Toll-like Receptors and Type I Interferons

Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and trigger innate immune responses. A subset of TLRs recognizes viral components and induces antiviral responses. Whereas TLR4 recognizes viral components at the cell surface, TLR3, TLR7, TLR8, and TLR9 recognize viral nucleic acids on endosomal membrane. After ligand recognition, these members activate their intrinsic signaling pathways and induce type I interferon. In this review, we discuss the recent findings of the viral recognition by TLRs and their signaling pathways. Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and trigger innate immune responses. A subset of TLRs recognizes viral components and induces antiviral responses. Whereas TLR4 recognizes viral components at the cell surface, TLR3, TLR7, TLR8, and TLR9 recognize viral nucleic acids on endosomal membrane. After ligand recognition, these members activate their intrinsic signaling pathways and induce type I interferon. In this review, we discuss the recent findings of the viral recognition by TLRs and their signaling pathways. Type I interferon (IFN), 2The abbreviations used are: IFN, interferon; IRF, IFN regulatory factor; RLH, RIG-I-like helicase; TLR, Toll-like receptor; ds, double-stranded; ss, single-stranded; TIR, Toll/IL-1 receptor; IL, interleukin; IRAK, IL-1R-associated kinase; TRAF, tumor necrosis factor receptor-associated factor; TAK, transforming growth factor-β-activated kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; cDC, conventional dendritic cell; pDC, plasmacytoid dendritic cell; RIP, receptor-interacting protein; LPS, lipopolysaccharide.2The abbreviations used are: IFN, interferon; IRF, IFN regulatory factor; RLH, RIG-I-like helicase; TLR, Toll-like receptor; ds, double-stranded; ss, single-stranded; TIR, Toll/IL-1 receptor; IL, interleukin; IRAK, IL-1R-associated kinase; TRAF, tumor necrosis factor receptor-associated factor; TAK, transforming growth factor-β-activated kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; cDC, conventional dendritic cell; pDC, plasmacytoid dendritic cell; RIP, receptor-interacting protein; LPS, lipopolysaccharide. which was first discovered by Isaacs and Lindenmann in 1957 (1Isaacs A. Lindenmann J. Proc. R. Soc. Lond. B Biol. Sci. 1957; 147: 258-267Crossref PubMed Google Scholar), derives its name from a function to “interfere” in viral replication. Type I IFNs are encoded by more than 13 IFN-α subfamily genes, a single IFN-β gene, and others, such as IFN-ω, -ϵ, and -κ. Type I IFNs are the key cytokines that mediate antiviral responses. Secreted IFNs bind and activate the type I IFN receptor (a heterodimer of IFNAR1 and IFNAR2) in an autocrine and paracrine manner. This binding leads to the activation of IFN-stimulated gene factor 3 (ISGF3; a heterotrimer of signal transducer and activator of transcription 1 (STAT1), STAT2, and IFN regulatory factor 9 (IRF9)), which translocates to the nucleus and induces the transcription of hundreds of effector molecules, called IFN-inducible genes (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). These effector molecules directly influence protein synthesis, cell growth, and survival, in the process of establishing an antiviral state. In addition to the induction of effector proteins, type I IFNs also induce maturation of dendritic cells, enhance antibody responses in B cells, mediate induction of CD8+ T cell responses, and recruit lymphocytes and monocytes to inflamed sites by inducing chemokines. Thus, type I IFNs mediate both innate immune responses and the subsequent development of adaptive immunity to viruses (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). The induction of type I IFNs is triggered by pattern recognition receptors. These receptors recognize conserved molecular patterns characteristic of microorganisms, which are not generated by the host and are essential for microbial survival. The pattern recognition receptors involved in the induction of type I IFNs are divided into two categories: Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs). RLHs are expressed ubiquitously and are localized in the cytosol, where they recognize dsRNA produced upon viral infection. On the other hand, TLRs are located on cell surfaces or in endosomes, where they detect viral components or viral nucleic acid (3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1408) Google Scholar). Here we review the induction of type I IFNs by TLRs, especially focusing on their signaling pathways. TLRs, a family of evolutionarily conserved pathogen recognition receptors, play a pivotal role in innate immunity. To date, the TLR family consists of 13 mammalian members. The cytoplasmic portions of TLRs show high similarity to that of the interleukin-1 receptor (IL-1R) family and are now called the Toll/IL-1 receptor (TIR) domain. A TIR domain is required to initiate intracellular signaling. The extracellular regions of TLRs and IL-1R are markedly different. Whereas IL-1R possesses an Ig-like domain, TLRs contain leucine-rich repeats in their extracellular domains. TLRs are pattern recognition receptors that sense a wide range of microorganisms, such as bacteria, fungi, protozoa, and viruses. Each TLR has its own intrinsic signaling pathway and induces specific biological responses against microorganisms such as dendritic cell maturation, cytokine production, and the development of adaptive immunity (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). The activation of TLR signaling pathways originates from the cytoplasmic TIR domains. Each TLR mediates distinctive responses in association with a different combination of four TIR domain-containing adapters (MyD88, TIRAP/MAL, TRIF, and TRAM) through the homophilic interaction of TIR domains (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). The association of TLRs with MyD88, which is utilized by all TLRs except TLR3, recruits IL-1R-associated kinase (IRAK)-1 and IRAK-4 and tumor necrosis factor receptor-associated factor 6 (TRAF6). IRAK-1 and TRAF6 then dissociate from this receptor complex and associate with another complex composed of transforming growth factor-β-activated kinase (TAK1) and TAK1-binding proteins 1 (Tab1) and 2 (Tab2). This complex formation leads to the activation of TAK1, which in turn activates the transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) through the canonical IκB kinase (IKK) complex and the mitogen-activated protein kinase pathway, respectively. The kinase activity of the IKK complex is modulated by its IKKγ subunit, the transcription factor NF-κB essential modulator (NEMO). NF-κB activates multiple proinflammatory cytokine genes, including tumor necrosis factor α, IL-6, and IL-1β. In addition to this common pathway, called a MyD88-dependent pathway (Fig. 1), some TLR family members specifically involved in virus recognition have unique signaling pathways to induce type I IFNs (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). TLR3—TLR3 recognizes a synthetic analog of viral dsRNA, polyinosinic acid-cytidylic acid (poly(I·C)), and viral dsRNAs derived from dsRNA viruses such as reovirus or ssRNA viruses such as West Nile virus, respiratory syncytial virus, and encephalomyocarditis virus (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). Although TLR3 is expressed on the cell surface of fibroblasts, it localizes to endosomes in conventional dendritic cells (cDCs), requiring acidification of vesicles for its signaling (4Matsumoto M. Funami K. Tanabe M. Oshiumi H. Shingai M. Seto Y. Yamamoto A. Seya T. J. Immunol. 2003; 171: 3154-3162Crossref PubMed Scopus (617) Google Scholar). The three-dimensional structure of human TLR3 leucine-rich repeat motifs demonstrated direct binding of the TLR3 ectodomain to poly(I·C) (5Choe J. Kelker M.S. Wilson I.A. Science. 2005; 309: 581-585Crossref PubMed Scopus (492) Google Scholar). TLR3 interacts with CD14 and c-Src for ligand uptake and signal transduction (6Lee H.K. Dunzendorfer S. Soldau K. Tobias P.S. Immunity. 2006; 24: 153-163Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 7Johnsen I.B. Nguyen T.T. Ringdal M. Tryggestad A.M. Bakke O. Lien E. Espevik T. Anthonsen M.W. EMBO J. 2006; 25: 3335-3346Crossref PubMed Scopus (156) Google Scholar). TLR3 signaling activates the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB via the adapter molecule TRIF (8Yamamoto 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 (2479) Google Scholar, 9Hoebe 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 (1028) Google Scholar) (Fig. 1). TRIF interacts with noncanonical IKKs TBK1 (also called NAK or T2K) and IKKι (also called IKKϵ) through TRAF3 and NAK-associated protein 1 (NAP1), which mediate phosphorylation of IRF3 (10Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Nature. 2006; 439: 208-211Crossref PubMed Scopus (705) Google Scholar, 11Hacker H. Redecke V. Blagoev B. Kratchmarova I. Hsu L.C. Wang G.G. Kamps M.P. Raz E. Wagner H. Hacker G. Mann M. Karin M. Nature. 2006; 439: 204-207Crossref PubMed Scopus (733) Google Scholar, 12Sasai M. Oshiumi H. Matsumoto M. Inoue N. Fujita F. Nakanishi M. Seya T. J. Immunol. 2005; 174: 27-30Crossref PubMed Scopus (106) Google Scholar, 13Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1347) Google Scholar). Activated IRF3 translocates into the nucleus and induces expression of IFN-β. TBK1 and, to a lesser extent, IKKι are responsible for TRIF-mediated IFN-β induction (14Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (460) Google Scholar, 15Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (307) Google Scholar). In addition to TBK1/IKKι, phosphatidylinositol 3-kinase and its downstream kinase Akt are necessary for full activation of IRF3 (16Sarkar S.N. Peters K.L. Elco C.P. Sakamoto S. Pal S. Sen G.C. Nat. Struct. Mol. Biol. 2004; 11: 1060-1067Crossref PubMed Scopus (306) Google Scholar). TRAF1 and TRAF4 interact with TRIF and negatively regulate TRIF-mediated signaling pathways (17Su X. Li S. Meng M. Qian W. Xie W. Chen D. Zhai Z. Shu H.B. Eur. J. Immunol. 2006; 36: 199-206Crossref PubMed Scopus (51) Google Scholar, 18Takeshita F. Ishii K.J. Kobiyama K. Kojima Y. Coban C. Sasaki S. Ishii N. 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Although NF-κB activation by poly(I·C) is abrogated in TRAF6-deficient fibroblasts, TRIF-mediated signaling pathways were not impaired in TRAF6-deficient macrophages (22Gohda J. Matsumura T. Inoue J. J. Immunol. 2004; 173: 2913-2917Crossref PubMed Scopus (234) Google Scholar). TLR3 signaling was shown to promote cross-priming of T cells, a process that is necessary for the induction of virus-specific T cell responses. In mice, plasmacytoid DCs (pDCs) and CD8+ DCs appear to be the major adenomatous polyposis coli subtypes involved in priming antiviral cytotoxic T lymphocyte. When CD8+ DCs expressing high levels of TLR3 phagocytize the apoptotic bodies of virus-infected or dsRNA-loaded cells, the dsRNA in the apoptotic bodies is recognized by TLR3, triggering the maturation of immature CD8+ DCs that are required for the subsequent induction of antigen-specific CD4+ and CD8+ T cell responses. In addition, type I interferons released from virus-infected cells facilitate cross-priming (23Schulz O. Diebold S.S. Chen M. Naslund T.I. Nolte M.A. Alexopoulou L. Azuma Y.T. Flavell R.A. Liljestrom P. Reis e Sousa C. Nature. 2005; 433: 887-892Crossref PubMed Scopus (733) Google Scholar). In contrast to RLHs, cytoplasmic receptors for dsRNA, TLR3 plays a crucial role in the cross-priming of cytotoxic T lymphocytes against viruses that do not directly infect DCs. The functions of TLR3 have been elucidated in actual viral infections. TLR3-deficient mice showed susceptibility to mouse cytomegalovirus infection because of reduced interferon production (24Tabeta K. Georgel P. Janssen E. Du X. Hoebe K. Crozat K. Mudd S. Shamel L. Sovath S. Goode J. Alexopoulou L. Flavell R.A. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3516-3521Crossref PubMed Scopus (796) Google Scholar). However, TLR3-deficient mice were resistant to West Nile virus infection. This ssRNA flavivirus induces inflammatory responses in a TLR3-dependent manner that trigger a breakdown of the blood-brain barrier, which subsequently results in enhanced brain infection (25Wang T. Town T. Alexopoulou L. Anderson J.F. Fikrig E. Flavell R.A. Nat. Med. 2004; 10: 1366-1373Crossref PubMed Scopus (896) Google Scholar). These results are quite interesting, revealing that TLR3-mediated inflammatory responses to West Nile virus contribute to pathogenesis rather than to protection. TLR4—TLR4, the first mammalian homologue of the Drosophila Toll protein (26Medzhitov R. Preston-Hurlburt P. Janeway C.J. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4393) Google Scholar) recognizes lipopolysaccharide (LPS), which is a cell wall component of Gram-negative bacteria (27Poltorak 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. 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The N terminus of TRAM has a myristoylation site, mutation of which alters its normal membrane localization and abolishes TLR4 signaling, suggesting that TRAM acts as a bridging adapter between TLR4 and TRIF (33Rowe D.C. McGettrick A.F. Latz E. Monks B.G. Gay N.J. Yamamoto M. Akira S. O'Neill L.A. Fitzgerald K.A. Golenbock D.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6299-6304Crossref PubMed Scopus (208) Google Scholar). It has also been reported that phosphorylation of TRAM by PKCϵ is crucial for the activation of IRF3 and the induction of IFN-inducible genes (34McGettrick A.F. Brint E.K. Palsson-McDermott E.M. Rowe D.C. Golenbock D.T. Gay N.J. Fitzgerald K.A. O'Neill L.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9196-9201Crossref PubMed Scopus (108) Google Scholar). TRIF is also required for the induction of inflammatory cytokines in the TLR4 signaling pathway (8Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. 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Bacterial DNA, which contains unmethylated CpG motifs, is a strong activator of host immunity. In vertebrates, the frequency of CpG motifs is remarkably reduced, and the cysteine residues of CpG motifs are highly methylated, leading to abrogation of immunostimulatory activity. TLR9 mediates the recognition of CpG DNA (37Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5323) Google Scholar). CpG DNA motifs are also found in the genomes of DNA viruses. Mouse pDCs produce IFN-α by recognizing the CpG-containing DNA of herpes simplex virus type 2 (HSV-2) via TLR9 (38Lund J. Sato A. Akira S. Medzhitov R. Iwasaki A. J. Exp. Med. 2003; 198: 513-520Crossref PubMed Scopus (1001) Google Scholar). 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The synthetic imidazoquinoline-like molecules imiquimod (R837) and resiquimod (R848) have potent antiviral activities and are used clinically for the treatment of viral infections. Murine TLR7 and human TLR7 and TLR8 recognize imidazoquinoline compounds (41Hemmi H. Kaisho T. Takeuchi O. Sato S. Sanjo H. Hoshino K. Horiuchi T. Tomizawa H. Takeda K. Akira S. Nat. Immunol. 2002; 3: 196-200Crossref PubMed Scopus (2044) Google Scholar, 42Ito T. Amakawa R. Kaisho T. Hemmi H. Tajima K. Uehira K. Ozaki Y. Tomizawa H. Akira S. Fukuhara S. J. Exp. Med. 2002; 195: 1507-1512Crossref PubMed Scopus (411) Google Scholar). Furthermore, murine TLR7 has been shown to recognize guanosine analogs such as loxoribine, which has antiviral and anti-tumor activities (2Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8607) Google Scholar). Recently, TLR7 and human TLR8 have been shown to recognize guanosine- or uridine-rich ssRNA from viruses such as human immunodeficiency virus, vesicular stomatitis virus, and influenza virus (43Diebold S.S. Kaisho T. Hemmi H. Akira S. Reis E. Sousa C. Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2701) Google Scholar, 44Heil F. Hemmi H. Hochrein H. Ampenberger F. Kirschning C. Akira S. Lipford G. Wagner H. Bauer S. Science. 2004; 303: 1526-1529Crossref PubMed Scopus (3007) Google Scholar). The induction of type I IFNs by TLR7 and TLR9 depends entirely on MyD88 in pDCs (45Hemmi H. Kaisho T. Takeda K. Akira S. J. Immunol. 2003; 170: 3059-3064Crossref PubMed Scopus (298) Google Scholar) (Fig. 2). IRF7 is a transcription factor that is structurally related to IRF3 and is expressed constitutively in pDCs. IRF7 forms a signaling complex with MyD88 and TRAF6 in the cytoplasm (46Kawai T. Sato S. Ishii K.J. Coban C. Hemmi H. Yamamoto M. Terai K. Matsuda M. Inoue J. Uematsu S. 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K/B-type CpG DNA is rapidly transferred and degraded in the lysosome, whereas D/A-type CpG DNA is retained in the endosomes of pDCs. These results suggest that the endosomal retention of ligands in pDCs probably provides a platform for the interactions of signal-transducing molecules such as MyD88 and IRF7. However, this hypothesis does not fully explain the ligand specificity. If the endosomal retention of a ligand enhances its signaling, it is easy to assume that D/A-type CpG DNA induces robust production of inflammatory cytokines as well as IFN-α. As is generally known, D/A-type CpG DNA shows lower efficacy in the induction of proinflammatory cytokines than K/B-type CpG DNA. Furthermore, K/B-type CpG DNA even induces a certain amount of IFN-α from pDCs at lower concentrations (45Hemmi H. Kaisho T. Takeda K. Akira S. J. Immunol. 2003; 170: 3059-3064Crossref PubMed Scopus (298) Google Scholar). 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A. 2006; 103: 15136-15141Crossref PubMed Scopus (245) Google Scholar) (Fig. 2). A recent report showed that IRF1 specifically participates in induction of the IFN-β gene in this signaling pathway. IRF1 is induced by IFN-γ stimulation. After ligand stimulation, IRF1 interacts with MyD88, is activated by an unknown mechanism, and translocates into the nucleus to induce the expression of IFN-β, inducible nitric-oxide synthase, and IL-12 p35 (56Negishi H. Fujita Y. Yanai H. Sakaguchi S. Ouyang X. Shinohara M. Takayanagi H. Ohba Y. Taniguchi T. Honda K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15136-15141Crossref PubMed Scopus (245) Google Scholar). Furthermore, IRF1 is not essential for the TRIF-dependent pathway or the TLR9-mediated pathway in pDCs. Thus, the induction of type I IFNs by CpG DNA is quite distinct between cDCs and pDCs. The study of innate immunity has progressed rapidly over the last decade. TLR family members, which were initially considered to be receptors for bacterial components, have been shown to be involved in viral recognition and subsequent induction of type I IFNs, which are the most important antiviral agents. Simultaneously, pDCs have been identified as professional interferon-producing cells, which play crucial roles during viral infection. It has become clear that TLRs are the major receptors for the initiation of type I IFN production in pDCs (57Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). In the process of these studies, evidence has been obtained that non-professional interferon-producing cells, such as cDCs and epithelial cells, can produce type I IFNs in a TLR-independent manner. All of these results led to the discovery of RLH family members, the functions and signaling pathways of which are currently among the hottest topics in immunology. Although antiviral responses have been well clarified at the cellular level, it remains unknown when, how, and in what kind of cells TLR and RLH systems are used to exclude viruses. It will be necessary to examine the functions of TLRs and RLHs dynamically by using various viral infection models in the future.