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Toll-like receptors and innate antiviral responses Sagar A Vaidyay and Genhong Chengz Toll-like receptors (TLRs) have a unique and important role in detecting the presence of pathogenic infection. TLRs can recognize conserved structures from a wide variety of microorganisms, such as bacteria, mycobacteria, spirochetes and yeast. However, they are generally not thought to play a major role in viral infection. Several reports have now identified distinct viral ligands for the TLRs, and evidence is accumulating for a functional role of the TLRs in mediating antiviral effector mechanisms. Addresses Department of Microbiology, Immunology and Molecular Genetics, 8-240 Factor Building, 10833 Le Conte Avenue, University of California Los Angeles, Los Angeles, CA 90095, USA y UCLA-MSTP Graduate Program, University of California Los Angeles, Los Angeles, CA 90095, USA z e-mail: [email protected] Correspondence: Genhong Cheng
Current Opinion in Immunology 2003, 15:402–407 This review comes from a themed issue on Host–pathogen interactions Edited by Robert L Modlin and Peter Doherty 0952-7915/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00070-0
Abbreviations IFN interferon IL interleukin IL-1R interleukin 1 receptor IKK IkB kinase IRF interferon regulatory factor LPS lipopolysaccharide MyD88 myeloid differentiation factor 88 NF-jB nuclear factor kappa B poly I:C polyinosine-polycytidylic acid PAMP pathogen-associated molecular pattern PDC plasmacytoid dendritic cell RSV respiratory syncytial virus TBK TANK-binding kinase TIR Toll/IL-1 receptor TIRAP TIR adaptor protein TNF tumor necrosis factor TNFR TNF receptor TLR toll-like receptor
ciated molecular patterns (PAMPs) [1]. These invariant structures, which are found in microbial membranes, cell walls, proteins and DNA, can be viewed as molecular ‘signatures’ of the invading pathogen. Pathogen detection by TLRs triggers an immediate response mediated by macrophages, neutrophils, complement proteins and antimicrobial molecules. In addition, the production of unique cytokine combinations can mediate systemic responses, recruit additional leukocytes to sites of inflammation, and modulate aspects of the adaptive response. To date, ten TLRs have been cloned in mammals and each receptor appears to be involved in the recognition of a unique set of PAMPs that are distinct in chemical structure (reviewed in [1–4]). TLRs contain multiple leucine-rich repeats in their extracellular domain and an intracellular Toll/IL-1 receptor (TIR) domain, which is conserved in all TLRs. Receptor binding triggers a signaling cascade involving myeloid differentiation factor 88 (MyD88), IL-1Rassociated kinase (IRAK) and TNFR-associated factor 6 (TRAF6), which then leads to the activation of both the nuclear factor kappa B (NF-kB) and Jun amino-terminal kinase (JNK) signaling pathways [5]. In addition to NF-kB and JNK, TLRs are also able to activate other signaling cascades, including the p38, extracellular signal-related kinase (ERK), interferon regulatory factor (IRF3), and phosphatidylinositol-3 kinase (PI3K) pathways [3,6]. Activation of TLRs leads to major changes in gene expression, as commonly measured by production of inflammatory cytokines such as TNFa, IL-1, IL-6 and IL-12 [7]. Previously, TLRs were not thought to play a major role in viral infections. Instead, it had long been recognized that infection by virus induces the production of type I interferons (IFNa/b), cytokines that induce complex antiviral resistance mechanisms in infected and uninfected cells. Viral infection results in the auto-amplification of IFNa/b through a sophisticated system of transcription factors of the IRF family (reviewed in [8–11]). At the present time the initial mechanism by which the IRF proteins become activated by viral infection remains to be elucidated (see also Update). Here, we review the recent developments linking the TLRs to viral recognition and innate antiviral responses.
Introduction
Toll-like receptors as detectors of viral infection
In response to constant invasion by pathogens, metazoans have evolved complex and powerful systems for recognition of non-self. Toll-like receptors (TLRs) play a critical role in innate immunity by recognizing constitutive and conserved microbial components, termed pathogen-asso-
The first widely accepted report implicating the TLRs in viral detection came from Kurt-Jones and colleagues [12], who demonstrated that TLR4 was responsible for the response to the respiratory syncytial virus (RSV) fusion (F) protein. Purified F protein was shown to mediate
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cytokine production specifically through TLR4. In addition, in vivo experiments showed that RSV persisted longer and at higher titers in the lungs of TLR4-mutant mice. This was consistent with impaired recruitment of immune cells to the lung [13]. Soon thereafter, studies from other laboratories demonstrated that remarkably divergent ligands, such as lipids, proteins and matrix sugars, induced TLR4-dependent responses [14–16]. This raised some suspicions about possible contamination of the stimulus preparations with endotoxin, which can be detected by TLR4 at very low concentrations. However, a recent study from a different group has confirmed that the activation of NF-kB upon in vivo infection by RSV is dependent on TLR4 [17].
humans, both TLR7 and TLR8 have been postulated to mediate the response to imidazoquinolines [20]. In addition, TLR7 expression is highest on the interferonproducing plasmacytoid dendritic cells (PDCs) in humans, furthering the implication of TLR7 expression in antiviral responses [21–23]. However, the natural ligand for TLR7 is still unknown, and studies have yet to show a role for TLR7 in response to a specific viral infection. As the interest in the TLR field has greatly expanded, the list of TLR ligands grows ever longer. Several different groups have now demonstrated that viral proteins other than PAMPs can be detected by TLRs (see Figure 1). A report studying murine mammary tumor virus (MMTV) infection found decreased NF-kB activation in B cells of TLR4/mice [24]. In addition, MMTV viral envelope proteins were shown to co-purify with TLR4. TLR2 was shown to be required for induction of IL-6 by the hemagglutinin protein of the measles virus, and TLR2deficient macrophages are unable to produce IL-6 in response to measles infection [25]. Recent studies indicate that human cytomegalovirus (hCMV) may also activate TLR2, as UV-inactivated hCMV particles induced a dose-dependent production of IL-6 and IL-8 in wildtype, but not TLR2-deficient, macrophages [26].
Further evidence for the role of TLRs in viral detection came from the generation of TLR3-deficient mice by Alexopoulou and colleagues [18]. TLR3-deficient macrophages were shown to have greatly reduced responses to polyinosine-polycytidylic acid (poly I:C), which structurally mimics double-stranded RNA of viral origin. Poly I:C has long been used to synthetically induce IFNa/b expression, and this report linked the TLRs directly to viral effector cytokines. Although many studies have gone on to characterize the biochemical pathways activated by poly I:C, the role of TLR3 in viral infection in vivo remains a key avenue of research to be explored.
While the search goes on for more ligands, it is critical that additional studies look deeper and address the true functional relevance of this response. Many of the recent studies have been conducted in simple overexpression systems and have not examined how TLR–virus interactions might contribute to the in vivo antiviral response. Without such information it is at least conceivable (although unlikely) that the virus may be activating TLRs
Imidazoquinoline compounds are therapeutics that have antiviral properties (by inducing the expression of IFNa) and were recently shown to signal through TLR7 [19]. TLR7-deficient cells are unresponsive to imiquimod and R-848, but produce normal levels of TNFa and IL-6 upon stimulation with lipopolysaccharide (LPS) or CpG. In Figure 1
TLR2 putative ligands
TLR3 putative ligands [25]
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[18] [18]
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Several viral proteins and virus-related molecules have recently been described as TLR ligands. www.current-opinion.com
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for its own purposes, for example, the upregulation of coreceptors used for viral entry.
Toll-like receptor-mediated antiviral gene program Although some researchers have focused on the TLRs in host–pathogen interactions, others have taken a different route and used the TLR recognition system to study the signaling networks leading to specific gene expression. Early gene expression studies showed similar induction of inflammatory genes by various TLR ligands; however, careful analysis revealed some key differences. In particular, TLR4 ligands could induce a limited number of genes in MyD88-deficient cells, whereas TLR2 and TLR9 agonists produced no gene-expression response [27]. This was termed the MyD88-independent pathway, and it is characterized by genes such as IFN-inducible protein 10 (CXCL10, also known as IP10) and immunoresponsive gene 1 (IRG1), as well as by the upregulation of co-receptors on dendritic cells [28]. Additional studies showed that the MyD88-independent pathway could also induce IFNb production and STAT1 activation, but the mechanism by which gene expression was controlled remained unresolved [29]. Studies in our laboratory initially began using microarrays to understand global expression differences between the TNFR superfamily and the TLR family in B cells, using CD40L and LPS as ligands, respectively [30]. Our results indicate that CD40L and LPS activate the same sets of genes for their overlapping biological functions, such as cell survival, cell proliferation and immunological isotype switching. However, LPS induces a unique subset of genes that had been previously characterized as antiviral or interferon-inducible. Notable examples include Mx1, a GTPase-like protein which was one of the earliest genes demonstrated to provided resistance to influenza virus, and IRF7, a transcription factor that is critical in amplifying the IFNa/b response [31,32]. Subsequent analyses showed that these antiviral genes identified in B cells were specifically inducible in macrophages by poly I:C and LPS, the ligands for TLR3 and TLR4 respectively, but not by other common TLR ligands. In addition, only poly I:C and LPS could induce nuclear translocation of IRF3. On the basis of the induction kinetics and sensitivity to the protein synthesis inhibitor cycloheximide, we further grouped these TLR3/4-specific genes into primary and secondary response genes. Using dominant-negative approaches in the RAW 264.7 macrophage cell line, we showed that both the NF-kB and IRF3 pathways are required for poly I:C- and LPS-induced upregulation of several primary response genes, including IFNb. However, overexpression of IRF3 was sufficient to allow induction of antiviral genes by bacterial peptidoglycan, the ligand for TLR2. These data support the conclusion that, although NF-kB Current Opinion in Immunology 2003, 15:402–407
is required for TLR-dependent gene activation, IRF3 is the principal component mediating the TLR3/4 specificity of antiviral gene expression (see Figure 2). Further gene expression analysis with IFNa/b blocking antibodies and IFNa/b receptor 1 (IFNAR1)-deficient cells showed that several other TLR3/4-specific genes important for host defense are secondarily activated by autocrine/paracrine secretion of the primary response gene product, IFNb. Thus, our studies indicate that there exists a signaling network that leads to the automatic and sequential activation of specific genes (a gene program) in response to TLR3 and TLR4 ligands, but not by stimulation of other TLRs or CD40. This antiviral gene program involves a subset of immediate early response genes that are regulated by IRF3, and a group of secondary response genes activated by the production of IFNb [30].
Connecting the Toll-like receptor antiviral signaling pathway It has long been established that stimulation with TLR4 ligands in MyD88-deficient cells can activate the NFkB pathway as well as the mitogen-activated protein kinase (MAPK) cascades, albeit with delayed kinetics [33]. Biochemical characterizations of signaling using various TLR ligands in MyD88-deficient cells showed that the MyD88-independent pathway was unique to TLR3 and TLR4, but was not activated by TLR2, TLR5, TLR7 or TLR9 [3]. Key studies from Kawai and colleagues [28] have also demonstrated that IRF3 can be activated in a MyD88-independent fashion by stimulation of TLR4. This thereby brought about the hypothesis that a novel adaptor existed for TLR4 that would mediate activation of IRF3 and upregulation of IFNb. TIR domain-based homology searches identified a novel TIR adaptor protein, TIRAP (also called Mal), which was shown to mediate TLR signaling through interaction of its TIR domain [34,35]. However, the recent generation of TIRAP-deficient mice indicated that TIRAP is critical for TLR2- and TLR4-dependent expression of inflammatory cytokines, in a role similar to that of MyD88 (see Figure 2; [36,37]). Of note, stimulation of TLR7 and TLR9 in PDCs has also been shown to induce production of IFNs, presumably in a MyD88dependent manner not involving IRF3 [21,38]. The role of this pathway in antiviral responses is currently under investigation. Recent identification of a novel protein, TIR domaincontaining adapter-inducing IFNb (TRIF, also called TICAM-1), by two groups [39,40], suggests that this protein may be the critical adaptor of the MyD88-independent pathway. Overexpression of TRIF not only induces high levels of an IFNb reporter, but TRIF was also shown to co-immunoprecipitate with endogenous IRF3. TRIF has thus far been shown to associate with TLR3, and it is currently a matter of conjecture how www.current-opinion.com
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Figure 2
TLR4
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• IFNβ, IP10, RANTES • Mx1, IRF7, TLR3
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Simplified theoretical model of the signaling pathways leading to TLR-specific antiviral gene expression in macrophages.
TLR4 activates IRF3 (see Figure 2). Clearly, studies using TRIF-deficient mice are much awaited.
Functional role of the Toll-like receptors in viral infection Although a great deal has been published regarding the biochemistry of TLR recognition and signaling, our understanding of the functional role of the TLRs in viral pathogenesis is still in its infancy. Currently, RSV is the only viral disease model that has been shown to have a reproducible TLR-dependent immune response in vivo [12,13,17]. Although we can be sure that studies with other viruses are in progress, some information regarding the functional role of the TLRs and virus can be indirectly derived by other important observations. For example, it was recently shown that the poxvirus A52R gene encodes a protein that can inhibit TLR signaling, and this was found to associate with IRAK2 and TRAF6 [41]. www.current-opinion.com
One might wonder why a virus would carry around a TLR blocking protein; however, the contribution of A52R to viral virulence is less convincing. Another study showed that infection of human macrophages with influenza A virus causes upregulation of TLR1, TLR2, TLR3 and TLR7 — again implying that these receptors may be used in the antiviral response [42]. Studies in our laboratory have used the murineherpesgamma68 (MHV68) model of viral infection in order to investigate the relationship between the TLRs and antiviral responses. We have shown that in vitro stimulation of TLR3 or TLR4 in macrophages could inhibit viral replication in an autocrine and paracrine fashion [30]. Although this response was determined to be dependent on secreted IFNb, the detailed mechanisms by which the antiviral response functions are unknown. Many of the antiviral genes upregulated are still minimally Current Opinion in Immunology 2003, 15:402–407
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characterized, and it is unclear how they function to inhibit the viral life cycle. Whatever the final output of the TLR antiviral response, several lines of evidence suggest that this pathway is subject to considerable modulation. Servant and colleagues [43] recently showed that IRF3 phosphorylation occurs on different residues in response to TLR3 or TLR4 ligands. It remains to be determined whether residue-specific IRF3 phosphorylation has a function in regulation of the antiviral response. Also, we have found that TLR3 ligands induce stronger and prolonged activation of the antiviral gene program in comparison to TLR4 ligands [44]. This correlates with the positive upregulation of TLR3 mRNA by IFNb after either TLR3 or TLR4 stimulation, suggesting that the TLRs have the ability to further regulate their own responses.
co-precipitate with IRF3 and IRF7. In addition, an elegant RNAi approach showed that the simultaneous knockdown of IKKe and TBK1 could inhibit interferon signaling and viral replication. Experiments examining role of these molecules in TLR-signaling are currently underway.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions A growing number of viral proteins have been shown to induce TLR-dependent inflammatory responses and cytokine production. In addition, it is becoming clearer that some TLRs have the capability to induce antiviral responses by selective activation of IRF3 and IFNa/b production. As our insights grow deeper into the complex biology of the TLRs, there now exist many additional avenues to be pursued in the study of host–virus interactions. The identification of the TLR7 agonist and potent antiviral compound, imiquimod, indicates that the TLRs may also be excellent candidates for the high-throughput screening of chemotherapeutics, which can synthetically activate antiviral effector responses. Such novel treatment strategies might be extremely useful clinically, as many diseases and therapies lead to immunosuppression with enhanced susceptibility to viral infection. To genuinely appreciate the role of the TLRs in antiviral responses, however, immunologists-turned-virologists (or vice versa) must first answer several formidable questions. If TLRs truly detect viruses, then how is this recognition mediated? Double-stranded RNA viruses aside, what are the conserved structures recognized and why haven’t they mutated? TLRs are involved in critical aspects of the adaptive response, is this also the case for viral infection? Finally, what are the similarities and differences between the TLR-mediated responses to virus versus other pathogens, such as bacteria? Of course these hypotheses await verification by functional analyses in TLR-deficient mice. Let the infections begin.
Update Recent work by two independent groups has demonstrated that the IkB kinase (IKK)-related kinases, IKKe and TANK-binding kinase (TBK1), are involved in events upstream of IRF3/IRF7 activation by viral infection [45,46]. Biochemical studies showed that both IKKe and TBK1 could phosphorylate IRF3 and IRF7, as well as Current Opinion in Immunology 2003, 15:402–407
10. Garcia-Sastre A: Mechanisms of inhibition of the host interferon alpha/beta-mediated antiviral responses by viruses. Microbes Infect 2002, 4:647-655. 11. Levy DE, Marie I, Prakash A: Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr Opin Immunol 2003, 15:52-58. 12. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ et al.: Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 2000, 1:398-401. 13. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA: Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 2001, 75:10730-10737. 14. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000, 6:435-442. 15. Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, Chow JC, Strauss JF III: The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001, 276:10229-10233. 16. Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA: Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol 2000, 164:13-17. 17. Haeberle HA, Takizawa R, Casola A, Brasier AR, Dieterich HJ, Van Rooijen N, Gatalica Z, Garofalo RP: Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung www.current-opinion.com
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involves alveolar macrophages and toll-like receptor 4-dependent pathways. J Infect Dis 2002, 186:1199-1206. 18. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA: Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001, 413:732-738. 19. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S: Small anti-viral compounds activate immune cells via the TLR7 MyD88dependent signaling pathway. Nat Immunol 2002, 3:196-200. By generating TLR7-deficient mice, this paper demonstrates that TLR7 is required for cytokine production by the imidazoquinoline compounds imiquimod and R-848. In addition, responses to the imidazoquinolines was found to be completely MyD88-dependent. 20. Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, Lipford G, Bauer S: Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 2002, 3:499. 21. Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE, Oesterich JL, Gorden KB, Qiu X, McKane SW et al.: Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol 2002, 218:74-86. 22. Dzionek A, Inagaki Y, Okawa K, Nagafune J, Rock JJ, Sohma Y, Winkels G, Zysk M, Yamaguchi Y, Schmitz JJ: Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions(1). Hum Immunol 2002, 63:1133-1148. 23. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 2002, 168:4531-4537. 24. Rassa JC, Meyers JL, Zhang Y, Kudaravalli R, Ross SR: Murine retroviruses activate B cells via interaction with toll-like receptor 4. Proc Natl Acad Sci USA 2002, 99:2281-2286. 25. Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V, Schneider-Schaulies S: Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 2002, 76:8729-8736. 26. Compton T, Kurt-Jones EA, Boehme CW, Belko J, Latz E, Golenbock DT, Finberg RW: Human cytomegalovirus activates inflammatory cytokine responses via CD14 and toll-like receptor 2. J Virol 2003, in press. 27. Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S: Endotoxininduced maturation of MyD88-deficient dendritic cells. J Immunol 2001, 166:5688-5694. 28. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, Akira S: Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001, 167:5887-5894. 29. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ et al.: TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/ beta-dependent gene expression in macrophages. Nat Immunol 2002, 3:392-398. 30. Doyle S, Vaidya S, O’Connell R, Dadgostar H, Dempsey P, Wu T, Rao G, Sun R, Haberland M, Modlin R et al.: IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 2002, 17:251-263. This study identifies a subset of antiviral genes activated by stimulation with TLR3 and TLR4 ligands. IRF3 was shown to be the critical factor mediating selective upregulation of antiviral gene expression, and pretreatment with TLR3 and TLR4 ligands could potently inhibit viral replication. The authors suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs, and are capable of activating innate antiviral responses through IRF3.
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Toll-like receptors and innate antiviral responses Sagar A Vaidyay and Genhong Chengz Toll-like receptors (TLRs) have a unique and important role in detecting the presence of pathogenic infection. TLRs can recognize conserved structures from a wide variety of microorganisms, such as bacteria, mycobacteria, spirochetes and yeast. However, they are generally not thought to play a major role in viral infection. Several reports have now identified distinct viral ligands for the TLRs, and evidence is accumulating for a functional role of the TLRs in mediating antiviral effector mechanisms. Addresses Department of Microbiology, Immunology and Molecular Genetics, 8-240 Factor Building, 10833 Le Conte Avenue, University of California Los Angeles, Los Angeles, CA 90095, USA y UCLA-MSTP Graduate Program, University of California Los Angeles, Los Angeles, CA 90095, USA z e-mail: [email protected] Correspondence: Genhong Cheng
Current Opinion in Immunology 2003, 15:402–407 This review comes from a themed issue on Host–pathogen interactions Edited by Robert L Modlin and Peter Doherty 0952-7915/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00070-0
Abbreviations IFN interferon IL interleukin IL-1R interleukin 1 receptor IKK IkB kinase IRF interferon regulatory factor LPS lipopolysaccharide MyD88 myeloid differentiation factor 88 NF-jB nuclear factor kappa B poly I:C polyinosine-polycytidylic acid PAMP pathogen-associated molecular pattern PDC plasmacytoid dendritic cell RSV respiratory syncytial virus TBK TANK-binding kinase TIR Toll/IL-1 receptor TIRAP TIR adaptor protein TNF tumor necrosis factor TNFR TNF receptor TLR toll-like receptor
ciated molecular patterns (PAMPs) [1]. These invariant structures, which are found in microbial membranes, cell walls, proteins and DNA, can be viewed as molecular ‘signatures’ of the invading pathogen. Pathogen detection by TLRs triggers an immediate response mediated by macrophages, neutrophils, complement proteins and antimicrobial molecules. In addition, the production of unique cytokine combinations can mediate systemic responses, recruit additional leukocytes to sites of inflammation, and modulate aspects of the adaptive response. To date, ten TLRs have been cloned in mammals and each receptor appears to be involved in the recognition of a unique set of PAMPs that are distinct in chemical structure (reviewed in [1–4]). TLRs contain multiple leucine-rich repeats in their extracellular domain and an intracellular Toll/IL-1 receptor (TIR) domain, which is conserved in all TLRs. Receptor binding triggers a signaling cascade involving myeloid differentiation factor 88 (MyD88), IL-1Rassociated kinase (IRAK) and TNFR-associated factor 6 (TRAF6), which then leads to the activation of both the nuclear factor kappa B (NF-kB) and Jun amino-terminal kinase (JNK) signaling pathways [5]. In addition to NF-kB and JNK, TLRs are also able to activate other signaling cascades, including the p38, extracellular signal-related kinase (ERK), interferon regulatory factor (IRF3), and phosphatidylinositol-3 kinase (PI3K) pathways [3,6]. Activation of TLRs leads to major changes in gene expression, as commonly measured by production of inflammatory cytokines such as TNFa, IL-1, IL-6 and IL-12 [7]. Previously, TLRs were not thought to play a major role in viral infections. Instead, it had long been recognized that infection by virus induces the production of type I interferons (IFNa/b), cytokines that induce complex antiviral resistance mechanisms in infected and uninfected cells. Viral infection results in the auto-amplification of IFNa/b through a sophisticated system of transcription factors of the IRF family (reviewed in [8–11]). At the present time the initial mechanism by which the IRF proteins become activated by viral infection remains to be elucidated (see also Update). Here, we review the recent developments linking the TLRs to viral recognition and innate antiviral responses.
Introduction
Toll-like receptors as detectors of viral infection
In response to constant invasion by pathogens, metazoans have evolved complex and powerful systems for recognition of non-self. Toll-like receptors (TLRs) play a critical role in innate immunity by recognizing constitutive and conserved microbial components, termed pathogen-asso-
The first widely accepted report implicating the TLRs in viral detection came from Kurt-Jones and colleagues [12], who demonstrated that TLR4 was responsible for the response to the respiratory syncytial virus (RSV) fusion (F) protein. Purified F protein was shown to mediate
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cytokine production specifically through TLR4. In addition, in vivo experiments showed that RSV persisted longer and at higher titers in the lungs of TLR4-mutant mice. This was consistent with impaired recruitment of immune cells to the lung [13]. Soon thereafter, studies from other laboratories demonstrated that remarkably divergent ligands, such as lipids, proteins and matrix sugars, induced TLR4-dependent responses [14–16]. This raised some suspicions about possible contamination of the stimulus preparations with endotoxin, which can be detected by TLR4 at very low concentrations. However, a recent study from a different group has confirmed that the activation of NF-kB upon in vivo infection by RSV is dependent on TLR4 [17].
humans, both TLR7 and TLR8 have been postulated to mediate the response to imidazoquinolines [20]. In addition, TLR7 expression is highest on the interferonproducing plasmacytoid dendritic cells (PDCs) in humans, furthering the implication of TLR7 expression in antiviral responses [21–23]. However, the natural ligand for TLR7 is still unknown, and studies have yet to show a role for TLR7 in response to a specific viral infection. As the interest in the TLR field has greatly expanded, the list of TLR ligands grows ever longer. Several different groups have now demonstrated that viral proteins other than PAMPs can be detected by TLRs (see Figure 1). A report studying murine mammary tumor virus (MMTV) infection found decreased NF-kB activation in B cells of TLR4/mice [24]. In addition, MMTV viral envelope proteins were shown to co-purify with TLR4. TLR2 was shown to be required for induction of IL-6 by the hemagglutinin protein of the measles virus, and TLR2deficient macrophages are unable to produce IL-6 in response to measles infection [25]. Recent studies indicate that human cytomegalovirus (hCMV) may also activate TLR2, as UV-inactivated hCMV particles induced a dose-dependent production of IL-6 and IL-8 in wildtype, but not TLR2-deficient, macrophages [26].
Further evidence for the role of TLRs in viral detection came from the generation of TLR3-deficient mice by Alexopoulou and colleagues [18]. TLR3-deficient macrophages were shown to have greatly reduced responses to polyinosine-polycytidylic acid (poly I:C), which structurally mimics double-stranded RNA of viral origin. Poly I:C has long been used to synthetically induce IFNa/b expression, and this report linked the TLRs directly to viral effector cytokines. Although many studies have gone on to characterize the biochemical pathways activated by poly I:C, the role of TLR3 in viral infection in vivo remains a key avenue of research to be explored.
While the search goes on for more ligands, it is critical that additional studies look deeper and address the true functional relevance of this response. Many of the recent studies have been conducted in simple overexpression systems and have not examined how TLR–virus interactions might contribute to the in vivo antiviral response. Without such information it is at least conceivable (although unlikely) that the virus may be activating TLRs
Imidazoquinoline compounds are therapeutics that have antiviral properties (by inducing the expression of IFNa) and were recently shown to signal through TLR7 [19]. TLR7-deficient cells are unresponsive to imiquimod and R-848, but produce normal levels of TNFa and IL-6 upon stimulation with lipopolysaccharide (LPS) or CpG. In Figure 1
TLR2 putative ligands
TLR3 putative ligands [25]
• double-stranded RNA • poly I:C
[26]
[18] [18]
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• Measles virus HA protein • hCMV particles
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[12] [24]
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• Natural ligand? • Imiquimod • R-848
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Several viral proteins and virus-related molecules have recently been described as TLR ligands. www.current-opinion.com
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for its own purposes, for example, the upregulation of coreceptors used for viral entry.
Toll-like receptor-mediated antiviral gene program Although some researchers have focused on the TLRs in host–pathogen interactions, others have taken a different route and used the TLR recognition system to study the signaling networks leading to specific gene expression. Early gene expression studies showed similar induction of inflammatory genes by various TLR ligands; however, careful analysis revealed some key differences. In particular, TLR4 ligands could induce a limited number of genes in MyD88-deficient cells, whereas TLR2 and TLR9 agonists produced no gene-expression response [27]. This was termed the MyD88-independent pathway, and it is characterized by genes such as IFN-inducible protein 10 (CXCL10, also known as IP10) and immunoresponsive gene 1 (IRG1), as well as by the upregulation of co-receptors on dendritic cells [28]. Additional studies showed that the MyD88-independent pathway could also induce IFNb production and STAT1 activation, but the mechanism by which gene expression was controlled remained unresolved [29]. Studies in our laboratory initially began using microarrays to understand global expression differences between the TNFR superfamily and the TLR family in B cells, using CD40L and LPS as ligands, respectively [30]. Our results indicate that CD40L and LPS activate the same sets of genes for their overlapping biological functions, such as cell survival, cell proliferation and immunological isotype switching. However, LPS induces a unique subset of genes that had been previously characterized as antiviral or interferon-inducible. Notable examples include Mx1, a GTPase-like protein which was one of the earliest genes demonstrated to provided resistance to influenza virus, and IRF7, a transcription factor that is critical in amplifying the IFNa/b response [31,32]. Subsequent analyses showed that these antiviral genes identified in B cells were specifically inducible in macrophages by poly I:C and LPS, the ligands for TLR3 and TLR4 respectively, but not by other common TLR ligands. In addition, only poly I:C and LPS could induce nuclear translocation of IRF3. On the basis of the induction kinetics and sensitivity to the protein synthesis inhibitor cycloheximide, we further grouped these TLR3/4-specific genes into primary and secondary response genes. Using dominant-negative approaches in the RAW 264.7 macrophage cell line, we showed that both the NF-kB and IRF3 pathways are required for poly I:C- and LPS-induced upregulation of several primary response genes, including IFNb. However, overexpression of IRF3 was sufficient to allow induction of antiviral genes by bacterial peptidoglycan, the ligand for TLR2. These data support the conclusion that, although NF-kB Current Opinion in Immunology 2003, 15:402–407
is required for TLR-dependent gene activation, IRF3 is the principal component mediating the TLR3/4 specificity of antiviral gene expression (see Figure 2). Further gene expression analysis with IFNa/b blocking antibodies and IFNa/b receptor 1 (IFNAR1)-deficient cells showed that several other TLR3/4-specific genes important for host defense are secondarily activated by autocrine/paracrine secretion of the primary response gene product, IFNb. Thus, our studies indicate that there exists a signaling network that leads to the automatic and sequential activation of specific genes (a gene program) in response to TLR3 and TLR4 ligands, but not by stimulation of other TLRs or CD40. This antiviral gene program involves a subset of immediate early response genes that are regulated by IRF3, and a group of secondary response genes activated by the production of IFNb [30].
Connecting the Toll-like receptor antiviral signaling pathway It has long been established that stimulation with TLR4 ligands in MyD88-deficient cells can activate the NFkB pathway as well as the mitogen-activated protein kinase (MAPK) cascades, albeit with delayed kinetics [33]. Biochemical characterizations of signaling using various TLR ligands in MyD88-deficient cells showed that the MyD88-independent pathway was unique to TLR3 and TLR4, but was not activated by TLR2, TLR5, TLR7 or TLR9 [3]. Key studies from Kawai and colleagues [28] have also demonstrated that IRF3 can be activated in a MyD88-independent fashion by stimulation of TLR4. This thereby brought about the hypothesis that a novel adaptor existed for TLR4 that would mediate activation of IRF3 and upregulation of IFNb. TIR domain-based homology searches identified a novel TIR adaptor protein, TIRAP (also called Mal), which was shown to mediate TLR signaling through interaction of its TIR domain [34,35]. However, the recent generation of TIRAP-deficient mice indicated that TIRAP is critical for TLR2- and TLR4-dependent expression of inflammatory cytokines, in a role similar to that of MyD88 (see Figure 2; [36,37]). Of note, stimulation of TLR7 and TLR9 in PDCs has also been shown to induce production of IFNs, presumably in a MyD88dependent manner not involving IRF3 [21,38]. The role of this pathway in antiviral responses is currently under investigation. Recent identification of a novel protein, TIR domaincontaining adapter-inducing IFNb (TRIF, also called TICAM-1), by two groups [39,40], suggests that this protein may be the critical adaptor of the MyD88-independent pathway. Overexpression of TRIF not only induces high levels of an IFNb reporter, but TRIF was also shown to co-immunoprecipitate with endogenous IRF3. TRIF has thus far been shown to associate with TLR3, and it is currently a matter of conjecture how www.current-opinion.com
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Figure 2
TLR4
TLR2
9
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TL R MyD88
MyD88 TIRAP
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NF-kB MAPK
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• Cytokines (TNFα, IL-6) • Surface markers (ICAM1)
• IFNβ, IP10, RANTES • Mx1, IRF7, TLR3
IRF3
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Simplified theoretical model of the signaling pathways leading to TLR-specific antiviral gene expression in macrophages.
TLR4 activates IRF3 (see Figure 2). Clearly, studies using TRIF-deficient mice are much awaited.
Functional role of the Toll-like receptors in viral infection Although a great deal has been published regarding the biochemistry of TLR recognition and signaling, our understanding of the functional role of the TLRs in viral pathogenesis is still in its infancy. Currently, RSV is the only viral disease model that has been shown to have a reproducible TLR-dependent immune response in vivo [12,13,17]. Although we can be sure that studies with other viruses are in progress, some information regarding the functional role of the TLRs and virus can be indirectly derived by other important observations. For example, it was recently shown that the poxvirus A52R gene encodes a protein that can inhibit TLR signaling, and this was found to associate with IRAK2 and TRAF6 [41]. www.current-opinion.com
One might wonder why a virus would carry around a TLR blocking protein; however, the contribution of A52R to viral virulence is less convincing. Another study showed that infection of human macrophages with influenza A virus causes upregulation of TLR1, TLR2, TLR3 and TLR7 — again implying that these receptors may be used in the antiviral response [42]. Studies in our laboratory have used the murineherpesgamma68 (MHV68) model of viral infection in order to investigate the relationship between the TLRs and antiviral responses. We have shown that in vitro stimulation of TLR3 or TLR4 in macrophages could inhibit viral replication in an autocrine and paracrine fashion [30]. Although this response was determined to be dependent on secreted IFNb, the detailed mechanisms by which the antiviral response functions are unknown. Many of the antiviral genes upregulated are still minimally Current Opinion in Immunology 2003, 15:402–407
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characterized, and it is unclear how they function to inhibit the viral life cycle. Whatever the final output of the TLR antiviral response, several lines of evidence suggest that this pathway is subject to considerable modulation. Servant and colleagues [43] recently showed that IRF3 phosphorylation occurs on different residues in response to TLR3 or TLR4 ligands. It remains to be determined whether residue-specific IRF3 phosphorylation has a function in regulation of the antiviral response. Also, we have found that TLR3 ligands induce stronger and prolonged activation of the antiviral gene program in comparison to TLR4 ligands [44]. This correlates with the positive upregulation of TLR3 mRNA by IFNb after either TLR3 or TLR4 stimulation, suggesting that the TLRs have the ability to further regulate their own responses.
co-precipitate with IRF3 and IRF7. In addition, an elegant RNAi approach showed that the simultaneous knockdown of IKKe and TBK1 could inhibit interferon signaling and viral replication. Experiments examining role of these molecules in TLR-signaling are currently underway.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions A growing number of viral proteins have been shown to induce TLR-dependent inflammatory responses and cytokine production. In addition, it is becoming clearer that some TLRs have the capability to induce antiviral responses by selective activation of IRF3 and IFNa/b production. As our insights grow deeper into the complex biology of the TLRs, there now exist many additional avenues to be pursued in the study of host–virus interactions. The identification of the TLR7 agonist and potent antiviral compound, imiquimod, indicates that the TLRs may also be excellent candidates for the high-throughput screening of chemotherapeutics, which can synthetically activate antiviral effector responses. Such novel treatment strategies might be extremely useful clinically, as many diseases and therapies lead to immunosuppression with enhanced susceptibility to viral infection. To genuinely appreciate the role of the TLRs in antiviral responses, however, immunologists-turned-virologists (or vice versa) must first answer several formidable questions. If TLRs truly detect viruses, then how is this recognition mediated? Double-stranded RNA viruses aside, what are the conserved structures recognized and why haven’t they mutated? TLRs are involved in critical aspects of the adaptive response, is this also the case for viral infection? Finally, what are the similarities and differences between the TLR-mediated responses to virus versus other pathogens, such as bacteria? Of course these hypotheses await verification by functional analyses in TLR-deficient mice. Let the infections begin.
Update Recent work by two independent groups has demonstrated that the IkB kinase (IKK)-related kinases, IKKe and TANK-binding kinase (TBK1), are involved in events upstream of IRF3/IRF7 activation by viral infection [45,46]. Biochemical studies showed that both IKKe and TBK1 could phosphorylate IRF3 and IRF7, as well as Current Opinion in Immunology 2003, 15:402–407
10. Garcia-Sastre A: Mechanisms of inhibition of the host interferon alpha/beta-mediated antiviral responses by viruses. Microbes Infect 2002, 4:647-655. 11. Levy DE, Marie I, Prakash A: Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr Opin Immunol 2003, 15:52-58. 12. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ et al.: Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 2000, 1:398-401. 13. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA: Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 2001, 75:10730-10737. 14. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000, 6:435-442. 15. Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, Chow JC, Strauss JF III: The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001, 276:10229-10233. 16. Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA: Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol 2000, 164:13-17. 17. Haeberle HA, Takizawa R, Casola A, Brasier AR, Dieterich HJ, Van Rooijen N, Gatalica Z, Garofalo RP: Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung www.current-opinion.com
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involves alveolar macrophages and toll-like receptor 4-dependent pathways. J Infect Dis 2002, 186:1199-1206. 18. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA: Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001, 413:732-738. 19. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S: Small anti-viral compounds activate immune cells via the TLR7 MyD88dependent signaling pathway. Nat Immunol 2002, 3:196-200. By generating TLR7-deficient mice, this paper demonstrates that TLR7 is required for cytokine production by the imidazoquinoline compounds imiquimod and R-848. In addition, responses to the imidazoquinolines was found to be completely MyD88-dependent. 20. Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, Lipford G, Bauer S: Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 2002, 3:499. 21. Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE, Oesterich JL, Gorden KB, Qiu X, McKane SW et al.: Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol 2002, 218:74-86. 22. Dzionek A, Inagaki Y, Okawa K, Nagafune J, Rock JJ, Sohma Y, Winkels G, Zysk M, Yamaguchi Y, Schmitz JJ: Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions(1). Hum Immunol 2002, 63:1133-1148. 23. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 2002, 168:4531-4537. 24. Rassa JC, Meyers JL, Zhang Y, Kudaravalli R, Ross SR: Murine retroviruses activate B cells via interaction with toll-like receptor 4. Proc Natl Acad Sci USA 2002, 99:2281-2286. 25. Bieback K, Lien E, Klagge IM, Avota E, Schneider-Schaulies J, Duprex WP, Wagner H, Kirschning CJ, Ter Meulen V, Schneider-Schaulies S: Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 2002, 76:8729-8736. 26. Compton T, Kurt-Jones EA, Boehme CW, Belko J, Latz E, Golenbock DT, Finberg RW: Human cytomegalovirus activates inflammatory cytokine responses via CD14 and toll-like receptor 2. J Virol 2003, in press. 27. Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S: Endotoxininduced maturation of MyD88-deficient dendritic cells. J Immunol 2001, 166:5688-5694. 28. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, Akira S: Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001, 167:5887-5894. 29. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ et al.: TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/ beta-dependent gene expression in macrophages. Nat Immunol 2002, 3:392-398. 30. Doyle S, Vaidya S, O’Connell R, Dadgostar H, Dempsey P, Wu T, Rao G, Sun R, Haberland M, Modlin R et al.: IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 2002, 17:251-263. This study identifies a subset of antiviral genes activated by stimulation with TLR3 and TLR4 ligands. IRF3 was shown to be the critical factor mediating selective upregulation of antiviral gene expression, and pretreatment with TLR3 and TLR4 ligands could potently inhibit viral replication. The authors suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs, and are capable of activating innate antiviral responses through IRF3.
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