Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity and disease resistance. The adaptor protein Tollip was identified initially as an intermediate in interleukin (IL)-1 signaling. Here we report that Tollip also associates directly with TLR2 and TLR4 and plays an inhibitory role in TLR-mediated cell activation. Inhibition by Tollip is mediated through its ability to potently suppress the activity of IL-1 receptor-associated kinase (IRAK) after TLR activation. In addition, we show for the first time that Tollip is a bona fidesubstrate for IRAK and is phosphorylated by IRAK upon stimulation with lipopolysaccharide or IL-1. Negative regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory mediators during inflammation and infection. Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity and disease resistance. The adaptor protein Tollip was identified initially as an intermediate in interleukin (IL)-1 signaling. Here we report that Tollip also associates directly with TLR2 and TLR4 and plays an inhibitory role in TLR-mediated cell activation. Inhibition by Tollip is mediated through its ability to potently suppress the activity of IL-1 receptor-associated kinase (IRAK) after TLR activation. In addition, we show for the first time that Tollip is a bona fidesubstrate for IRAK and is phosphorylated by IRAK upon stimulation with lipopolysaccharide or IL-1. Negative regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory mediators during inflammation and infection. Toll-like receptors (TLRs) 1TLR(s)Toll-like receptor(s)AP-1activator protein-1GSTglutathioneS-transferaseHAhemagglutininIL-1Rinterleukin-1 receptorIL-18Rinterleukin-18 receptorIRAKIL-1R-associated kinaseLPSlipopolysaccharideLucluciferaseMalMyD88-adaptor-likeMyD88myeloid differentiation protein 88NF-κBnuclear factor-κBPGNpeptidoglycanTIRToll/IL-1R homology domainTIRAPTIR domain-containing adaptor proteinTollipToll-interacting proteinTRAF6tumor necrosis factor receptor-associated factor 6UBAubiquitin-associated represent a family of phylogenetically conserved proteins that have been found in insects, plants, and mammals (1O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar, 2Anderson K.V. Curr. Opin. Immunol. 2000; 12: 13-19Crossref PubMed Scopus (527) Google Scholar, 3Zhang G. Ghosh S. J. Clin. Invest. 2001; 107: 13-19Crossref PubMed Scopus (620) Google Scholar). In addition to playing a critical role in the dorso-ventral axis formation in insects, TLRs are also involved in innate immunity and disease resistance (2Anderson K.V. Curr. Opin. Immunol. 2000; 12: 13-19Crossref PubMed Scopus (527) Google Scholar, 3Zhang G. Ghosh S. J. Clin. Invest. 2001; 107: 13-19Crossref PubMed Scopus (620) Google Scholar, 4Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3958) Google Scholar). In mammals, 10 members of the TLR family have been identified which are expressed by host immune cells most likely to come into direct contact with pathogens from the environment, such as dendritic cells, peripheral phagocytes, and mucosal epithelia (5Muzio M. Polentarutti N. Bosisio D. Prahladan M.K. Mantovani A. J. Leukocyte Biol. 2000; 67: 450-456Crossref PubMed Scopus (155) Google Scholar). Recent studies have revealed that a striking feature of TLRs is their ability to discriminate among different classes of pathogens (3Zhang G. Ghosh S. J. Clin. Invest. 2001; 107: 13-19Crossref PubMed Scopus (620) Google Scholar, 4Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3958) Google Scholar). For example, TLR4 detects lipopolysaccharide (LPS) from Gram-negative bacteria, whereas TLR2 recognizes peptidoglycan (PGN), lipoproteins, and zymosan from Gram-positive bacteria and yeast. TLRs alone or in combination are believed to mediate host immune responses to a large array of pathogens (6Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1693) Google Scholar). In addition to detecting exogenous stimuli, TLRs are also able to trigger cell activation in response to endogenous signals, such as heat shock protein 60 (7), fibronectin (8Okamura Y. Watari M. Jerud E.S. Young D.W. Ishizaka S.T. Rose J. Chow J.C. Strauss III, J.F. J. Biol. Chem. 2001; 276: 10229-10233Abstract Full Text Full Text PDF PubMed Scopus (980) Google Scholar), fibrinogen (9Smiley S.T. King J.A. Hancock W.W. J. Immunol. 2001; 167: 2887-2894Crossref PubMed Scopus (791) Google Scholar), and unknown factors from the injured myocardium (10Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (569) Google Scholar) and necrotic cells (11Li M. Carpio D.F. Zheng Y. Bruzzo P. Singh V. Ouaaz F. Medzhitov R.M. Beg A.A. J. Immunol. 2001; 166: 7128-7135Crossref PubMed Scopus (395) Google Scholar). Activation of TLRs by endogenous ligands presumably provides immune surveillance at sites of inflammation, but sustained activation may lead to development of chronic inflammatory disorders and autoimmune diseases. Toll-like receptor(s) activator protein-1 glutathioneS-transferase hemagglutinin interleukin-1 receptor interleukin-18 receptor IL-1R-associated kinase lipopolysaccharide luciferase MyD88-adaptor-like myeloid differentiation protein 88 nuclear factor-κB peptidoglycan Toll/IL-1R homology domain TIR domain-containing adaptor protein Toll-interacting protein tumor necrosis factor receptor-associated factor 6 ubiquitin-associated All mammalian TLRs are type I transmembrane proteins consisting of multiple copies of leucine-rich repeats in the extracellular domain and a conserved Toll/interleukin-1 receptor (IL-1R) homology (TIR) domain in the cytoplasmic tail (1O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar, 3Zhang G. Ghosh S. J. Clin. Invest. 2001; 107: 13-19Crossref PubMed Scopus (620) Google Scholar). Because TIR domains of TLRs, IL-1R, and IL-18R are highly homologous, it is believed that these receptors activate similar signaling pathways when stimulated with their cognate ligands (1O'Neill L.A. Greene C. J. Leukocyte Biol. 1998; 63: 650-657Crossref PubMed Scopus (499) Google Scholar). Consistent with this hypothesis, the downstream signaling components, myeloid differentiation protein 88 (MyD88), IL-1R-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6) are all utilized by these three groups of receptors to activate both nuclear factor (NF)-κB and mitogen-activated protein kinase signaling cascades (12Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L., Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 13Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 14Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway C.A., Jr. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar, 15Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar, 16Wesche H. Henzel W.J. Shillinglaw W., Li, S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). MyD88 functions as a crucial adaptor that links the receptors to IRAK after activation. IRAK in turn is phosphorylated, dissociates from the receptor, and associates with TRAF6 to signal activation of either NF-κB or mitogen-activated protein kinases. However, genetic studies revealed that neither MyD88 (17Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1736) Google Scholar), IRAK (18Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (234) Google Scholar), nor TRAF6 (19Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C., Ho, A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1089) Google Scholar) is absolutely essential for cell activation in response to stimulation with LPS, implying the existence of alternative signaling pathways. The recent description of TIR domain-containing adaptor protein (TIRAP) (20Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (832) Google Scholar), also known as Mal (MyD88 adaptor-like) (21Fitzgerald 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), probably explains the residual signaling observed in the absence of MyD88. Interestingly TIRAP/Mal also appears to be involved in signaling through TLR4 but not IL-1, thereby demonstrating a unique specificity in the adaptor molecules that participate in signaling. In addition to MyD88, another adaptor protein named Toll-interacting protein (Tollip) was recently found to associate with the cytoplasmic TIR domain of IL-1Rs after IL-1 stimulation (22Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar). Tollip forms a complex with IRAK in resting cells and inhibits IL-1-induced signaling by blocking IRAK phosphorylation. Because of the significant homology in the intracellular portion of TLRs, IL-1R, and IL-18R, we hypothesized that Tollip might also inhibit TLR-mediated signaling by interacting with TLRs through the TIR domain. Here we report that Tollip associates directly with TLR2 and TLR4 and inhibits TLR-mediated cellular responses by suppressing phosphorylation and kinase activity of IRAK. Furthermore, we find that Tollip is phosphorylated by activated IRAK, making it the first direct substrate for IRAK to be characterized. Human embryonic kidney epithelial 293 cells stably expressing the NF-κB-dependent luciferase reporter pBIIX-Luc (293-Luc) were developed as described previously (23Kopp E. Medzhitov R. Carothers J. Xiao C. Douglas I. Janeway C.A. Ghosh S. Genes Dev. 1999; 13: 2059-2071Crossref PubMed Scopus (276) Google Scholar). Mouse macrophage RAW264.7 cells were obtained from ATCC (Manassas, VA). Both cell lines were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 7% heat-inactivated fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. LPS from Escherichia coli 0111:B4, PGN fromStaphylococcus aureus, anti-FLAG M2 antibody, and M2 agarose beads were purchased from Sigma. Purified polyclonal rabbit antibodies against IRAK and hemagglutinin (HA) epitope were from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A-Sepharose and glutathione-conjugated agarose beads were from Amersham Biosciences, Inc. Mammalian expression vectors containing N-terminal FLAG-tagged human TLR2 and TLR4 that were made by inserting PCR-generated cDNA fragments lacking the signal sequence into pFLAG-CMV-1 (Sigma) were kindly provided by R. Medzhitov (Yale Medical School). The intracellular domains of TLR2 and TLR4 were amplified by PCR and cloned into pGBKT7 (Clontech, Palo Alto, CA). Plasmids for human CD14, FLAG-tagged human Tollip, HA-tagged Tollip, and its deletion mutants were constructed in-frame by inserting PCR-generated cDNA fragments into pCDNA3.1 (Invitrogen). FLAG-IRAK and HA-IRAK were generated by inserting cDNA into pCDNA3.1. Kinase-inactive FLAG-IRAK (K239A) and HA-IRAK (K239A) were made using the Quick from CA). All and epitope were generated by into at the of cDNA were by direct The expression containing human was kindly provided by K. Medical cells were into and using 6 with of or with of a containing a resistance were in Dulbecco's modified Eagle's medium with μg/ml were and for the expression of TLRs by and using anti-FLAG cell lines were and maintained in Dulbecco's modified Eagle's medium for RAW264.7 cells were at a of in were in using 6 with of pBIIX-Luc or and the of The of was by with the pCDNA3.1. cells were either or stimulated with 100 LPS or 10 μg/ml PGN for 6 were and for luciferase activity The were to protein of using a protein from For of or cells were into and the with the using after cells were in of containing a portion of the was for and FLAG-tagged proteins were from the for at with of of anti-FLAG M2 with the beads were in were by to and with the The were by Biosciences, and its N-terminal and mutants were made by human Tollip cDNA fragments in-frame into Biosciences, proteins were with for at and with Purified proteins were obtained by from the beads with 10 by in proteins were generated with to the In were as described previously F. L.A. J. Ghosh S. Science. 2000; PubMed Scopus (620) Google Scholar). of was for at with of or and of beads in of The beads were three with in and by The proteins were by For the of endogenous IRAK or cells were into and the with of of using after cells were either or stimulated with μg/ml LPS for 10 or and in of containing 2 and a of Cell were at for with of and of of protein A-Sepharose IRAK was as described previously using developed for detecting F. L.A. J. Ghosh S. Science. 2000; PubMed Scopus (620) Google Scholar). with and with kinase 2 10 the beads were in of kinase containing 10 of Biosciences, The were to at for and with of The beads were were by and by For phosphorylation of Tollip by IRAK, IRAK was first and immune were with and kinase in of kinase with 10 of and 2 of or its proteins were with of by and by Tollip with TLRs the TIR we developed cell lines and that stably human CD14, as as FLAG-tagged human TLR2 or and revealed significant of TLR expression the cell not Furthermore, expression of the TLRs the 293 cells highly to which not activate these cells not Tollip and TLRs were by these cell lines with expressing which is a that to TLR4 and is for signaling R. S. H. Y. K. K. M. J. Exp. Med. 1999; PubMed Scopus Google Scholar). The FLAG-tagged TLRs were using anti-FLAG by with with TLR2 of Tollip and TLR4 was also but was in the of and 6 of the that in the cytoplasmic tail of TLR4 by may be for to Tollip directly with the TIR domain of TLRs, in were intracellular of TLR2 and TLR4 were by in and with either or in both TLR2 and TLR4 to but not demonstrating that Tollip associates directly with the TIR domain of the of Tollip for with the TLRs, we the of TLR2 and TLR4 with mutants of Tollip as in A. TLR4 to either Tollip or mutants with an Tollip and Tollip 2 and of the whereas Tollip with was found TLR4 and Tollip or Tollip that the domain in the of Tollip and and 6 of were observed with TLR2 not of Tollip to is also mediated through a similar (22Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google phosphorylation and kinase activity of IRAK. of the of phosphorylated IRAK to the TLR4 receptor cells were in and with of of the and were as described in the to A. of kinase activity of IRAK by and cells were in and with the of after cells were either or stimulated with μg/ml LPS for 10 or in cell were using and protein A-Sepharose were and an of the was for to The of the proteins was in kinase at for by and phosphorylated IRAK was by is phosphorylated by activated IRAK. phosphorylation of Tollip by IRAK. cells were in and with of with or of after cells were either or stimulated with μg/ml LPS for Cell were with and protein A-Sepharose and kinase were as described in the to B. The were also with and antibodies to the expression of Tollip and phosphorylation of endogenous IRAK upon phosphorylation of by IRAK. cells were in and with were or stimulated with μg/ml LPS for and were with and protein A-Sepharose were in the of 2 of or proteins were with by and by of protein are the Tollip is phosphorylated by IRAK at the cells were in and with cells were stimulated with μg/ml LPS for and were with and protein A-Sepharose were in the of 2 of or its The of proteins in the kinase TLRs, IL-1R, and IL-18R are all known to either or upon stimulation with their cognate it is that downstream proteins might also to to be Tollip forms Tollip and it in found that Tollip to but not to alone that Tollip is of the domains involved in Tollip cells were with FLAG-tagged Tollip and the HA-tagged Tollip deletion mutants described in 2 and the the proteins was by All Tollip mutants with Tollip for the containing the domain and 2 of Tollip is mediated by both the and of the protein and may in formation of a protein both Tollip and MyD88 as to or TLRs to downstream neither in nor in mammalian revealed a complex these molecules not that MyD88 and Tollip in protein which is with that both molecules are to the after stimulation (22Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar). the role of Tollip in TLR-mediated we macrophage RAW264.7 cells with of Tollip with either NF-κB or luciferase reporter activity was after stimulation of the cells with Both NF-κB and were activated in response to LPS and PGN not and both were by Tollip, that Tollip is a of TLR2 and TLR4 signaling. of Tollip also NF-κB activation in and cells after stimulation with LPS or PGN not the by which Tollip inhibits TLR-mediated we its the of IRAK to the TLR and cells were with Tollip with either type or (K239A) IRAK. Consistent with of type IRAK in significant of the of phosphorylation was in the of Tollip K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google and and 10 of from the of TLR4 with anti-FLAG revealed that Tollip not to the of IRAK (K239A) to the TLR4 receptor complex in the or absence of and and 6 of the In of phosphorylated IRAK was by Tollip of expression and of the were observed in cells not In kinase were also to that of IRAK by Tollip was the of IRAK kinase and cells were with Tollip stimulated with LPS, and IRAK was and for its kinase IRAK was phosphorylated after LPS stimulation 2 and of the but this was in the of Tollip and of the a by which Tollip might TLR by blocking the of IRAK, which is for the of IRAK from the receptor complex X. M. C. K. Cao Z. Mol. Cell. Biol. 1999; PubMed Scopus Google X. M. Z. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). IRAK is a kinase with a domain Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar). stimulation with either IL-1 or IRAK is activated and phosphorylated 13Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google and 2 of both and However, the of IRAK In addition, recent studies have that the kinase activity of IRAK is for its role as an intermediate in IL-1 signaling J. Martin 1999; PubMed Scopus Google Scholar, B. Ray K. K. Volpe F. J. 1999; PubMed Scopus Google Scholar). However, we were to find that after LPS activation of IRAK is by a phosphorylation of Tollip in 6 and 6 of the and cells not of Tollip not in resting cells activated IRAK 6 of the this the and cells were either or stimulated with IRAK was from these cells and with in in kinase found that upon stimulation with LPS, with the phosphorylation of IRAK, 6 of the but not alone 6 of the was were also observed in cells stimulated with IL-1 not these that in addition to IRAK also of Tollip by activated IRAK may the of IRAK from Tollip, thereby IRAK to the inhibitory of Tollip and downstream signaling the domain of Tollip which is phosphorylated by IRAK, we N-terminal and mutants of Tollip with However, the of Tollip, which the is when expressed in kinase were with and Tollip domains with type in 6 the but not N-terminal of Tollip is phosphorylated by IRAK. the is also for the of Tollip with TLRs and IRAK K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar). However, we the that the domain of Tollip might also be phosphorylated by IRAK upon activation the of phosphorylation at the is that for type Tollip of the In this we that the adaptor protein Tollip TLR signaling pathways. initially as a protein involved in IL-1 we show that Tollip associate with TLR2 and TLR4 and an inhibitory role in TLR-mediated cell activation. Consistent with during the of the Y. E. Thomas L. O. M. J. Immunol. 2001; 167: PubMed Scopus Google Scholar) that Tollip with TLR2 and and of Tollip inhibits and NF-κB activation in human striking feature of Tollip is its ability to suppress the kinase activity of IRAK. lines of that the by which Tollip inhibits TLR signaling is by with IRAK, it from phosphorylated and activated the TLR receptor In studies using cells that phosphorylation of IRAK is for it to from the receptor and to downstream molecules to NF-κB activation X. M. C. K. Cao Z. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, X. M. Z. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). The ability of Tollip to directly with both the and kinase domains of IRAK may the inhibitory role of Tollip and not IRAK and dissociates from the studies that after stimulation IRAK also The of this phosphorylation is it is that phosphorylation of Tollip may its from IRAK and by is with the that Tollip a highly conserved or of to domain at the domains in proteins have been to to T. E.S. M.A. Chen J. Nat. Biol. 1998; PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar). the the domain of Tollip to IRAK resting and also associates with the TLRs and upon stimulation (22Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (453) Google Scholar). The of Tollip also be phosphorylated by IRAK 6 activated the receptor, IRAK Tollip, which may lead to the of Tollip from IRAK and the receptor the domain Tollip may lead to its by a The of Tollip signaling to by activated IRAK to to downstream Because IRAK also and after activation Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), it is to that phosphorylated Tollip the of IRAK by studies be to the of Tollip phosphorylation by IRAK. a of functions of Tollip the and of lacking Tollip, it is that the role of may be to immune cells in a in the absence of and the of cell signaling during inflammation and infection. is that this conserved inhibitory the host by the production of proinflammatory mediators and to Tollip expression may in both chronic and inflammatory such as inflammatory disease and and for the and members of the Ghosh for for of the are also to the the