Interferon Regulatory Factor-3-mediated Activation of the Interferon-sensitive Response Element by Toll-like receptor (TLR) 4 but Not TLR3 Requires the p65 Subunit of NF-κ

Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of IκB kinase 2 and a mutant form of IκB, which acts as a super-repressor of NF-κB, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-κB was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-κB. Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of IκB kinase 2 and a mutant form of IκB, which acts as a super-repressor of NF-κB, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-κB was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-κB. The discovery of human Toll-like receptors (TLRs) 1The abbreviations used are: TLRToll-like receptorLPSlipopolysaccharideTIRToll/interleukin-1 receptorIFNβinterferon βIRFinterferon regulatory factorISREinterferon-sensitive response elementIKKIκB kinaseMEFmouse embryonic fibroblasthTLRhuman TLRCBPcAMP-responsive element-binding protein-binding protein. has increased our understanding of the molecular basis to innate immunity. TLRs allow the host to differentiate between groups of pathogens and to tailor its initial response accordingly. At least 10 different TLRs occur in humans (1Dunne, A., and O'Neill, L. A. (2003) Science's STKE, http://stke.sciencemag.org/gci/content/full/sigtrans;2003/171/reGoogle Scholar). Most of their ligands have been assigned, the majority of which are so-called pathogen-associated molecular patterns. 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It is involved in the activation of the transcription factor interferon regulatory factor 3 (IRF-3), which binds the interferon-sensitive response element (ISRE) and induces a subset of genes, including IFNβ (2Hoshino K. Kaisho T. Iwabe T. Takeuchi O. Akira S. Int. Immunol. 2002; 14: 1225-1231Crossref PubMed Scopus (238) Google Scholar, 14Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 15Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (691) Google Scholar). Although TRIF is recruited to both TLR4 and TLR3, the recently described TRIF-related adaptor molecule (16Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (939) Google Scholar), also called TIR-containing adaptor molecule 2 (17Oshiumi H. Sasai M. 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In its inactive form IRF-3 is constitutively present in a latent cytoplasmic pool. Upon stimulation with poly(I-C), IRF-3 becomes phosphorylated in its C terminus, which presumably reveals the previously hidden dimerization domain (22Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (273) Google Scholar). LPS does not induce C-terminal phosphorylation but appears to causes N-terminal phosphorylation (23Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 24Servant M.J. Grandvaux N. Hiscott J. Biochem. Pharmacol. 2002; 64: 985-992Crossref PubMed Scopus (134) Google Scholar). The nature of this phosphorylation and the responsible kinase is still uncertain. In the case of the C-terminal phosphorylation two IκB kinase (IKK)-related proteins, IKKϵ and TANK-binding kinase 1, have recently been identified as possible components of the virus- and TLR3-activated kinase complex for IRF-3 (25Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1371) Google Scholar, 26Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2099) Google Scholar). In this study we have found a key role for the NF-κB subunit p65 in the IRF-3-mediated induction of the ISRE by TLR4 but not TLR3. p65 promoted transactivation of gene expression by IRF-3. TLR4 and TLR3 therefore differ in their mechanism of ISRE induction. Cell Culture, Plasmids, and Reagents—HEK293 and U373 cell lines were purchased from the Centre for Applied Microbiology & Research (Salisbury, UK), the HEK293-TLR3 cell line was kind gift of Katherine Fitzgerald (University of Massachusetts Medical School). The mouse embryonic fibroblasts (MEFs) with a targeted deletion in p65 and wild type MEFs were a kind gift from Ron Hay (University of St. Andrews, Fife, Scotland), and MEFs with a deletion of the β subunit of the interferon-α/β receptor on a 129SV/Ev genetic background and their parental MEFs were a gift from Otto Haller (University of Freiburg, Freiburg, Germany). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, and 100 units ml-1 gentamycin and maintained at 37 °C in a humidified atmosphere of 5% CO2, 200 mg ml-1 of the neomycin analog G418 was added to maintain HEK293-TLR3 cells. The cells were seeded at 0.5 × 105 to 105 ml-1 for experiments and treated as indicated in figure legends. LPS from Escherichia coli serotype O26:B6 and poly(I-C) were purchased from Sigma. The NF-κB luciferase construct bearing five repeats of the κB consensus was a gift from Dr. R. Hofmeister (Universitaet Regensburg, Regensburg, Germany). The ISRE luciferase construct, which has five repeats of the ISRE sequence from the ISG15 promoter, was purchased from Clontech (Palo Alto, CA). Constructs for the IRF-3 transactivation assay, p-55UASGLuc, Gal4-DBD, and Gal4-IRF-3 were a kind gift of Takashi Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). The chimeric CD4-TLR4 and CD4-TLR3 expressing vectors were a gift from Ruslan Medzhitov (Yale University School of Medicine). Mal was expressed from pDC304, and MyD88 was from pCDNA3.1. The plasmid encoding TRIF was a kind gift from Shizuo Akira (Osaka University, Osaka, Japan). The construct comprising hemagglutinin-tagged p65 and the IκB-SR expression vector were kind gifts from Keith Ray (GlaxoSmithKline, Stevenage, UK). Constructs encoding the dominant negative version IKK2, IKK2KA, and human TLR3 (hTLR3) were obtained from Tularik (San Francisco, CA). Expression constructs bearing IRF-3 and ΔNIRF-3 were kind gifts of John Hiscott (McGill University, Montreal, Canada). Transfection-based Reporter Gene Assays—HEK293, U373, and MEFs were seeded at 1-2 × 104 cells well-1 in 96-well plates, incubated overnight, and transfected using GeneJuice transfection reagent (Novagen, Madison, WI) according to the manufacturer's instructions with a total amount of 300 ng of DNA well-1 comprising 100 ng of reporter gene construct, plasmid DNA of interest, and empty vector as filler DNA. For transactivation assays 80 ng of p-55UASGLuc were applied in combination with 100 ng of either Gal4-DBD or Gal4-IRF-3 and additional plasmids as indicated in figure legends. The cells were lysed in passive lysis buffer (Promega, Southampton, UK) for 15 min. At 24 or 48 h post-transfection, the extracts were monitored for firefly luciferase activity following standard protocols. 40 ng well-1Renilla reniformis luciferase construct was used as internal control for transfection efficiency. The activities were expressed as fold activation over unstimulated empty vector controls (ctrl). The experiments were carried out in triplicate with error bars indicating standard deviations. Electrophoretic Mobility Shift Assays—U373 cells were grown in 15-cm dishes and treated as indicated in figure legends, and their nuclear extracts were prepared as previously described with minor modifications (27Yamaguchi S. Murata Y. Nagaya T. Hayashi Y. Ohmori S. Nimura Y. Seo H. J. Mol. Endocrinol. 1999; 22: 81-90Crossref PubMed Scopus (33) Google Scholar). Specifically, the samples were homogenized using a Dounce homogenizer (80 strokes at 4 °C). Following the addition of the high salt buffer, the samples were rotated for 30 min at 4 °C. 10 μg of nuclear protein was incubated for 30 min with 10,000 cpm of double-stranded [γ-32P]ATP ISRE oligonucleotide (5′-GAT CGG GAA AGG GAA ACC GAA ACT GAA-3′) in hybridization buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 4% glycerol, and 100μg ml-1 nuclease free bovine serum albumin) containing 2 μg ml-1 of poly(dI-dC) as nonspecific competitor. Supershifting antibodies specific for p65 (SC-8008X) and IRF-3 (SC-9082X) were added to the nuclear extracts 1 h prior to hybridization with the oligonucleotide. The samples were kept on ice at all stages. Protein-DNA complexes were run on a 5% native polyacrylamide gel, and complex formation was detected with autoradiography. The TLR4 Adaptors MyD88 and Mal Enhance ISRE-dependent Gene Expression—We were interested in comparing IRF-3 activation by TLR4 and TLR3. To examine this process we used three systems comprising transfection of HEK293 cells with a CD4-TLR4 fusion protein, which is constitutively active, HEK293 cells rendered sensitive to poly(I-C) by stable transfection with TLR3, and addition of the ligands LPS and poly(I-C) to U373 cells, which express TLR3 and TLR4 constitutively. Fig. 1A demonstrates that transfection with a plasmid encoding a dominant negative version of IRF-3 blocked CD4-TLR4 and TLR3/poly(I-C)-stimulated induction of a reporter gene, luciferase, linked to five ISRE sites from the ISG15 promoter (left-hand panel) but did not inhibit the expression of luciferase linked to five NF-κB-binding sites (right-hand panel). These results indicate that the ISRE luciferase response involves IRF-3. We next probed the link between the adaptors MyD88, Mal, and TRIF with IRF-3. Fig. 1B demonstrates how overexpression of each of the proteins on their own affected ISRE- or κB-linked reporter gene expression. In agreement with other studies (10Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (1014) Google Scholar, 11Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1025) Google Scholar) expression of TRIF led to the strongest activation of the ISRE, causing a 200-fold induction over controls. By comparison, MyD88 and Mal had only marginal effects on the ISRE but were much better inducers of NF-κB when compared with TRIF. Overexpression of IRF-3 only induced the ISRE response. However, as shown in Fig. 1C, when concentrations of Mal or MyD88 unable to activate the ISRE were co-expressed with IRF-3, a clear synergy was observed in ISRE induction (Fig. 1C, left-hand panel). Mal enhanced the IRF-3 alone response by 5-fold, whereas MyD88 had a 7-fold effect. IRF-3 had no effect on NF-κB either alone or in combination with Mal or MyD88, as expected (Fig. 1C, right-hand panel). NF-κB Is Required for IRF-3 Activation by LPS but Not poly(I-C)—The synergism observed between MyD88 or Mal and IRF-3 with respect to ISRE activation suggested that TLR4 signaling to IRF-3 might involve NF-κB, because Mal and MyD88 are both NF-κB activators. To examine this we tested specific inhibitors of the NF-κB pathway for their effects on TLR4-mediated and, for comparison, TLR3-mediated signal transduction to the ISRE. We used IκB-SR, a mutant form of IκBα (S32A/S36A), which cannot undergo phosphorylation and acts as a super-repressor of NF-κB, and a kinase inactive mutant of IKK 2 (IKK2KA), which acts as a dominant negative inhibitor. Interestingly, both agents inhibited CD4-TLR4-stimulated ISRE and, as expected, NF-κB-mediated gene induction (Fig. 2A). Strikingly, neither had an effect on ISRE activation by poly(I-C) in TLR3-transfected HEK293 cells but, as expected, blocked NF-κB activation, although the inhibitory effect of IKK2KA reached a maximum of only 50% (Fig. 2B). Similar results were obtained when ISRE- and κB-dependent expression was measured in U373 cells. LPS-mediated activation of the ISRE was repressed by both IKK2KA and IκB-SR, whereas the effect of poly(I-C) was not impaired by either of these agents (Fig. 2C, left-hand panel). NF-κB activation by LPS and poly(I-C) in U373 was blocked by both IKK2KA and IκB-SR (right-hand panel) with IKK2KA again having a lesser effect than the IκB-SR on the poly(I-C) response. TLR4 Fails to Induce ISRE-dependent Gene Expression in the Absence of p65—We further tested the significance of NF-κB in the TLR4 response using p65-deficient cells. Fig. 3 (left-hand panel) demonstrates how CD4-TLR4 activates NF-κB in wild type MEFs, whereas the effect in p65-deficient MEFs was impaired, as expected. TLR3 signaling to NF-κB activated by poly(I-C) in MEFs transfected with TLR3 was decreased in p65-deficient cells. Importantly, a different result was obtained using the ISRE luciferase construct, as shown in Fig. 3 (right-hand panel). The ISRE response to TLR4 was almost abolished in the p65-deficient MEFs relative to wild type controls. We were unable to test LPS itself on MEFs because in wild type cells the activation of the ISRE was marginal (data not shown). The effect of poly(I-C), however, was similar in wild type and in p65-deficient cells. This result provides additional evidence that NF-κB, and in particular the p65 subunit, is required for signaling by TLR4 but not TLR3 to the ISRE. p65 Interacts with IRF-3 at the ISRE and Promotes Transactivation—We next tested whether p65 was part of the IRF-3-containing ISRE activation complex. We used the ISRE from the ISG15 promoter (as used in the ISRE luciferase construct) in an electrophoretic mobility shift assay on nuclear extracts from LPS- or poly(I-C)-stimulated U373 cells. As shown in Fig. 4A, we observed complex formation at the ISRE oligonucleotide following stimulation with either LPS (lane 4) or poly(I-C) (lane 7). Incubation of the extract with an IRF-3-specific antibody abolished DNA binding, confirming the presence of IRF-3 in each complex (lanes 5 and 8). Importantly, a p65-specific antibody prevented ISRE complex formation in extracts from LPS-treated cells (lane 6) but had only a marginal effect on extracts from poly(I-C)-treated cells (lane 9). The supershifting ability of the p65-specific antibody was confirmed with a κB site-containing oligonucleotide and LPS-stimulated U373 and THP-1 nuclear cell extracts (data not shown).Fig. 4Interaction between NF-κB and IRF-3.A, nuclear extracts prepared from 105 U373 cells treated for 6 h with LPS (1 μg/ml) or poly(I-C) (25 μg/ml) or left untreated were analyzed in a electrophoretic mobility shift assay using an oligonucleotide comprising the ISRE from the ISG15 promoter. The arrow indicates protein/DNA complexes. The extracts were preincubated with antibodies against IRF-3 or p65 as indicated. These results are representative of three separate experiments. B, 106 HEK293 cells were co-transfected with a construct bearing the Gal4 upstream activation sequence fused to the luciferase reporter gene (100 ng), an expression vector for Gal4-IRF-3 (80 ng), or the Gal4 DNA-binding domain on its own Gal4-DBD (80 ng) and expression vector for p65 in increasing amounts (5, 25, and 50 ng). C, 106 HEK293 cells transfected with Gal4 luciferase construct (100 ng), the expression vector for Gal4-IRF-3 (80 ng), and p65 (50 ng) were additionally transfected with expression vectors for the dominant negative form of IKK2 (IKK2KA 20 ng) and the IκB-SR (20 ng). D, 0.5× 106 wild type MEFs (wt) or MEFs with bearing a deletion of IFNAR1 (IFNAR1-/-) were seeded in 96-well microtiter plates and transfected with 100 ng of the ISRE-or the κB luciferase constructs and with either CD4-TLR4 (60 ng) or hTLR3 (0.5 ng) for 24 h. The cells transfected with hTLR3 were stimulated with 1μg/ml poly(I-C) for 7 h. E, IFNAR1-/- MEFs were co-transfected with ISRE luciferase, CD4-TLR4, or hTLR3, respectively, and increasing doses of IκB-SR (10 ng and 30 ng). Unstimulated cells transfected with an empty vector were used as control (ctrl). In all experiments cell lysates were prepared 24 h after transfection and assayed for luciferase reporter gene activity, and the obtained values were normalized against the R. reniformis luciferase activity for transfection efficiency. Luciferase activity in cells transfected with a Gal4-DBD expression vector was in all cases below the control values of cells transfected with the Gal4-IRF-3 construct. The data shown are the means ± standard deviations of triplicate determinations. The results shown are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also addressed what role p65 might have on the functioning of IRF-3. For this we used an IRF-3 transactivation assay. This assay involved transfecting cells with a plasmid encoding a fusion protein comprising the transactivation domain of IRF-3 fused to the DNA-binding domain of Gal4. When activated, this fusion protein drives a Gal4-controlled luciferase reporter gene co-transfected into the cells. Fig. 4B demonstrates that increasing expression of p65 in HEK293 cells dose-dependently promoted IRF-3-mediated transactivation. This effect was blocked by the IκB-SR but not the dominant negative IKK2KA (Fig. 4C). Overexpressed IκB-SR presumably sequesters p65 in the cell, thereby preventing it from enhancing IRF-3 transactivation activity. Taken together these data indicate that p65 and IRF-3 interact in a complex at the ISRE as a consequence of TLR4 but not TLR3 signaling and that p65 promotes transactivation by IRF-3. Finally, we wanted to exclude the possibility that activation of the ISRE in our experiments is predominantly driven via a type I interferon feedback loop through activation of ISGF3. We therefore transfected MEFs bearing a targeted disruption of the β subunit of the interferon-α/β receptor (IFNAR1-/- MEFs) and their corresponding wild type MEFs with the κB and ISRE luciferase constructs and tested TLR3 and TLR4 responses. TLR4 and TLR3 signaling to either ISRE or NF-κB was comparable in both IFNAR1-/--deficient and wild type cells (Fig. 4D), indicating that the ISRE construct serves primarily as an IRF-3 read-out. Consistent with our data in HEK293 and U373 cells, upon co-transfection with the IκB-SR only the TLR4-stimulated ISRE response was decreased, whereas TLR3 signaling remained unaltered (Fig. 4E). It should be noted that the parental MEFs of the IFNAR1-/- MEFs, although clearly evident, were less responsive compared with the wild type MEFs, which were the corresponding controls for the p65-deficient cells. The results from this study indicate a difference between TLR4- and TLR3-mediated activation of the ISRE. TLR4 has an absolute requirement for NF-κB in this process, with p65 occurring in the ISRE binding complex with IRF-3, whereas the TLR3-induced ISRE response is NF-κB-independent. We first suspected NF-κB involvement in the pathway to IRF-3 from TLR4 when we observed that Mal or MyD88 synergize with overexpressed IRF-3 in ISRE activation. Both of these adaptors target NF-κB. NF-κB involvement was confirmed by the inhibitory effect of IKK2KA and IκB-SR on the TLR4 response and most compellingly in cells from p65-deficient mice, which were unresponsive in terms of TLR4 signaling to the ISRE but were normal for TLR3. The necessity for cooperation between NF-κB and IRF-3 has been outlined in other studies (14Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 28Kim T. Kim T.Y. Lee W.G. Yim J. Kim T.K. J. Biol. 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Additionally, in an earlier study it has been shown that the necessity for p65 at the IFNβ promoter could be overcome with a chimera comprising the N-terminal DNA-binding domain of IRF-3 and the N-terminal p65 transactivation domain (14Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). This again argues for a direct cooperation between IRF-3 and p65 at the ISRE, with the role of p65 to promote transactivation. In support of this model we found that the inhibition of the NF-κB pathway in IFNAR1-/- MEFs cells blocks TLR4 signaling to the ISRE. Additionally, we detected both p65 and IRF-3 in the LPS/TLR4-induced activation complex at the ISRE. An interaction between p65 and IRF-3 at the ISRE could be mediated by the IRF-3 co-activators cAMP-responsive element-binding protein-binding protein (CBP) or p300. IRF-3 relies on recruitment of these co-activators not only to reduce export from the nucleus but moreover for transcriptional activation (15Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (691) Google Scholar, 31Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (66) Google Scholar). The histone acetylase function of CBP is vital for binding to the ISRE (32Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). CBP has been shown to interact with both IRF-3 and p65 (31Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (66) Google Scholar, 33Hottiger M.O. Felzien L.K. Nabel G.J. 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However, because of the availability of p65-deficient cells, we focused on the role of p65 in this study. The mechanism by which LPS activates IRF-3 via TLR4 is still uncertain, although other studies have also indicated differences between LPS and viral stimuli or poly(I-C) in this process (23Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 37Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 38Doyle S.E. O'Connell R. Vaidya S.A. Chow E.K. Yee K. Cheng G. J. Immunol. 2003; 170: 3565-3571Crossref PubMed Scopus (155) Google Scholar). LPS appears to cause N-terminal phosphorylation of IRF-3, whereas the activation induced by poly(I-C) is more potent and results in phosphorylation of the C terminus (23Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 37Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). It is possible that IRF-3 phosphorylated in its C terminus does not require p65 for it to enhance transcription, whereas N-terminally phosphorylated IRF-3 does. Recently, two independent studies have implicated two members of the IKK family, IKKϵ and TANK-binding kinase 1, as crucial mediators in virus- or poly(I-C)-induced IRF-3 activation and have suggested that IKKϵ is an activating kinase for IRF-3 and IRF-7 (25Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1371) Google Scholar, 26Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. 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One possibility we are exploring is that p65 and IRF-3 interact directly, or via CBP as stated above, and is required for these responses. Although the signaling pathways induced by TLR4 and TLR3 therefore differ, both lead to ISRE activation allowing for IFNβ expression. Clearly, the ISRE can be activated by different transcription factor complexes including IRF-7 and ISGF3, a trimeric complex comprising STAT1, STAT2, and IRF-9 (22Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (273) Google Scholar, 43Bluyssen A.R. Durbin J.E. Levy D.E. Cytokine Growth Factor Rev. 1996; 7: 11-17Crossref PubMed Scopus (118) Google Scholar, 44Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). Its promiscuity may allow for fine-tuning of responses in different biological contexts. It is also possible that the p65/IRF-3 dimer targets an additional set of genes, increasing the repertoire of responses to LPS. LPS induces IRF-3- and NF-κB-dependent production of IFNβ, which in an autocrine manner via the IFNAR1 receptor and ISFG3 activates the ISRE (8Toshchakov V. Jones B.W. Perera P.Y. Thomas K. Cody M.J. Zhang S. Williams B.R. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (682) Google Scholar, 38Doyle S.E. O'Connell R. Vaidya S.A. Chow E.K. Yee K. Cheng G. J. Immunol. 2003; 170: 3565-3571Crossref PubMed Scopus (155) Google Scholar). Inhibition of NF-κB could block LPS-stimulated IFNβ production and hence interfere with this positive feedback loop. However, our study points to a more direct effect because cells deficient in the β-chain of the IFNα/β receptor remain responsive to TLR4 and TLR3 signaling. Given our other data demonstrating that p65-depleted cells are impaired in the ISRE response to TLR4 but not TLR3 and that p65 could be detected in the LPS-activated ISRE-binding complex and could promote transactivation by IRF-3, we conclude that LPS directly induces a p65-IRF-3 complex for ISRE activation, whereas the poly(I-C) response is direct only toward IRF-3. In conclusion, our study provides the first demonstration that p65 is absolutely required for activation of the ISRE by TLR4 but not TLR3. The emerging differences in signaling by TLR4 and TLR3 as well as other TLRs will continue to improve our understanding of the fine-tuning of the innate immune response. We thank Dr. John Hiscott (Montreal, Canada), Dr. Takashi Fujita (Tokyo, Japan), Dr. Ron Hay (Fife, Scotland), and Dr. Otto Haller (Freiburg, Germany) for reagents.