used for hTLR3: 5′-TAA ACT GAA CCA TGC ACT CT-3′ and 5′-TAT GAC GAA AGG CAC CTA TC-3′; hIL-12p40: 5′-AGG ACC AGA AAG AAC CCA AA-3′ and 5′-AGC AGG TGA AAC GTC CAG A-3′; hIFN-β: 5′-GCA GCA GTT CCA GAA GGA G-3′; hIFN-β reverse: 5′-GCC AGG AGG TTC TCA ACA AT-3′ and hOASE: 5′-AGG TGG TAA AGG GTG GCT CC-3′ and 5′-ACA ACC AGG TCA GCG TCA GAT-3′.

Immunoprecipitation and immunoblot analysis

To assess IRF-8 and SHP2 phosphorylation levels, cell extracts from MDDCs were incubated overnight with 1 μg polyclonal anti–IRF-8 (Santa Cruz Biotechnology) or anti-phosphotyrosine (pTyr; Millipore) Abs in denaturing conditions (28). To evaluate IRF-8 and SHP2 interaction, cell extracts from MDDCs were incubated overnight with 1 μg polyclonal anti–IRF-8 Abs in nondenaturing conditions (35). Immunocomplexes were then incubated with Ultralink immobilized protein A/G-Sepharose (Pierce Biotechnology, Rockford, U.K.) for 2 h at room temperature. After extensive washing, immunoprecipitates were eluted by boiling the beads for 5 min in SDS sample buffer. Eluted proteins were separated by SDS-PAGE and subjected to Western blotting with anti–IRF-8, anti-pTyr, and anti-SHP2 Abs (Santa Cruz Biotechnology).

Flow cytometry

mAbs used for flow cytometric analysis were PE-conjugated anti-human CD1a, FITC-conjugated anti-human CD14, and PE-conjugated anti-human CD80 and CD86 (BD Biosciences). Samples were analyzed on an FACS-Calibur (BD Biosciences).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (38). Briefly, MDDC cells were cross-linked with 1% formaldehyde and quenched in 0.125 M glycine. Cell lysates were sonicated and immunoprecipitated with normal rabbit serum (BD Biosciences) or anti–IRF-1 or anti–IRF-8 Abs (Santa Cruz Biotechnology). The immunoprecipitated DNA was eluted and amplified by real-time PCR using an ABI 7700 (Applied Biosystems). Values were normalized to corresponding input control and expressed as fold enrichment relative to normal rabbit serum for each experiment. The sequence of specific primers used for amplification of the hTLR3 gene surrounding the IRF-E binding sites was as follows: 5′-GAA ACG CCT CTC TGA GGT TG-3′ and 5′-CCA GAC ATT TCA TCA GGG AAG-3′. Real-time qPCR was performed as described above. The primers gave a unique product at 154 bp.

Statistical analysis

Student t tests were used to calculate differences between the groups. Differences in p values ≤0.05 were considered statistically significant.

Results

Differential effects of IRFs on TLR3 promoter activity

Multiple cis-acting elements have been previously characterized within the hTLR3 gene promoter, including a functionally relevant IRF-E located close to the transcription start site. This element has been shown to constitutively bind IRF-2 and recruit IRF-1 after IFN stimulation (21). In this study, we analyzed the transcriptional activity of these factors on the promoter activity in cells over-expressing IRF proteins. Luciferase assays showed that IRF-1 overexpression promoted a 25-fold increase of hTLR3 activity, whereas IRF-2 and IRF-8 did not show any effect (Fig. 1A). Because IRF-2 and IRF-8 have been regarded as repressors or activators of IRF-1–induced transcriptional activity on most target genes (15), we analyzed their role on IRF-1–mediated TLR3 regulation by transfecting combinations of their expression vectors. Interestingly, we found that hTLR3 activity was synergistically stimulated by IRF-1 and IRF-2 cotransfection. Conversely, IRF-8 totally abrogated IRF-1–induced hTLR3 transcription as well as the synergistic activation induced by IRF-1 and IRF-2. The specificity of the IRF effect was demonstrated by mutation of the IRF-E that resulted in complete loss of IRF-1–induced upregulation of the promoter activity (Fig. 1B, middle panel) as compared with the WT promoter (Fig. 1B, left panel). As an additional control of specificity, we performed parallel experiments with the IL-12p40 promoter, known to be synergistically upregulated by cotransfection of IRF-1 and IRF-8 (39). As expected, transfection of either IRF-1 or IRF-8 stimulated IL-12p40 promoter activity, and their coexpression led to a synergistic activation of the promoter (Fig. 1B, right panel).

FIGURE 1.

FIGURE 1

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IRF-8 inhibits IRF-1–induced TLR3 transcriptional activity. A, HEK 293T cells were cotransfected with the human WT TLR3 promoter and IRF-1–, IRF-2–, and IRF-8–expressing vectors alone or in combination (ratio 1:1:1). B, HEK 293T cells were transfected with IRF-1, IRF-8, or combination of expressing vectors and the human WT TLR3 promoter, the same carrying the mutated IRF-E or the IL-12p40 promoter. C, HEK 293T cells were cotransfected with the murine TLR3 promoter and IRF-1, IRF-8, or combination of expressing vectors. D, HEK 293T cells were cotransfected with IRF-1, IRF-8, or combination of expressing vectors in different ratios and the human TLR3-promoter. E, THP-1 monocytic cells were electroporated with IRF-1– and IRF-8–expressing vectors alone or in combination and the human TLR3 promoter. Twenty-four hours later, firefly and Renilla luciferase activities were determined, and data were normalized by activity of Renilla luciferase vector. Results are representative of at least three independent experiments performed in duplicates. Means are ± SD. n = 3. *p < 0.05 versus CTR, **p < 0.05 versus IRF-1.

Of note, when we performed the same transfection experiments with the mouse TLR3 promoter construct, we found that, as for the human counterpart, IRF-1 stimulated mouse TLR3 expression (20-fold), and IRF-8 exerted an inhibitory role on the IRF-1–induced TLR3 promoter activity. Unexpectedly, IRF-8 alone stimulated the basal activity of the mouse promoter ~4-fold (Fig. 1C). Dose-response experiments indicated that IRF-8 inhibitory role was dose dependent, and even low doses of IRF-8 (IRF-1/IRF-8 ratio 5:1) were able to suppress the IRF-1–mediated transcriptional activity. At a ratio IRF-1/IRF-8 of 1:1, promoter activity was completely abolished (Fig. 1D).

Importantly, luciferase assays performed in human myeloid THP-1 cells confirmed that IRF-8 suppressed the IRF-1–induced TLR3 promoter activity in a more physiologic context (Fig. 1E). Of note, IRF-8 alone significantly stimulated the hTLR3 promoter in these cells (Fig. 1E).

Collectively, these results indicate that IRF-8 exerts an inhibitory role on IRF-1–induced TLR3 transcriptional activity both in human and mouse cell systems. We chose to focus on the regulation of the human promoter for the remaining part of this report.

IRFs bind the IRF-E present on the hTLR3 promoter

To further dissect the functional characteristics of the IRF-1 and IRF-8 effect on hTLR3 transcription via the IRF-E, we performed DNA affinity binding assays. Experiments were performed by incubating nuclear extracts from HEK 293T cells transfected with IRF-1 and IRF-8 alone or in combination in an equal molar ratio 1:1 with WT and mutant IRF-E/TLR3-biotinylated oligonucleotides. The isolated complexes were then examined by immunoblotting with specific Abs against IRF-1 and IRF-8. As shown in Fig. 2, both IRF-1 and IRF-8 directly bound to the WT oligonucleotide, but not the mutated sequence, confirming the specificity of the binding. Interestingly, when IRF-1 and IRF-8 were cotransfected, IRF-1 binding was decreased, whereas IRF-8 binding was increased as compared with factors transfected alone (Fig. 2). These results suggest that the two factors may compete for binding to the IRF-E. We also evaluated the binding of IRF-2 and did not observe any difference in the binding ability when we cotransfected IRF-2 with IRF-1 or IRF-8 (data not shown).

FIGURE 2.

FIGURE 2

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IRF-8 binds to the IRF-E present on the human TLR3 promoter. HEK 293T nuclear extracts were incubated with biotinylated oligonucleotide corresponding to a WT or mutated IRF-E consensus binding site in the TLR3 promoter before precipitation with streptavidin beads. The DNA-bound IRF-1 and IRF-8 were detected by immunoblotting with specific Abs. Direct immunoblot of extracts is indicated as Input. Representative data of one from three independent experiments are shown.

IRF-1 and IRF-8 regulate endogenous TLR3 expression in MDDCs

To assess the functional relevance of the IRF-mediated TLR3 regulation in a physiological context, we performed experiments in human MDDCs because TLR3 is selectively expressed in mDCs, the best professional APCs, and is specifically involved in DC cross-priming activity during viral infections (4). CD14+ monocytes were magnetically isolated from HD cultured with hrIL-4 and hrGM-CSF up to 7 d to obtain immature MDDCs. Control FACS analysis was routinely performed, confirming that although the CD14 marker expression was downregulated soon after 2 d of culture, CD1a and costimulatory molecule CD80-, CD86-, and HLA-DR–enhanced expression was reached during differentiation (data not shown). We first analyzed TLR3 expression by real-time PCR during the differentiation from CD14+ to MDDCs. As shown in Fig. 3A, TLR3 expression was undetectable in monocytes, but increased during their differentiation into MDDCs, with a peak at 5 d that rapidly declined after 7 d of cultures.

FIGURE 3.

FIGURE 3

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IRF-8 inhibits IRF-1–induced TLR3 transcriptional activity in MDDCs. A, CD14+ monocytes were magnetically isolated, cultured with hrIL-4 and hrGM-CSF up to 10 d, and allowed to differentiate into MDDCs. Every 2 d, cells were collected, total RNA was isolated, and cDNAs were analyzed by real-time qPCR for TLR3 and GAPDH expression. Data are normalized on GAPDH values and expressed as means ± SD. n = 3. *p < 0.05. B, MDDCs at day 5 were nucleofected with endotoxin-free IRF-1 or IRF-8 expression vectors or combinations at 1:1 or 1:2 ratios. Twenty-four hours later, cells were collected, total RNA was isolated, and cDNAs were analyzed by real-time qPCR for TLR3 and GAPDH expression. Results are representative of at least three independent experiments performed in duplicate. Means are ± SD. n = 3. *p < 0.05 versus CTL, **p < 0.05 versus IRF-1. C, MDDCs were obtained as in A but every 2 d transfected with poly-IC–Fugene 6 complexes and collected after 24 and 72 h. Total RNA was isolated, and cDNAs were analyzed by real-time qPCR for TLR3 and GAPDH expression. Data are normalized on GAPDH values and expressed as means ± SD. n = 3. Results are representative of at least three independent experiments. *p < 0.05.

Then, we performed nucleofection experiments in primary MDDCs at day 5 of culture to assess the role of IRF-1 and IRF-8 on TLR3 expression. Consistent with previous results, IRF-1 stimulated endogenous TLR3 expression 5-fold versus control, whereas IRF-8 totally abrogated the IRF-1 effect in a dose-dependent manner (Fig. 3B). These results confirm that IRF-8 also exerts a negative role on the IRF-1–stimulated TLR3 expression in the physiological context of MDDCs.

Regulation of TLR3 gene expression was then analyzed in human conventional DCs stimulated with TLR3 ligand. MDDCs at 2, 5, and 7 d of culture were left untreated or transfected with poly-IC and collected 24 and 72 h posttreatment. As shown in Fig. 3C, 24 h of poly-IC treatment stimulated TLR3 expression at each day of culture; of note, at day 5, when TLR3 was maximally expressed, it was also strongly upregulated by poly-IC, whereas 72 h later, its expression suddenly dropped, and it was no longer stimulated by ligand treatment.

IRF-1 and IRF-8 differentially bind to the hTLR3 promoter in MDDCs

To gain further insights into the mechanism by which IRF-8 controls IRF-1–induced TLR3 expression in MDDCs, we performed Western blot and DNA affinity binding assays, focusing our attention on day 5 of culture, when DCs are well differentiated and TLR3 is maximally expressed and upregulated by 24 h of poly-IC treatment, and after 72 h of stimulation, when TLR3 expression totally dropped. Western blot analysis indicated that IRF-1 and IRF-8 expression was barely detected in MDDCs (Fig. 4A). Conversely, IRF-1 and IRF-8 expression as well as their binding to IRF-E/TLR3 were strongly increased after poly-IC stimulation, although after 24 h of stimulation, IRF-1 bound much more strongly as compared with IRF-8. By contrast, after 72 h of poly-IC treatment, expression and binding of IRF-1 were both significantly reduced, whereas those of IRF-8 substantially increased. However, IRF-2 was found to be constitutively expressed and bound (Fig. 4A).

FIGURE 4.

FIGURE 4

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IRF-1 and IRF-8 differentially bind TLR3 promoter. CD14+ monocytes were magnetically isolated from HD, cultured with hrIL-4 and hrGM-CSF up to 8 d, and allowed to differentiate into MDDCs. At day 5 of culture, cells were treated with poly-IC and collected 24 and 72 h later. A, Nuclear extracts were incubated with biotinylated oligonucleotide corresponding to a WT or mutated IRF-E consensus binding site in the TLR3 promoter before DNA affinity binding assay. The DNA-bound IRF-1, IRF-8, IRF-2, p300, CBP, and HDAC3 were detected by immunoblotting. Direct immunoblot of extracts is indicated as Input. Representative data from one of three independent experiments are shown. B, Chromatin was immunoprecipitated using Abs against IRF-1 and IRF-8. Anti-IgG Ab was used as a negative control. Quantification of binding of IRF-1– and IRF-8–specific or IgG Abs was performed by qPCR using primers surrounding the IRF-E and is shown normalized on relative input. Data are shown as means ± SD of relative binding of replicates and are representative of three experiments. n = 3. *p < 0.05.

A number of DNA binding transcription factors including IRFs increase or repress transcription by recruiting to the target promoter coactivators as histone acetylases or repressors as HDACs. In particular, it has been reported that IRF-1 recruits CBP/p300 as coactivators but recruits IRF-8 HDAC3 as corepressors (25). Therefore, to further dissect the mechanism underlying the stimulatory and inhibitory effect on TLR3 transcription of IRF-1 and IRF-8, respectively, DNA affinity binding assays were performed with Abs against CBP/p300 and HDAC3. As shown in Fig. 4A, at early time points after poly-IC stimulation, binding of p300 and CBP was significantly increased, whereas the HDAC3 signal was barely detectable. By contrast, after 72 h, a substantial increase in HDAC3 binding and reduced CBP/p300 binding were observed.

The in vivo relevance of these findings was determined by ChIP analysis. After DNA immunoprecipitation with specific IRF-1 and IRF-8 Abs, real-time PCR amplification of the TLR3 genes surrounding the IRF-E site showed that upon 24 h of poly-IC treatment, the binding of both IRF-1 and IRF-8 was increased. Conversely, at 72 h poststimulation, IRF-1 binding was substantially decreased, whereas that of IRF-8 significantly increased (Fig. 4B). These results together with luciferase functional assays strongly indicate that IRF-8 acts as a repressor of IRF-1–mediated stimulation and that a balance between IRF-1 and IRF-8 bound to the TLR3 gene promoter determines modulation of TLR3 expression in poly-IC–stimulated MDDCs.

IRF-8 phosphorylation controls TLR3 expression and the MDDC cytokine profile

Because transcriptional activity of IRF-8 is strictly dependent on its phosphorylation status, the phosphorylated form being unable to bind DNA (28, 29), we sought to analyze IRF-8 tyrosine phosphorylation levels following poly-IC stimulation of MDDCs. To assess for IRF-8 tyrosine phosphorylation, immunoprecipitation assays were performed under denaturing conditions. As shown in Fig. 5A, coimmunoprecipitation analysis with anti–IRF-8 blotted with anti-pTyr Abs reveals that IRF-8 was actively tyrosine phosphorylated at 24 h after poly-IC treatment (lane 2) when TLR3 was maximally stimulated (Fig. 3C). Conversely, after 72 h, when TLR3 expression was repressed (Fig. 3C), a significant decrease of IRF-8 tyrosine phosphorylation was observed (Fig. 5A, lane 6 versus lane 2). Similar results were obtained when coimmunoprecipitation analysis was performed, immunoprecipitating with anti-pTyr and blotting with anti–IRF-8 Abs (Fig. 5B).

FIGURE 5.

FIGURE 5

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NSC-87877 SHP2 inhibitor affects IRF-8 phosphorylation. CD14+ monocytes were magnetically isolated and cultured with hrIL-4 and hrGM-CSF to differentiate into MDDCs. At day 5 of culture, cells were pretreated with NSC-87877 inhibitor for 2 h, transfected with poly-IC–Fugene 6 complexes, and collected 24 and 72 h later. Proteins were extracted and immunoprecipitated (IP) in denaturing (A, B) and nondenaturing (C) conditions and immunoblotted (IB) with specific Abs. D, HEK 293T cells were cotransfected with the human WT TLR3 promoter and IRF-1–, IRF-8–, and IRF-8 12Y-expressing vectors alone or in combination (ratio 1:1:1). Twenty-four hours later, firefly and Renilla luciferase activities were determined, and data were normalized by activity of Renilla luciferase vector. E, MDDCs at day 5 were nucleofected with endotoxin-free IRF-1 or IRF-8 12Y expression vectors or combinations at 1:1 ratio. Twenty-four hours later, cells were collected, total RNA was isolated, and cDNAs were analyzed by real-time qPCR for TLR3 and GAPDH expression. Results are representative of at least three independent experiments performed in duplicate. Means are ± SD. n = 3. *p < 0.05 versus CTL, **p < 0.05 versus IRF-1.

Because it has been reported that IRF-8 is a substrate for SHP2, a ubiquitously expressed intracellular tyrosine phosphatase and that SHP2 negatively regulates poly-IC–induced TLR3-activated signaling (31–33), we investigated whether IRF-8 and SHP2 could interact in MDDCs. Coimmunoprecipitation experiments performed in nondenaturing conditions (Fig. 5C) with anti–IRF-8 Abs probed with anti-SHP2 Abs showed that IRF-8 specifically interacts with SHP2 in MDDCs and that poly-IC stimulation increased this interaction. Then, we investigated SHP2 phosphorylation/activation status by coimmunoprecipitation assays in denaturing conditions with anti-pTyr Abs. Interestingly, SHP2 becomes strongly tyrosine phosphorylated only at 72 h of poly-IC stimulation (Fig. 5B).

In keeping with the hypothesis that SHP2-mediated dephosphorylation of IRF-8 could be responsible for negative regulation of poly-IC–stimulated TLR3 expression, we performed MDDC culture at day 5, pretreated with the SHP2 inhibitor NSC-87877 (34), and then stimulated with poly-IC for 24 and 72 h, and IRF-8 phosphorylation status was analyzed by coimmunoprecipitation analysis with anti–IRF-8 and anti–pTyr Abs in both denaturing and nondenaturing conditions. Interestingly, treatment with the SHP2 inhibitor increased IRF-8 phosphorylation at 72 h of poly-IC stimulation when IRF-8 was detected as no longer phosphorylated in control cultures (lane 8 versus lane 6) (Fig. 5A, 5B). Of note, treatment with NSC-87877 did not modify the IRF-8/SHP2 association (Fig. 5C), whereas it affected SHP2 phosphorylation at 72 h after TLR3 stimulation when SHP2 is active (Fig. 5B).

These results show the occurrence of an IRF-8 phosphorylation modulation during poly-IC stimulation of MDDCs that correlated with a modulation in TLR3 expression. Consistently, reporter gene assays in HEK 293T cells (Fig. 5D) using an IRF-8 mutant in which all 12 tyrosine residues were mutated to phenylalanine (IRF-8 12Y) (28) indicated that 12Y-mut IRF-8 was no longer able to suppress the TLR3 transcription induced by IRF-1. More importantly, nucleofection experiments in MDDCs indicated that the 12Y-mut IRF-8 was unable to inhibit endogenous TLR3 expression stimulated by IRF-1 (Fig. 5E).

Next, we assessed whether treatment with the SHP2 inhibitor NSC-87877 also resulted in TLR3 expression modulation. Real-time PCR analysis of TLR3 expression in MDDCs at day 5 of culture (Fig. 6A) showed that NSC-87877 treatment at 24 h of poly-IC stimulation did not alter significantly the high TLR3 expression; in contrast, a significant recovery of TLR3 expression was detected in both control and poly-IC–treated cultures after 72 h when TLR3 expression dropped. These results indicate that IRF-8–mediated TLR3 inhibition is dependent on its phosphorylation status, which is modified by SHP2 activity.

FIGURE 6.

FIGURE 6

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NSC-87877 SHP2 inhibitor restores TLR3 expression, MDDC activation markers, and cytokines. A, CD14+ monocytes were magnetically isolated and cultured with hrIL-4 and hrGM-CSF to differentiate into MDDCs. At day 5 of culture, cells were pretreated with NSC-87877 inhibitor for 2 h, transfected with poly-IC–Fugene 6 complexes, and collected 24 and 72 h later. Total RNA was subjected to qPCR for TLR3 and GAPDH expression. Data are normalized on GAPDH values and expressed as means ± SD. Results are representative of at least three independent experiments. B, The expression of DC differentiation markers was determined by FACS analysis. Representative data from one of three independent experiments are shown. C–E, Total RNA was subjected to qPCR as in A for IL-12p40, IFN-β, and 2′-5′-OASE, respectively. *p < 0.05.

We also analyzed the DC cell-surface expression levels of HLA-DR, CD86, and CD80 activation markers in the presence of the SHP2 inhibitor. We found that NSC-87877 treatment significantly increased HLA-DR expression at 24 h after poly-IC stimulation, and its expression was further increased at 72 h (Fig. 6B). CD86 as well as CD80 (not shown) expression was also significantly increased at 24 h when cells were stimulated with poly-IC and treated with NSC-87877. In contrast, after 72 h of treatment, their expression was not affected by NSC-87877 treatment (Fig. 6B). These results suggest that prolonged SHP2 inhibition overall promotes MDDC differentiation with different extents and kinetics depending on the differentiation marker considered.

Finally, to assess whether the negative regulation of TLR3 by IRF-8 also had an effect on TLR3 response, we determined cytokine expression profile of MDDCs during poly-IC stimulation at different time points in the presence of SHP2 inhibitor. As shown in Fig. 6C, poly-IC induced a substantial increase in the expression of both IL-12p40 and IFN-β after 24 h of stimulation, whereas after 72 h of treatment, their synthesis dropped, perfectly resembling TR3 expression (Figs. 3C, 6A). Inhibition of SHP2 phosphatase activity by NSC-87877 led to a significant increase in the expression of IL-12p40 at both 24 and 72 h of poly-IC treatment (Fig. 6C). Similarly, IFN-β expression was recovered in the presence of NSC-87877 but only after 72 h of stimulation (Fig. 6D). Accordingly, expression of an IFN-induced gene (i.e., 2′,5′-OASE) was modulated by NSC-87877 with a kinetic similar to that observed for IFN-β and TLR3 expression (Fig. 6E).

SHP2 specifically inhibits TLR3 transcription by dephosphorylating IRF-8 in myeloid and nonmyeloid cells

It has been documented that NSC-87877 also inhibits SHP1 (34), a protein tyrosine phosphatase structurally and functionally related to SHP2, which is expressed in DCs as SHP2. To exclude a role for SHP1 in TLR3 expression pathway, we performed experiments with cell-active SHP2 selective inhibitor SPI-112Me (37). Real-time PCR analysis of TLR3 expression in MDDCs at day 5 of culture (Fig. 7A) showed that treatment with SPI-112Me had no effect on basal TLR3 expression at any time point. At 24 h of poly-IC stimulation, the high TLR3 expression was not altered in contrast with after 72 h, when TLR3 expression dropped, and a complete recovery of TLR3 expression was detected specifically in poly-IC–treated cultures. These results clearly indicate that IRF-8–mediated TLR3 inhibition during poly-IC stimulation is specifically dependent on SHP2 phosphatase activity in MDDCs.

FIGURE 7.

FIGURE 7

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IRF-8 downregulates TLR3 expression by specifically involving SHP2. A, CD14+ monocytes were magnetically isolated and cultured with hrIL-4 and hrGM-CSF to differentiate into MDDCs. At day 5 of culture, cells were pretreated with SPI-112Me inhibitor for 2 h, transfected with poly-IC–Fugene 6 complexes, and collected 24 and 72 h later. B, A549 cells were transfected with IRF-1, IRF-8, or combination expressing vectors and then transfected with poly-IC–Fugene 6 complexes. C, A549 cells were pretreated with NSC-87877 inhibitor for 2 h, transfected with IRF-8 expressing vectors, and then transfected with poly-IC–Fugene 6 complexes. Cells were collected 18–24 h posttransfection. Total RNA was subjected to qPCR for TLR3 and GAPDH expression. Data are normalized on GAPDH values and expressed as means ± SD. n = 3. *p < 0.05 versus CTR, **p < 0.05 versus IRF-1.

Furthermore, we analyzed the effect of IRF-8 phosphorylation on TLR3 expression in the A549 lung epithelial cell line that endogenously expresses TLR3 but does not express SHP-1. First, we performed transfection experiments with IRF-8 to assess whether this transcription factor was able to repress the endogenous IRF-1– and poly-IC–induced TLR3 expression as in MDDCs. As shown in Fig. 7B, ectopic expression of IRF-8 significantly down-regulated endogenous as well as IRF-1– and poly-IC–induced TLR3 expression, thus providing evidence that IRF-8 also negatively regulates basal and stimulated TLR3 expression in nonmyeloid cells. We then performed experiments in IRF-8–expressing A549 cells stimulated with poly-IC and treated or not with NSC-87877. Interestingly, addition of the SHP2 inhibitor totally restored the IRF-8–suppressed poly-IC–stimulated TLR3 expression (Fig. 7C). Altogether these results, ruling out a possible role of SHP1, clearly indicate that SHP2 is specifically involved in IRF-8–mediated negative regulation of poly-IC–induced TLR3 expression in a myeloid and nonmyeloid context (Fig. 8).

FIGURE 8.

FIGURE 8

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Proposed model for TLR3 promoter regulation by IRFs in MDDCs. In absence of signal, TLR3 is constitutively expressed and bound by IRF-2. In response to specific inducers such as poly-IC or viral dsRNA that reach the endosome compartment, a signaling cascade via TLR3 stimulation is initiated that results in the induction of IRF-1 and constitutively phosphorylated IRF-8 that can also form heterocomplexes with IRF-2 and CBP/p300 coactivators that activate TLR3 gene expression. A further late phase involves the specific induction of active SHP2 phosphatase that dephosphorylates IRF-8, leading to dissociation of the heterocomplexes from the DNA. Dephosphorylated IRF-8 in turn becomes an active repressor that binds directly to the DNA-recruiting HDAC3 as corepressor and downregulates TLR3 gene expression.

Discussion

mDCs hold a central position in alerting the host immune system to infection by bridging innate and adaptive immune responses. Stimulation of TLRs on mDCs triggers the activation of signaling cascades, leading to the induction of immune and proinflammatory genes. Although full activation of mDCs is necessary for elimination of invading pathogens, inappropriate production of IFNs and/or proinflammatory cytokines promotes the development of immunopathological conditions such as endotoxin shock and autoimmune responses (1, 2, 9). The intensity and duration of TLR response must be, therefore, tightly controlled. Several negative regulatory mechanisms acting along the signaling pathway elicited by TLRs have been described, with degradation or destabilization of signal transduction factors being the principal ones (9). Although several studies report the identification of gene regulatory elements controlling cell-type specificity and high inducibility of TLR gene transcription, only limited attention has been given to negative transcriptional regulation of TLR genes (10).

In the current study, we present, for the first time, to our knowledge, evidence of a transcriptional mechanism for negative regulation of TLR gene expression. In particular, we show that TLR3 downregulation, occurring during DC differentiation and promptly after ligand stimulation, is finely controlled by transcriptional mechanisms attended by IRF-8 that negatively regulates IRF-1–triggered TLR3 expression.

Previously, basal as well as IFN-β–induced activation of TLR3 gene expression has been reported as dependent on an highly conserved IRF consensus sequence present both in murine and human promoters, which binds IRF-2 and recruits IRF-1 post-stimulation (21). In accordance with these observations and extending them, we show in this study that TLR3 expression is specifically stimulated by IRF-1, whereas IRF-2 alone did not exert any effect. Moreover, we found that IRF-1 and IRF-2 synergistically stimulate TLR3 expression, highlighting a positive role also for IRF-2 in TLR3 expression. Our transfection studies also demonstrate that IRF-8 dose dependently inhibited IRF-1– and IRF-1 plus IRF-2–mediated TLR3 promoter stimulation. This IRF-mediated regulation of TLR3 expression was observed in multiple cell systems including A549 cells that express TLR3 endogenously, in the monocytic THP-1 cells, and, importantly, in the physiological context of human MDDCs that, as reported, express the highest TLR3 within human myeloid cells (21).

Of note, whereas IRF-8 per se had no activity on the human promoter in HEK 293T cells and in MDDCs, it significantly stimulated TLR3 expression in THP-1 cells. Because in THP-1 and freshly isolated CD14+ cells TLR3 expression is undetectable, it is possible that surrounding sequences containing regulatory sites can allow the formation of different IRF-8–containing active complexes during myeloid differentiation. Similarly, differences in proximal promoter regions previously implicated in species-specific regulation of TLR3 (21) can account for our results showing that IRF-8 stimulated 4-fold the basal activity of the mouse promoter. Accordingly, it has been previously shown that DCs isolated from IRF-8 KO mice did not express a detectable amount of TLR3 mRNA (16).

To better define the role of IRFs in modulating TLR3 expression in MDDCs, we performed a detailed TLR3 expression profile and show that both basal and induced TLR3 upregulation is modulated along the MDDC differentiation, being maximally expressed at 5 d when poly-IC maximally stimulated TLR3 expression and then promptly declining. Interestingly, the data presented in this study show that modulation of TLR3 expression mirrors well differential IRF expression and binding to the promoter. Indeed, MDDCs constitutively express IRF-2 bound to IRF-E/TLR3. Conversely, IRF-1 expression, as well as its binding to the TLR3 promoter, substantially increases upon stimulation. This finding correlated with an increased expression of TLR3, indicating that IRF-1 plays a ligand-dependent role when recruited upon stimulation, whereas IRF-2 constitutively regulates TLR3 expression. IRF-8 binding was induced by TLR3 ligation at an early time point, even if at a lower extent as compared with IRF-1. Later on, IRF-1 expression and binding as well as TLR3 expression declined, whereas IRF-8 expression and binding to IRF-E/TLR3 increased on time. The relevance of this finding has been strengthened by measuring the in vivo binding of these factors by ChIP analysis that confirmed an IRF-1 versus IRF-8 differential binding to the TLR3 promoter depending on the stimulation time. These findings, together with our functional assays demonstrating that IRF-8 abrogates IRF-1 stimulatory activity on the TLR3 promoter, indicate that IRF-8 controls TLR3 downregulation occurring in MDDCs during prolonged ligand stimulation. Importantly, we identify p300/CBP and HDAC3 as good candidates acting, respectively, as coactivator and corepressor in IRF-1– and IRF-8–mediated effects on TLR3 expression. Indeed, we found that at an early time point when the IRF-1 binding is maximal, p300/CBP are recruited to the promoter, whereas later on, when IRF-8 is specifically bound, HDAC3 deacetylase is recruited.

Furthermore, our findings identify the molecular mechanism responsible for the IRF-8–mediated TLR3 repression by demonstrating that it is dependent upon IRF-8 phosphorylation status. DNA binding and activity of IRF-8 has been strictly related to its phosphorylation status in that specific tyrosine phosphorylation prevents direct IRF-8 binding to DNA while increasing its binding as a heterodimer with IRF-1 and IRF-2; conversely, dephosphorylated IRF-8 dissociates from heterodimers and can bind target DNA alone (23, 28, 29). In this paper, we show that IRF-8 is highly tyrosine phosphorylated after 24 h of poly-IC stimulation, when TLR3 is maximally expressed; after that, 72 h later, when TLR3 is downregulated, IRF-8 is dephosphorylated, indicating that a further delayed response to TLR3 stimulation involves induction of specific phosphatases. Interestingly, it has been recently reported that IRF-8 is a substrate of tyrosine phosphatase SHP2 and that SHP2 negatively regulates poly-IC–induced TLR3-activated signaling in murine macrophages (31–33). Our data support the view that SHP2 contributes to the control of TLR3 transcriptional regulation, as it is activated later in TLR3 stimulation, it physically interacts with IRF-8, and this interaction results in IRF-8 dephosphorylation. Consistently, we found that pharmacological inhibition of the SHP2 phosphatase activity reverts IRF-8 dephosphorylation status and, more significantly, TLR3 downmodulation. In particular, our data indicate that both NSC-87877 and selective SPI-112Me SHP2 inhibitors exert the same effect in MDDCs, excluding a possible role for SHP1 in IRF-8–mediated TLR3 expression regulation. However, it has to be pointed out that we cannot exclude a role for IRF-1 phosphorylation status in TLR3 expression regulation. Indeed, IRF-1 has been shown to be a substrate for SHP1 (21), although, to our knowledge, a direct role for SHP2 in IRF-1 dephosphorylation has never been demonstrated.

Moreover, data obtained in A549 cells that do not express SHP1 showing that IRF-8 dephosphorylation is specifically exerted by SHP2 highlight a specific role for SHP2 in the mechanism of shutting down TLR3 activation by IRF-8 also in nonmyeloid cells.

Finally, our data also support a negative role of SHP2 in activation/maturation of poly-IC–stimulated MDDCs because inhibition of SHP2 pushed forward DC activation marker expression and, importantly, also affected MDDC cytokine profile expression in that inhibition of SHP2 activity increased IL-12p40, IFN-β, and 2′,5′-OASE expression at a late time point after TLR3 stimulation.

From our results, we can infer the following scenario: during MDDC differentiation and in response to specific inducers, such as poly-IC, a signaling cascade is initiated that results in the induction of IRF-1 and constitutively phosphorylated IRF-8 that can also form heterocomplexes with IRF-2 and CBP/p300 coactivators, inducing TLR3 gene expression (Fig. 8). A further delayed response to stimulation involves the specific induction of active SHP2 phosphatase that acts on IRF-8, leading to dissociation of heterocomplexes from DNA. Dephosphorylated IRF-8 in turn becomes an active repressor that binds directly to the DNA, recruiting HDAC3 as a corepressor and downregulating TLR3 gene expression (Fig. 8).

TLR3 has been postulated to be a key player in immunity against some viral infections. This notion is supported by in vitro and in vivo studies demonstrating the pivotal role of TLR3 in host responses against a number of viruses (e.g., West Nile, respiratory syncytial, SIV, and influenza A) (4). Data presented in this study thus have multiple implications that may be also of clinical relevance. If TLR3 upregulation mediates antiviral responses induced by TLR3-triggered DCs, then altering IRF-8–IRF-E–TLR3 interactions (e.g., by pharmacological inhibitors of SHP2) could be a useful tool in modulating specific antiviral responses. In this respect, we previously reported that IRF-8 represses the IRF-1–mediated activation of HIV-1 long terminal repeat (35), supporting the hypothesis that repression of HIV-1 transcription by IRF-8 may be implicated in the establishment and maintenance of pro-viral quiescence in latently infected cells. Supporting this hypothesis, microarray data identified IRF-8 as a candidate gene involved in HIV latency (40). Based on current results, it would therefore be of interest to investigate whether modulation of SHP2 activity could also affect long terminal repeat transcription and HIV-1 replication through IRF-8 phosphorylation.

Besides its role in antiviral immunity, TLR3 also has a number of critical functions in nonimmune cells, such as human cancer cells (41). Moreover, a new TLR3-dependent route that induces the synthesis of CD4+ T cell proinflammatory cytokines has been recently reported, extending TLR3 functions to autoimmune diseases (42). Therefore, it is possible that regulatory mechanisms described in this study can be also extended to different cell types.

In conclusion, our data identify new mechanisms in the control of TLR3 gene expression that are dependent on IRF stimulation and regulation by SHP2 tyrosine phosphatase. These can be exploited as new tools for therapeutic interventions aimed at modulating TLR3 expression during infection, inflammation, and cancer.

Acknowledgments

This work was supported in part by grants from the Istituto Superiore di Sanitá-National Institutes of Health Scientific Cooperation agreement (to A.B.), the Italian AIDS Project (to A.B.), the Italian Ministry of Health (Special Program in Oncology) (to A.B.), Istituto Superiore di Sanitá-Alliance Against Cancer Program 3 (to A.B.), and in part by National Institutes of Health Grant P01CA118210 (to J.W.).

We thank Dr. Elizabeth A. Eklund (Northwestern University, Chicago, IL) for providing the CS463-Shp2pSX, ICSBPpcDNAamp, and Y2-351F ICSBPpCDNAamp vectors. We also thank Sabrina Tocchio for editorial work and Roberto Gilardi for help with the figures.

Abbreviations used in this article

CBP

CREB-binding protein

ChIP

chromatin immunoprecipitation

DC

dendritic cell

h

human

HD

healthy donor

HDAC3

histone deacetylase 3

HEK

human embryonic kidney

IRF

IFN regulatory factor

IRF-E

IFN regulatory factor element

KO

knockout

mDC

myeloid dendritic cell

MDDC

monocyte-derived dendritic cell

OASE

oligoadenylate synthase

pDC

plasmacytoid dendritic cell

poly-IC

polyinosinic-polycytidylic acid

pTyr

phosphotyrosine

qPCR

quantitative PCR

SHP2

Src homology 2 domain-containing protein tyrosine phosphatase-2

WT

wild-type

12Y

Y2-351F

Footnotes

Disclosures

The authors have no financial conflicts of interest.

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