Toll-like Receptor Signaling

The innate immune response in vertebrates is the first line of defense against invading microorganisms. The main players in innate immunity are phagocytes such as neutrophils, macrophages, and dendritic cells. These cells can discriminate between pathogens and self by utilizing signals from the Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; IL, interleukin; IL-1R, interleukin-1 receptor; TIR, Toll/IL-1R; LRR, leucine-rich repeat; IRAK, IL-1RI-associated protein kinase; TAK, transforming growth factor (TGF)-β-activated kinase; TAB1, TAK1-binding protein 1; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; IKK, IκB kinase kinase; MAP, mitogen-activated protein; DD, death domain; LPS, lipopolysaccharide; siRNA, small interfering RNA; PI, phosphatidylinositol; IFN, interferon; DN, dominant-negative form.1The abbreviations used are: TLR, Toll-like receptor; IL, interleukin; IL-1R, interleukin-1 receptor; TIR, Toll/IL-1R; LRR, leucine-rich repeat; IRAK, IL-1RI-associated protein kinase; TAK, transforming growth factor (TGF)-β-activated kinase; TAB1, TAK1-binding protein 1; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; IKK, IκB kinase kinase; MAP, mitogen-activated protein; DD, death domain; LPS, lipopolysaccharide; siRNA, small interfering RNA; PI, phosphatidylinositol; IFN, interferon; DN, dominant-negative form. (1Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3866) Google Scholar, 2Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2589) Google Scholar, 3Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (5994) Google Scholar, 4Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (469) Google Scholar). TLRs recognize conserved motifs predominantly found in microorganisms but not in vertebrates. Stimulation of TLRs causes an immediate defensive response, including the production of an array of antimicrobial peptides and cytokines. Accumulating evidence has shown that individual TLRs can activate overlapping as well as distinct signaling pathways, ultimately giving rise to distinct biological effects. Here, I will review our current understanding of the TLR signaling pathways. Toll was initially identified in insects as a receptor essential for dorsoventral polarity during embryogenesis. Subsequent studies revealed that it also plays an essential role in insects in the innate immune response against fungal infection (5Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2930) Google Scholar). Mammalian homologues of Toll were subsequently identified through expressed sequence tag and genomic sequence data base searches. To date, 10 members of the TLR family have been identified in mammals. TLRs are members of a larger superfamily of interleukin-1 receptors (IL-1Rs) that share significant homology in their cytoplasmic regions. In particular, TLRs and members of the IL-1R family share a conserved stretch of ∼200 amino acids in their cytoplasmic region known as the Toll/IL-1R (TIR) domain. The region of homology in the TIR motif is confined to three conserved boxes that contain amino acids crucial for signaling. In contrast, the extracellular regions are quite diverse. The extracellular portion of the TLRs contains a leucine-rich repeat (LRR) motif whereas that of the IL-1Rs contains three immunoglobulin domains. The LRR domains consist of varying numbers of repeats, each 24–29 amino acids in length, containing the motif XXLXLXX and other conserved leucines. It is thought that these LRR domains are directly involved in the recognition of a variety of pathogens. The major ligands recognized by individual TLRs are summarized in Fig. 1. The IL-1R and TLR family signal via shared downstream signaling molecules (6O'Neill L.A. Curr. Top. Microbiol. Immunol. 2002; 270: 47-61Crossref PubMed Scopus (248) Google Scholar). They include the adaptor molecule MyD88, IL-1RI-associated protein kinases (IRAKs), the transforming growth factor (TGF)-β-activated kinase (TAK1), TAK1-binding protein 1 (TAB1) and 2 (TAB2), and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The generally accepted scenario of the IL-1/TLR signaling pathway is shown in Fig. 2. Triggering of the IL-1R or TLR causes the adaptor protein MyD88 to be recruited to the receptor complex, which in turn promotes association with the IL-1R-associated kinases IRAK4 and IRAK1. During the formation of this complex, IRAK4 is activated, leading to the hyperphosphorylation of IRAK-1, which then induces the interaction of TRAF6 with the complex. The association of IRAK-4·IRAK-1·TRAF6 causes some conformational change in one or more of these factors, leading to their disengagement from the receptor complex. The IRAK-4·IRAK-1·TRAF6 complex then interacts at the membrane with another preformed complex consisting of TAK1, TAB1, and TAB2. This interaction induces phosphorylation of TAB2 and TAK1, which then translocate together with TRAF6 and TAB1 to the cytosol. TAK1 is subsequently activated in the cytoplasm, leading to the activation of IKK. Inactive IKK sequesters NF-κB in the cytoplasm, but activation leads to phosphorylation and degradation of IκB and consequent release of NF-κB. Activation of TAK1 also results in the activation of MAP kinases and c-Jun NH2-terminal kinase (JNK). I will discuss these molecules and their interactions in more detail below. MyD88 —MyD88 was originally isolated as a myeloid differentiation primary response gene that is rapidly induced upon IL-6-stimulated differentiation of M1 myeloleukemic cells into macrophages (7Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 1095-1097PubMed Google Scholar). MyD88 consists of an N-terminal death domain (DD) separated from its C-terminal TIR domain by a short linker sequence. MyD88 was subsequently cloned as an adapter molecule that functions to recruit IRAK to the IL-1 receptor complex following IL-1 stimulation (8Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar, 9Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (967) Google Scholar). The association between MyD88 and IRAK is mediated through a DD-DD interaction. MyD88 also forms homodimers through DD-DD and TIR-TIR domain interactions and exists as a dimer when recruited to the receptor complex. When the C-terminal TIR domain of MyD88 is expressed by itself, it acts as a dominant-negative inhibitor of TLR4 and IL-1R signaling by preventing IRAK association with the receptors. Thus, MyD88 functions as an adapter linking IL-1R/TLRs with downstream signaling molecules harboring DD. IRAK Family—Four different IRAKs (IRAK-1, IRAK-2, IRAK-M, and IRAK-4) have been identified in mammals (9Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (967) Google Scholar, 10Janssens S. Beyaert R. Mol. Cell. 2003; 11: 293-302Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 11Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (763) Google Scholar, 12Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 13Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (526) Google Scholar, 14Suzuki N. Suzuki S. Duncan G.S. Millar D.G. Wada T. Mirtsos C. Takada H. Wakeham A. Itie A. Li S. Penninger J.M. Wesche H. Ohashi P.S. Mak T.W. Yeh W.C. Nature. 2002; 416: 750-756Crossref PubMed Scopus (647) Google Scholar). The expression patterns of these members differ; IRAK-1 and IRAK-4 are expressed in all tissues, IRAK-2 has a narrower cellular distribution, and IRAK-M expression is mainly restricted to cells of a myeloid origin. All IRAKs contain an N-terminal DD and a central Ser/Thr kinase domain (KD). Although the kinase activity of IRAK-1 increases strongly following IL-1 stimulation, IRAK-1 kinase activity is not required for its signaling function, because overexpression of a kinase-defective mutant of IRAK-1 is observed to strongly induce NF-κB activation in cells otherwise deficient for IRAK-1. Upon stimulation, IRAK-1 is recruited to the receptor through a homophilic interaction with the DD of MyD88. MyD88 also binds to IRAK-4 and thereby facilitates IRAK-4 phosphorylation of critical residue(s) in the kinase activation loop of IRAK-1, triggering IRAK-1's own kinase activity. Once activated, IRAK-1 likely autophosphorylates residues in its N terminus. TRAF6 is also recruited to the receptor complex via interaction with IRAK-1. Three TRAF6 binding motifs (Pro-X-Glu-X-X-aromatic/acidic residue) are found in IRAK-1, as well as one in IRAK-M and two in IRAK-2 (15Ye H. Arron J.R. Lamothe B. Cirilli M. Kobayashi T. Shevde N.K. Segal D. Dzivenu O.K. Vologodskaia M. Yim M. Du K. Singh S. Pike J.W. Darnay B.G. Choi Y. Wu H. Nature. 2002; 418: 443-447Crossref PubMed Scopus (519) Google Scholar). However, in contrast to IRAK-1 and IRAK-4, IRAK-2 and IRAK-M do not possess any detectable kinase activity. This is presumably because they have an asparagine and serine residue, respectively, in their kinase domains in place of an aspartate residue shown to be critical for the kinase activity of other IRAKs. IRAK-1-deficient mice and cell lines showed diminished cytokine production in response to IL-1 and LPS; nevertheless some response remained, suggesting that IRAK-2 or IRAK-M might compensate to some extent for the lack of IRAK-1 (16Kanakaraj P. Schafer P.H. Cavender D.E. Wu Y. Ngo K. Grealish P.F. Wadsworth S.A. Peterson P.A. Siekierka J.J. Harris C.A. Fung-Leung W.P. J. Exp. Med. 1998; 187: 2073-2079Crossref PubMed Scopus (176) Google Scholar, 17Thomas J.A. Allen J.L. Tsen M. Dubnicoff T. Danao J. Liao X.C. Cao Z. Wasserman S.A. J. Immunol. 1999; 163: 978-984PubMed Google Scholar). IRAK-4-deficient mice have been generated and showed almost complete unresponsiveness to IL-1, LPS, or other bacterial components, demonstrating that IRAK-4 is a key player in the IL-1R/TLR signaling (14Suzuki N. Suzuki S. Duncan G.S. Millar D.G. Wada T. Mirtsos C. Takada H. Wakeham A. Itie A. Li S. Penninger J.M. Wesche H. Ohashi P.S. Mak T.W. Yeh W.C. Nature. 2002; 416: 750-756Crossref PubMed Scopus (647) Google Scholar). Recently, patients with inherited IRAK-4 deficiency have been identified. These patients failed to respond to IL-1, IL-18, or the stimulation of at least five TLRs (TLR2, TLR3, TLR4, TLR5, TLR9) (18Picard C. Puel A. Bonnet M. Ku C.L. Bustamante J. Yang K. Soudais C. Dupuis S. Feinberg J. Fieschi C. Elbim C. Hitchcock R. Lammas D. Davies G. Al-Ghonaium A. Al-Rayes H. Al-Jumaah S. Al-Hajjar S. Al-Mohsen I.Z. Frayha H.H. Rucker R. Hawn T.R. Aderem A. Tufenkeji H. Haraguchi S. Day N.K. Good R.A. Gougerot-Pocidalo M.A. Ozinsky A. Casanova J.L. Science. 2003; 299: 2076-2079Crossref PubMed Scopus (760) Google Scholar). Data with IRAK-M knock-out mice have revealed that IRAK-M serves as a negative regulator of IL-1R/TLR signaling (19Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar). IRAK-M-deficient macrophages produced significantly higher cytokine levels in response to a variety of IL-1R/TLR ligands. Furthermore, IRAK-M-deficient macrophages did not become hyporesponsive following repeated exposure to LPS, suggesting that IRAK-M plays an essential role in endotoxin tolerance. TRAF6 —TRAFs constitute a family of evolutionarily conserved adaptor proteins. To date, six members of the TRAF family have been identified in mammals (20Chung J.Y. Park Y.C. Ye H. Wu H. J. Cell Sci. 2002; 115: 679-688Crossref PubMed Google Scholar). The TRAF proteins are characterized by the presence of a coiled-coil TRAF-N domain and a conserved C-terminal TRAF domain. The TRAF-C domain mediates self-association and interactions with upstream receptors and signaling proteins. The N-terminal portion of most TRAF proteins also contains a RING finger/zinc finger region essential for downstream signaling events. TRAF6 acts as the signaling mediator for both the TNF receptor superfamily and the IL-1R/TLR superfamily. TRAF6 directly interacts with CD40 and TRANCE-R, which are members of the TNF receptor superfamily. TRAF6 is indirectly coupled to IL-1/TlR receptor activation and is recruited into the signaling complex via its association with IRAK (21Ishida T. Mizushima S. Azuma S. Kobayashi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 22Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1103) Google Scholar). TRAF6 Downstream Signaling Pathway—The activation of both NF-κB and AP-1 by TRAF6 involves a MAP 3-kinase known as TAK1 and two adaptor proteins, TAB1 and TAB2. TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family (23Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1166) Google Scholar). Studies using small interfering RNA (siRNA) to inhibit TAK1 expression show that TAK1 is essential for both IL-1- and TNF-α-induced NF-κB activation. Two TAK1-binding proteins, TAB1 and TAB2, have been identified (24Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 25Jing Z. Ninomiya-Tsuji J. Qian Y. Matsumoto K. Li X. Mol. Cell. Biol. 2002; 22: 7158-7167Crossref PubMed Scopus (235) Google Scholar). When ectopically co-expressed, TAB1 augments the kinase activity of TAK1, indicating that TAB1 functions as an activator of TAK1. TAB2 is associated with the cell membrane in unstimulated conditions, but upon stimulation it translocates to the cytosol, where it functions as an adaptor linking TAK1 to TRAF6, thereby facilitating TAK1 activation. It has been shown recently that ubiquitination plays an important role in TAK1 activation (26Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar, 27Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J.I. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1596) Google Scholar). IKK activation by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A as well as the TAK1·kinase complex. TRAF6 can interact through its RING finger domain with Ubc13 in the Ubc13·Uev1A complex. This Ubc·TRAF6 complex catalyzes the formation of a Lys-63-linked polyubiquitin chain, which triggers TAK1 activation through a unique proteasome-independent mechanism. Tollip—Tollip (Toll-interacting protein) was originally cloned as a protein interacting with the IL-1 receptor accessory protein (28Burns K. Clatworthy J. Martin L. C. B. A. K. J. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus Google Scholar). also with and In forms a complex with members of the IRAK thereby preventing NF-κB activation. Upon activation are recruited to the in the of IRAK-1 and its from the the IRAK which to the of from IRAK-1 and its ubiquitination and is to to immune cells in a and to the of cell signaling during and infection G. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is a protein that was cloned through a against is conserved different least two members of the family have been identified in mammals. 1 and its from share amino and 1 and 2 share Studies using to inhibit 1 expression have shown that 1 is required for NF-κB activation and gene expression Z. H. J. Li X. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). 2 has also been shown to be involved in IL-1R/TLR signaling D.A. E. M.A. J. Immunol. 2002; PubMed Scopus Google Scholar). IL-1 stimulation, IRAK-1 and 2 to expression of a 2 was shown to inhibit IL-1- and but not activation of the 1 and 2 are to a role in facilitating the release of IRAK from the are activated in IL-1R/TLR signaling. The activation of 3-kinase through an interaction of the homology domain of the 3-kinase with a domain in the cell receptor containing the motif this 3-kinase binding motif is in a of in TLRs and 6 but not in TLRs or has been shown to directly to the of the 3-kinase L. N. L. M. N. Ulevitch R.J. Nat. Immunol. 2000; PubMed Scopus Google Scholar). 3-kinase binding acids is also found in the of MyD88. stimulation has been shown to in phosphorylation of MyD88 and formation of a complex M. K. M. K. M. J. Immunol. 2003; PubMed Scopus Google Scholar). However, the role of 3-kinase activation in TLR signaling to be mice have been generated and found to be in their to IL-1 and the T. Takeda K. Matsumoto M. H. M. K. S. Immunity. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The response to was shown to also be T. T. Takeda K. S. Immunity. 1999; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, macrophages were shown to be to other including and demonstrating the essential role of MyD88 in the response to all TLR Takeda K. K. T. S. Immunol. 2000; PubMed Scopus Google Scholar, A. K. T. K. M. P.F. S. J. Immunol. 2000; PubMed Scopus Google Scholar, H. Takeuchi K. S. H. J. Exp. Med. 2000; PubMed Scopus Google Scholar, Ozinsky A. Hawn T.R. S. Aderem A. Nature. 2001; PubMed Scopus Google Scholar, H. Kaisho T. Takeuchi S. H. K. T. H. Takeda K. S. Nat. Immunol. 2002; PubMed Scopus Google Scholar). Although MyD88 is to be involved in signaling L. Medzhitov R. Flavell R.A. Nature. 2001; PubMed Scopus Google not to the pathway to any significant because the response to was not in is unique the TLRs in that it a conserved residue in its cytoplasmic which in other TLRs has been shown to be essential for signaling. is an residue in this in TLR3, conserved between and It is with other TLR stimulation induces the production of of I and but of such as and suggesting that not the pathway for signaling. Although MyD88 plays a critical role in TLR is a in the signaling by and by other bacterial Activation of NF-κB and MAP kinases by is in or However, activation of MAP kinases and NF-κB in macrophages, it is with that in This that the response to both and pathways, each of which leads to the activation of MAP kinases and NF-κB. studies showed that including a are induced in macrophages in response to Subsequent studies have revealed that the pathway the factor T. Takeuchi T. Inoue J. P.F. S. K. S. J. Immunol. 2001; PubMed Scopus Google Scholar). or phosphorylation and translocates from the to the in the activation of I with mice and mice showed that in response to is to K. S. J. Nat. Immunol. 2002; PubMed Scopus Google Scholar, S.A. R. H. Wu G. R. G. Immunity. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Kaisho T. T. Takeuchi S. Immunol. 2002; PubMed Scopus (235) Google Scholar). TIR protein also was identified by data base T. Medzhitov R. Nat. Immunol. 2001; 2: PubMed Scopus Google Scholar, K.A. C.A. G. E. A. P. D. D.E. J.E. L.A. Nature. 2001; PubMed Scopus Google Scholar). was shown to with but not with other of a dominant-negative of was shown to inhibit NF-κB activation and dendritic cell of using a also of by it was initially that might in the However, studies with mice subsequently revealed that is not involved in the pathway and that it acts as an adaptor in the signaling downstream of and TLR4 M. S. H. H. S. Kaisho T. K. Takeuchi Kobayashi M. T. Takeda K. S. Nature. 2002; PubMed Scopus Google Scholar, T. Flavell R.A. Medzhitov R. Nature. 2002; PubMed Scopus Google Scholar). another adaptor molecule or has been identified M. S. K. Takeuchi K. Takeda K. S. J. Immunol. 2002; PubMed Scopus Google Scholar, H. Matsumoto M. K. T. T. Nat. Immunol. 2003; PubMed Scopus Google Scholar). of but not MyD88 or the dominant-negative of but not or the response to indicating the role of in signaling. also was observed to signaling activated by other TLR family which a role for in the signaling of mice showed the essential role of in the of and TLR4 signaling M. S. H. K. Kaisho T. H. Takeuchi M. M. Takeda K. S. Science. 2003; PubMed Scopus Google Scholar). mice the response to in of cytokine production that was to be mediated by the signaling mainly the pathway because induces activation of NF-κB and MAP kinases in macrophages almost to the extent as in of a dominant-negative of MyD88 or not the response to To date, interaction between MyD88 or and upon stimulation has been Recently, a that activation of NF-κB and MAP kinases involves an pathway the signaling Z. M. H. Li X. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, is a likely adaptor to recruit the complex to In three TRAF6 binding motifs in its terminus. data base of TIR has revealed two adaptor These that different adaptor molecules are involved in signaling and that their be the for of the to different TLRs Although the signaling downstream of the different TLRs were initially thought to be it is that individual TLRs activate different of signaling and distinct biological effects. This of signaling is most likely the of the of adaptor the of mice these adaptor molecules to this complete understanding of the signaling by each TLR has in the of against and all members in our for and and E. for