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. Two of the best studied TLRs are TLR4, which recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, and TLR3, which recognizes the viral double-stranded RNA mimic poly(I-C) (2Hoshino K. Kaisho T. Iwabe T. Takeuchi O. Akira S. Int. Immunol. 2002; 14: 1225-1231Crossref PubMed Scopus (238) Google Scholar, 3Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1458) Google Scholar, 4Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6478) Google Scholar, 5Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar). TLRs are defined by external leucine-rich repeats and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain (1Dunne, A., and O'Neill, L. A. (2003) Science's STKE, http://stke.sciencemag.org/gci/content/full/sigtrans;2003/171/reGoogle Scholar). Upon stimulation the TLRs recruit TIR domain-containing adaptor molecules via homotypic interactions with receptor TIR domains. This initiates signal transduction culminating in the activation of transcription factors and an increase in immune and inflammatory gene expression. A key question concerns specificity in signal transduction by different TLRs, because although common gene sets are induced by TLRs, specific patterns of gene expression have been demonstrated for TLR2, TLR3, and TLR4, which may provide a molecular basis for the tailoring of innate immune response (6Huang Q. Liu D. Majewski P. Schulte L.C. Korn J.M. Young R.A. Lander E.S. Hacohen N. Science. 2001; 294: 870-875Crossref PubMed Scopus (667) Google Scholar, 7Doyle S. Vaidya S. O'Connell R. Dadgostar H. Dempsey P. Wu T. Rao G. Sun R. Haberland M. Modlin R. Cheng G. Immunity. 2002; 17: 251-263Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar, 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). Toll-like receptor lipopolysaccharide Toll/interleukin-1 receptor interferon β interferon regulatory factor interferon-sensitive response element IκB kinase mouse embryonic fibroblast human TLR cAMP-responsive element-binding protein-binding protein. Myeloid differentiation factor 88 (MyD88) was the first TIR domain-containing adaptor to be described. It is a general adaptor for TLRs, and loss of MyD88 prevents signal transduction through most of the TLRs. The only known exceptions are TLR3 and TLR4, which both initiate a so-called “MyD88-independent” pathway (5Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar, 9Kawai T. Takeuchi O. Fujita T. Inoue J. Muhlradt P.F. Sato S. Hoshino K. Akira S. J. Immunol. 2001; 167: 5887-5894Crossref PubMed Scopus (903) Google Scholar). This alternative pathway is mediated by another adaptor termed TIR domain-containing adaptor-inducing IFNβ (TRIF) or TIR-containing adaptor molecule 1 (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, 12Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2527) Google Scholar, 13Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar). 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. Shida K. Fujita T. Matsumoto M. Seya T. J. Biol. Chem. September 30, 2003; (10.1074/jbc.M305820200)PubMed Google Scholar), which activates IRF-3 as well as NF-κB, is specific for TLR4 signaling and presumably acts upstream of TRIF. TLR4 also recruits MyD88 and an additional adaptor named MyD88 adapter-like (Mal) or TIR domain-containing adapter protein, which is also required for TLR2 signaling (18Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1005) Google Scholar, 19Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (832) Google Scholar, 20Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar, 21Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar). Both Mal and MyD88 would appear to be involved in the rapid activation of NF-κB by TLR4, whereas TRIF, in addition to regulating IRF-3, mediates later activation (12Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2527) Google Scholar, 13Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar, 20Yamamoto M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar, 21Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar). 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 and and from the for the was of of The mouse embryonic fibroblasts with a targeted deletion in p65 and a from of and with a deletion of the β subunit of the receptor a and their a from of The cells in with 2 and and in a of of the was to The cells to for and as in LPS from and poly(I-C) from The NF-κB bearing repeats of the was a from R. The ISRE which has repeats of the ISRE from the was from for the IRF-3 transactivation and a of Fujita of The and a from Medzhitov of Mal was from and MyD88 was from The TRIF was a from Akira The p65 and the expression from the dominant negative and human TLR3 from bearing IRF-3 and of Hiscott and cells in and to the with a of of of gene of and as transactivation of in with of or and additional as in The cells in for At or the for was used as for The as activation The in with cells in and as in and their as previously described with S. Y. T. Y. S. Y. H. J. Mol. 1999; PubMed Scopus Google Scholar). the a the addition of the the for 10 of was for with of double-stranded ISRE in 1 and 2 of as specific for p65 and IRF-3 to the 1 to with the The a and complex was detected with The TLR4 MyD88 and Mal in IRF-3 activation by TLR4 and TLR3. this we used of cells with a protein, which is constitutively cells to poly(I-C) by with TLR3, and addition of the ligands LPS and poly(I-C) to which TLR3 and TLR4 that with a a dominant negative of IRF-3 blocked and induction of a to ISRE from the but not the expression of to These results indicate that the ISRE response IRF-3. We the between the and TRIF with IRF-3. of of the their or gene expression. In with (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 expression of TRIF to the activation of the a induction MyD88 and Mal only the ISRE but of NF-κB with TRIF. of IRF-3 only induced the ISRE as in of Mal or MyD88 to activate the ISRE with IRF-3, a was in ISRE induction Mal the IRF-3 response by whereas MyD88 a IRF-3 NF-κB or in with Mal or as NF-κB for IRF-3 by LPS but between MyD88 or Mal and IRF-3 with to ISRE activation that TLR4 signaling to IRF-3 NF-κB, because Mal and MyD88 are both NF-κB this we specific of the NF-κB pathway for their for signal transduction to the We used a mutant form of which phosphorylation and acts as a super-repressor of NF-κB, and a kinase inactive mutant of 2 which acts as a dominant negative both ISRE as gene induction an ISRE activation by poly(I-C) in cells as blocked NF-κB although the of a of only results and expression was in activation of the ISRE was by both and whereas the of poly(I-C) was not by of these NF-κB activation by LPS and poly(I-C) in was blocked by both and with a the the poly(I-C) TLR4 to in the of the of NF-κB in the TLR4 response 3 activates NF-κB in whereas the in was as TLR3 signaling to NF-κB activated by poly(I-C) in with TLR3 was in a different was the ISRE as in 3 The ISRE response to TLR4 was in the to We to LPS because in cells the activation of the ISRE was not The of poly(I-C), was in and in This additional that NF-κB, and in the p65 is required for signaling by TLR4 but not TLR3 to the p65 with IRF-3 the ISRE and p65 was of the ISRE activation complex. We used the ISRE from the used in the ISRE in an from or in we complex the ISRE stimulation with LPS or poly(I-C) of the with an the of IRF-3 in complex and a ISRE complex in from cells but only a from cells The of the was with a and and not between NF-κB and from cells for with LPS or poly(I-C) or in a an the ISRE from the The The with IRF-3 or p65 as These results are of cells with a bearing the upstream activation to the gene an expression for or the domain its and expression for p65 in and cells with the expression for and p65 with expression for the dominant negative form of and the or with bearing a deletion of in and with of the the and with or for The cells with with poly(I-C) for with ISRE or and of and cells with an used as In and for gene and the the R. for in cells with a expression was in the of cells with the The are the of The results are of We also role p65 have the of IRF-3. this we used an IRF-3 transactivation This involved cells with a a the transactivation domain of IRF-3 to the domain of this a gene the that expression of p65 in cells promoted IRF-3-mediated This was blocked by the but not the dominant negative presumably p65 in the from IRF-3 transactivation these indicate that p65 and IRF-3 in a complex the ISRE as a of TLR4 but not TLR3 signaling and that p65 transactivation by IRF-3. Finally, we to the that activation of the ISRE in our is via a interferon through activation of We therefore bearing a targeted of the β subunit of the receptor and their with the and ISRE and TLR3 and TLR4 TLR4 and TLR3 signaling to ISRE or NF-κB was in both and cells that the ISRE as an IRF-3 with our in and with the only the ISRE response was whereas TLR3 signaling It be that the of the although with the which the for the The results from this study indicate a between and activation of the TLR4 has an for NF-κB in this with p65 in the ISRE complex with IRF-3, whereas the ISRE response is We first NF-κB in the pathway to IRF-3 from TLR4 we that Mal or MyD88 with IRF-3 in ISRE Both of these NF-κB. NF-κB was by the of and the TLR4 response and most in cells from which in of TLR4 signaling to the ISRE but for TLR3. The for between NF-κB and IRF-3 has been in S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. 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This for a between IRF-3 and p65 the with the role of p65 to In of this we found that the of the NF-κB pathway in cells TLR4 signaling to the we detected both p65 and IRF-3 in the activation complex the between p65 and IRF-3 the ISRE be mediated by the IRF-3 cAMP-responsive element-binding protein-binding or IRF-3 of these not only to from the but for activation M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (691) Google Scholar, W. M. T. S. K. H. S. Fujita T. J. Biochem. PubMed Scopus Google Scholar). The of is for to the ISRE W. M. I. Fujita T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). has been to with both IRF-3 and p65 W. M. T. S. K. H. S. Fujita T. J. Biochem. PubMed Scopus Google Scholar, EMBO J. 1998; 17: PubMed Scopus Google Scholar, Williams A.J. A.S. Moore S. Y. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and to recruit the two transcription factors to M. Y. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. G. Maniatis T. E. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of p65 with IRF-3 have a and the or of IRF-3 to of the complex. It is possible that of NF-κB, as or also to this complex and ISRE and we are their because of the of we the role of p65 in this The mechanism by which LPS activates IRF-3 via TLR4 is still although have also between LPS and viral or poly(I-C) in this (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, M.J. Grandvaux N. tenOever B.R. D. Lin R. Hiscott J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, O'Connell R. Vaidya K. Cheng G. J. Immunol. 2003; PubMed Scopus Google Scholar). LPS appears to N-terminal phosphorylation of IRF-3, whereas the activation induced by poly(I-C) is and results in phosphorylation of the C (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, M.J. Grandvaux N. tenOever B.R. D. Lin R. Hiscott J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). It is possible that IRF-3 phosphorylated in its C does not p65 for to whereas phosphorylated IRF-3 two have two of the IKKϵ and TANK-binding kinase 1, as in virus- or IRF-3 activation and have that IKKϵ is an kinase for IRF-3 and (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). This to be from these have which is a for both IKKϵ and TANK-binding kinase was not to be of IRF-3 from also a role for in the IRF-3 activation by poly(I-C). to LPS has been that ISRE induced by LPS in was with which a role for IKKϵ in IRF-3 with the that LPS does not C-terminal phosphorylation J.C. K. J. Biol. Chem. 2003; Scholar). It been that TRIF is involved in IKKϵ activation 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 and is required for IRF-3 activation by as by the of LPS to induce IRF-3 dimerization in cells (12Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2527) Google Scholar, 13Hoebe K. Du X. Georgel P. Janssen E. Tabeta K. Kim S.O. Goode J. Lin P. Mann N. Mudd S. Crozat K. Sovath S. Han J. Beutler B. Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar). be to this and is that is additional in adaptor in this because LPS still activate the ISRE in the of Mal and MyD88 M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar, 21Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar), TRIF 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 known as TIR-containing adaptor molecule 2 (17Oshiumi H. Sasai M. Shida K. Fujita T. Matsumoto M. Seya T. J. Biol. Chem. September 30, 2003; (10.1074/jbc.M305820200)PubMed Google or TIR domain-containing adapter J. Biol. Chem. 2003; Scholar), may provide the signal to NF-κB in the of MyD88 and Mal required for ISRE activation by TLR4. The of TLR2 to activate IRF-3 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 is presumably to the that via MyD88 and Mal and does not TRIF M. Sato S. Hemmi H. Sanjo H. Uematsu S. Kaisho T. Hoshino K. Takeuchi O. Kobayashi M. Fujita T. Takeda K. Akira S. Nature. 2002; 420: 324-329Crossref PubMed Scopus (821) Google Scholar, 21Horng T. Barton G.M. Flavell R.A. Medzhitov R. Nature. 2002; 420: 329-333Crossref PubMed Scopus (689) Google Scholar). IRF-3 activation LPS stimulation has also been demonstrated in transactivation and in L. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. M. K. Suhara W. Fukuhara Y. Fujita T. 2002; PubMed Scopus Google Scholar). LPS these is still not we are is that p65 and IRF-3 or via as and is required for these Although the signaling induced by TLR4 and TLR3 therefore both to ISRE activation for IFNβ expression. the ISRE be activated by different transcription factor including and a complex and (22Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (273) Google Scholar, J.E. D.E. PubMed Scopus Google Scholar, J.E. Science. PubMed Scopus Google Scholar). may allow for of in different It is also possible that the an additional of genes, the of to LPS induces and of which in an via the receptor and activates the ISRE 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, O'Connell R. Vaidya K. Cheng G. J. Immunol. 2003; PubMed Scopus Google Scholar). of NF-κB IFNβ and with this our study to a because cells in the of the receptor to TLR4 and TLR3 our that cells are in the ISRE response to TLR4 but not TLR3 and that p65 be detected in the ISRE-binding complex and transactivation by IRF-3, we that LPS induces a complex for ISRE whereas the poly(I-C) response is only IRF-3. In our study the first that p65 is required for activation of the ISRE by TLR4 but not TLR3. The in signaling by TLR4 and TLR3 as well as TLRs to our understanding of the of the innate immune We Hiscott Fujita and for

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