Recognition of microbial infection by Toll-like receptors Elizabeth Kopp and Ruslan Medzhitov

396 Recognition of microbial infection by Toll-like receptors Elizabeth Kopp and Ruslan Medzhitov The Toll-like receptors (TLRs) of the innate immune system detect host invasion by pathogens and initiate immune responses. All of the TLRs use the adaptor to transduce a signal; however, two newly identified signaling molecules, TIRAP and TRIF, interact with a subset of the TLRs, suggesting a signaling specificity that may be relevant to the type of infection. Activation of the TRIF pathway, for example, leads to the production of antiviral gene products via the transcription factor, IRF3. In vivo experiments in TLR-deficient mice underscore the importance of TLRs in overcoming infection. Addresses Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA e-mail: Ruslan.Medzhitov@yale.edu 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 Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00080-3 Abbreviations DC dendritic cell IFN interferon IL interleukin interleukin-1-receptor-associated kinase IRF interferon-regulated factor LPS lipopolysaccharide MAP mitogen-activated protein PAMP pathogen-associated molecular pattern TIR Toll/interleukin-1 receptor TIRAP TIR-associated protein TLR Toll-like receptor TRAF TNF-receptor-associated factor TRIF TIR-domain-containing adapter inducing IFN-b Introduction The field of innate immunity has recently received much attention. It is now well established that the activation of adaptive responses requires direction from the innate immune system [1]. In particular, the family of innate immune signaling receptors, known as the Toll-like receptors (TLRs), has proven to be essential in the detection and signaling of infection [2,3]. The mammalian TLRs comprise a family of germlineencoded transmembrane receptors. These receptors recognize conserved microbial structures called PAMPs (pathogen-associated molecular patterns), which are invariant within a given class of microorganism. Many PAMPs have now been recognized and their respective TLRs identified; these include peptidoglycan (which binds to TLR2), synthetic double-stranded RNA (TLR3), lipopolysaccharide (LPS; TLR4), flagellin (TLR5), and CpG DNA motifs associated with bacterial DNA (TLR9). Upon ligation, TLRs signal intracellularly via their cytoplasmic Toll/ interleukin-1 receptor (TIR) domains to promote the transcription of genes involved in immune activation [3]. Over the past year, developments in the TLR field have focused on three main areas: identification of additional TLR ligands including putative endogenous ligands, further elucidation of components of individual TLR signaling pathways, and in vivo studies of the involvement of TLRs in the resistance to infections. As a result of these studies, we are now addressing the questions of how TLRs control adaptive responses and whether TLRs can convey qualitative information about the type of infection and thereby orchestrate a response appropriate to the invading pathogen. Recent studies have identified additional ligands for TLRs, and these have been summarized in other reviews [3]. It is worth noting, however, that some TLRs (TLR4, for example) can be triggered by several structurally dissimilar PAMPs, and that TLRs appear to recognize molecular features of bacteria, fungi and viruses, but apparently not of multicellular parasites. TLRs are said to detect microbial non-self because the PAMPs they detect are unlike molecular signatures of the host; TLR ligation therefore indicates the presence of an infection and initiates a signaling cascade that results in clearance of the microorganism. This obvious point is made here to emphasize that stimulation of TLRs in the absence of infection (i.e. via endogenous ligands) could result in the maturation of dendritic cells (DCs) presenting host peptides and the subsequent priming of T cells against self (autoimmunity). In support of this hypothesis, it was shown that B cells of autoimmune-prone mice can be activated via the inappropriate dual engagement of both the B-cell receptor and TLR9 [4 ]. In this case, immune complexes containing chromatin are taken up by B cells and delivered to endosomes where they can activate TLR9. This interaction is clearly unintended and results in deleterious consequences. Recently, several putative endogenous TLR ligands have been described and all appear to signal through TLR2 and TLR4. These include the heat shock proteins HSP60, HSP70 and gp96, the inducible host antibiotic b-defensin, and oligosaccharides from the breakdown of www.current-opinion.com

Recognition of microbial infection by TLRs Kopp and Medzhitov 397 endogenous extracellular matrix (ECM) [5,6]. Most of these studies were performed using recombinant protein preparations from which LPS is notoriously difficult to remove [7]. This technical problem complicates the assessment is the induction of TLR4 via an endogenous ligand or via a tightly bound (LPS) contaminant Nevertheless, ostensibly, Hsps could engage TLRs upon release from necrotic cells and thereby induce an inflammatory response. Likewise, the breakdown of ECM implies tissue damage resulting in an inflammatory response, and b-defensin is produced by cells that have already been activated and is thought to participate in a positive feedback loop. Although it is theoretically possible that these molecules activate TLRs, it is difficult to imagine the benefit to the host of such a situation. We favor a hypothesis that, if TLRs do indeed recognize endogenous ligands, injury in the absence of infection would stimulate genes involved in tissue repair, not the activation of antigenspecific adaptive immunity [8]. Alternatively, chaperonins such as Hsps and other endogenous ligands may inadvertently stimulate TLRs by virtue of their ability to bind them. Indeed, the endoplasmic reticulum (ER) chaperone gp96 stabilizes TLR2 and TLR4 during their export to the cell surface [9]. In this scenario, gp96 is not a ligand for TLR2; instead, TLR2 is a substrate for gp96. As TLRs are active when overexpressed or artificially forced to dimerize, it is possible that Hsps released from necrotic cells could produce the same effect by binding to and concentrating TLRs on the cell surface. Much attention has been given to the ability of PAMPs to induce the maturation of DCs, which are now well recognized as the most important of the professional antigen presenting cells. In addition to secreting cytokines and presenting peptides to T cells, mature DCs provide T cells with a required second signal by expressing co-stimulatory molecules on their surface. The maturation of DCs relies on TLR ligation; TLRs, therefore, essentially permit an adaptive response [1,10]. We now know that ligation of DC TLRs regulates the adaptive immune response in two ways. In addition to controlling the expression of costimulatory molecules on DCs, TLRs induce DCs to relieve suppression of effector T cells by regulatory T cells [11]. Regulatory or suppressor T cells (T regs) are CD4 þ CD25 þ cells that suppress the activity of self-reactive T cells in the periphery. Suppression can be relieved, however, by the production by mature DCs of soluble factors, in particular, IL-6 [11]. Thus, by restricting IL-6 production to TLR-stimulated DCs, suppression is only relieved in the presence of infection. Pathogen-specificT cells may then be activated by these same TLR-induced DCs expressing co-stimulatory molecules in the context of foreign peptide presentation. TLR signaling specificities One indication that TLRs direct the type of adaptive response to a particular pathogen would be that there are differences in the signal transduction pathways between individual TLRs. The canonical signaling pathway for TLRs following PAMP ligation involves the interaction of the adaptor molecule,, with the TLR. has both a TIR domain and a death domain, and the recruitment of to a TLR occurs via a TIR TIR homotypic interaction. The death domain of then binds the death domain of a serine/threonine kinase, usually interleukin-1-receptor-associated kinase (), and the signal is propagated via a specific member of the TNF-receptor-associated factor (TRAF) family,, ultimately leading to the activation of NF-kB and mitogenactivated protein (MAP) kinases, and the transcription of immunologically-relevant genes (Figure 1). Several genes have now been discovered; -1 and -4 both have kinase activity [12,13], whereas -2 and -M do not [14,15]. -M appears to operate as a negative regulator of the TLR signal transduction pathway and might contribute to the phenomenon of LPS tolerance, in which cells chronically exposed to LPS become refractory to further stimulation [16 ]. -1, -2, and -4 may cooperate with one another to transduce a signal; only -4 appears to have a non-redundant role in NF-kB activation, however [17 ]. All of the TLRs, as well as the IL-1 receptor and the IL-18 receptor, are thought to use to transduce signals. As a result, -deficient mice have been an invaluable tool for analyzing global TLR loss-of-function [18]. Studies with these mice revealed that there are differences in signal transduction pathways between individual TLRs. LPS (a TLR4 ligand), for example, can induce the upregulation of maturation markers on DCs from -deficient mice, but cannot stimulate the release of cytokines from these cells [19]. Interestingly, NF-kB is induced in response to LPS, although with delayed kinetics [19,20], indicating that cytokine production is not strictly dependent on NF-kB activity. To date, the biological consequences of this delayed activation are unknown. For other TLRs with known ligands, namely TLR2 [21], TLR5 (E Kopp and R Medzhitov, unpublished observations) and TLR9 [19,22], is essential for both upregulation of maturation markers and induction of cytokine production. In addition, TLR4 (but not TLRs1, 2, 5, or 6) could induce interferonregulated factor 3 (IRF3), a transcription factor essential for the production of IFN-b and the antiviral response [23]. These results led to the speculation that another adaptor molecule, presumably containing a TIR domain, participates in signaling downstream of TLR4. Indeed, we and others identified and cloned a second TIR-associated protein (TIRAP, also called Mal) and showed that a dominant-negative form of TIRAP could selectively inhibit TLR4 signaling, but not TLR9 signaling [24,25]. TIRAP-deficient mice were generated and were found to be impaired in NF-kB and MAP kinase activation, and cytokine production downstream of TLR1, TLR2 and www.current-opinion.com

398 Host-pathogen interactions Figure 1 TLR5 TLR7, TLR8, TLR9 TLR2/1, TLR2/6 TLR3 TLR4 TIRAP TRIF TRIF TIRAP IFN-β IRF3 IFN-β Delayed IRF3 IFN-β Delayed TIR domain Death domain Kinase domain Current Opinion in Immunology Schematic of mammalian TLR signaling pathways. All TLRs are thought to signal through a pathway to induce NF-kB and MAP kinases. The -dependent pathway downstream of TLR4 and TLR2 also requires TIRAP. TRIF interacts with TLR3 and induces IFN-b by activating IRF3. Ligands for TLR7, TLR8, TLR9 and TLR4 also induce IFN-b, although it is unclear whether TRIF is involved in these pathways. The TLR3 and TLR4 pathways can induce NF-kB and MAP kinases in the absence of with delayed kinetics. Question marks indicate the possible presence of additional signaling molecules. TLR6 in addition to TLR4 (Figure 1). However, the expression of DC maturation markers and the induction of IRF3 was intact in response to LPS, strongly suggesting that the -independent pathway did not involve TIRAP [26,27 ]. Indeed, the signaling phenotype of these mice is very similar to that of the / mice downstream of TLR4; impaired cytokine production but normal induction of co-stimulatory molecules on dendritic cells and no defect in IRF3 activation. A third TIR-containing adaptor molecule, TIR-domaincontaining adapter inducing IFN-b (TRIF, also called TIR-containing adaptor molecule [TICAM-1]) has now been characterized [28,29 ]. TRIF was identified by sequence searches and by a yeast two-hybrid screen using TLR3. Preliminary evidence implicates TRIF in the induction of IRF3 by TLR3 and possibly by other TLRs (Figure 1). When overexpressed, TRIF strongly induces the IFN-b promoter, whereas TIRAP and do not. Dominant-negative constructs of TRIF inhibit the NF-kB activation of TLRs 2,3,4 and 7, and the IFN-b promoter activation by TLR3. In addition, TRIF can bind TLR3 (and possibly TLR2) directly [28,29 ].Confirmation of the pathways affected by TRIF must await the generation of TRIF-deficient mice. In the case of TLR3 signaling, however, the use of TRIF to transduce a signal to antiviral genes is conceptually appealing, as TLR3 responds to poly I:C, a synthetic mimic for double-stranded viral RNA. The role of TLRs in host defense in vivo Although the demonstration that PAMPs could activate distinct signaling pathways in DCs and other cells was strongly suggestive of an important role for TLRs in combating infection, it has been difficult to prove this in vivo with individual TLR-deficient strains; so far, these studies have only been conducted in TLR2 /, TLR4 /, / and 4 / mice. This difficulty may be due in part to the possibility that a microbial infection might stimulate several TLRs, creating some redundancy in the detection system. Nevertheless, now that we have a better idea of the ligands specific to each TLR and their individual signaling pathways, it is easier to understand the in vivo results. and other intracellular signaling molecules The -dependent pathway is critical to the production of inducible inflammatory cytokines, such as IL-12, TNF and IL-6. Furthermore, several TLR signal transduction pathways appear to utilize as their sole receptor-proximal adaptor. It could be predicted, then, that -deficient animals would be particularly susceptible to infection by microbes bearing PAMPs that utilize these TLRs. Gram-positive bacteria, for example, which could be expected to signal through TLRs 2, 6 and 9, should therefore be relatively unchecked in knockout mice. Indeed, is essential for clearance www.current-opinion.com

Recognition of microbial infection by TLRs Kopp and Medzhitov 399 of both Listeria monocytogenes [30,31] and Stapholococcus aureus [32] in vivo. In addition, / mice have a reduced resistance to the intracellular protozoan Toxoplasma gondii, suggesting that TLRs are also involved in recognizing this pathogen [33]. Based on the model of signal transduction pathways leading from TLR to effector response, -4 / animals would be expected to have a similar phenotype to / animals with respect to the production of cytokines necessary for the clearance of specific pathogens in vivo. This prediction appears to be true, as these animals resist LPS-induced septic shock (resulting from massive cytokine production) and succumb to S. aureus infection. They also produce reduced levels of IFN-g in response to lymphocytic choriomeningitis virus (LCMV) infection in vivo ([17 ], see also Update). TLR2 Immune defects have also been observed in TLR2- mutant mice. TLR2 signals as a heterodimer with TLR1 or TLR6 and recognizes a diverse set of microbial products from gram-negative and gram-positive bacteria, fungi, spirochetes and mycobacteria. TLR2-deficient mice are more susceptible than wild-type mice to several of these pathogens, including the gram-positive bacteria S. aureus [32] and Streptococcus pneumoniae [34,35]. In addition, TLR2-deficient mice are less resistant to high-dose challenge with Mycobacterium tuberculosis than wild-type animals [36]. In one study, a quarter of the patients suffering from the mycobacterial disease lepromatous leprosy had a particular polymorphism in a conserved region of the intracellular domain of TLR2. The mutation was found neither in control subjects nor in those suffering from tuberculoid leprosy [37]. TLR2 is also required for recognition of the OspA lipoprotein from Borrelia burgdorferi. Expression of the TLR2 binding partner, TLR1 was diminished in patients that made low antibody titers to OspA following vaccination, suggesting that TLR2 TLR1 heterodimers are required for mounting a productive OspA-specific antibody response against this antigen [38]. In vivo studies have substantiated the important role of TLR2 in the innate host response to Borrelia, as TLR2 / animals incur a much higher spirochete burden than wild-type animals. However, other TLRs may also play a role, as these mice still develop normal antibody responses against Borrelia [39]. TLR4 TLR4-deficient mice and TLR4 lack-of-function mice have been used to study the in vivo responses of different types of microbes. These studies have shown that TLR4 plays a role in host defense against bacterial, mycobacterial, fungal and viral infections. The lack of TLR4 correlates with defective clearance of aerosolized Haemophilus influenzae, illustrated by lower levels of cytokines and chemokines in alveolar lavage and defective neutrophil recruitment in the lung [40]. TLR4 is important for host defense against Salmonella infection [41] and is also required to control Mycobacterium tuberculosis infection [42], although another study found TLR2 to be the dominant pathway for controlling a high dose infection [36]. TLR4-null animals are more susceptible to Candida albicans infection and exhibit reduced secretion of the chemokines KC and macrophage inflammatory protein (MIP)-2 in addition to a subsequent decrease in neutrophil recruitment relative to wild-type animals [43]. TLR4-null mice have impaired responses to respiratory syncytial virus (RSV) by measurements of NK cell function, IL-12 production and viral clearance [44 46]. By contrast, these mice had little defect in their responses to influenza virus infection [44,45]. Conclusions and future directions In vitro and in vivo studies have now directly demonstrated that TLRs recognize microbial components, stimulate the maturation of dendritic cells and the production of cytokines and chemokines by leukocytes, relieve the suppression of regulatory T cells, and are important factors in host defense against several identified pathogens. The fact that antiviral compounds can activate TLR7 and TLR8 [47,48], and that synthetic double-stranded RNA can trigger TLR3, implies a role for these TLRs in detection and signaling of viral infections. If their functions do not overlap it will be interesting to observe their relative roles in the in vivo host response to viral infection. The generation of TRIF / mice will be important in determining the prevalence of this adaptor in signaling downstream of TLRs other than TLR3. It is possible that double knockouts of / /TRIF / or -4 / /TRIF / may prove to be the functional equivalent of complete loss-offunction for all TLRs. The question of whether TLRs can qualitatively inform the adaptive response of the type of infection is still open ended. The discovery of differences in the signal transduction pathways of individual TLRs suggests that they may induce different effector responses. However, genechip analysis of cells treated with specific PAMPshas revealed that gene expression is largely redundant between TLRs. One exception is the induction of antiviral gene expression via TLR3 and TLR4 [49,50 ]. TLR7 and TLR8, which respond to antiviral compounds, might also be expected to induce a specific antiviral response. Update An interesting recent report describes three children with ongoing life-long pyogenic bacterial infections [51]. The authors identified loss-of-function mutations in the -4 gene in all of these children, thus illustrating in humans the importance of proper TLR signaling in combating these infections. Although the children are experiencing fewer bacterial infections as they get older, www.current-opinion.com

400 Host-pathogen interactions and have been able to clear viral, fungal and parasitic infections, caution should be used when drawing general conclusions about the types of infections TLRs or the TIR pathways control on the basis of these patients. There are other signaling pathways downstream of TLRs that don t require -4. Importantly, the activation of IRF-3 and the induction of IFN-a and IFN-b are not dependent on -4, and therefore this important antiviral pathway is not affected in these patients. Furthermore, unlike laboratory mice with targeted gene deletions living in controlled conditions, these children have received intense medical treatment throughout their lifetimes. Indeed, the fact that so few patients with -4 deficiency exist indicates the essential role of TLRs in innate immunity. Acknowledgements The authors would like to thank Tiffany Horng for help in preparing the figure. 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. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R: Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001, 2:947-950. 2. Medzhitov R: Toll-like receptors and innate immunity. Nat Rev Immunol 2001, 1:135-145. 3. Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol 2003, 21:335-376. 4. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A: Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 2002, 416:603-607. This paper demonstrates that engagement of TLR9 on B cells by chromatin-containing immune complexes can lead to the activation of autoreactive B cells. 5. Beg AA: Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol 2002, 23:509-512. 6. Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ et al.: Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 2002, 298:1025-1029. 7. Gao B, Tsan MF: Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages. J Biol Chem 2003, 278:174-179. 8. Li M, Carpio DF, Zheng Y, Bruzzo P, Singh V, Ouaaz F, Medzhitov RM, Beg AA: An essential role of the NF-kappa B/Tolllike receptor pathway in induction of inflammatory and tissuerepair gene expression by necrotic cells. J Immunol 2001, 166:7128-7135. 9. Randow F, Seed B: Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat Cell Biol 2001, 3:891-896. 10. Kaisho T, Akira S: Dendritic-cell function in Toll-like receptorand -knockout mice. Trends Immunol 2001, 22:78-83. 11. Pasare C, Medzhitov R: Toll pathway-dependent blockade of CD4RCD25R T cell-mediated suppression by dendritic cells. Science 2003, 299:1033-1036. 12. Cao Z, Henzel WJ, Gao X: : a kinase associated with the interleukin-1 receptor. Science 1996, 271:1128-1131. 13. Li S, Strelow A, Fontana EJ, Wesche H: -4: a novel member of the family with the properties of an -kinase. Proc Natl Acad Sci USA 2002, 99:5567-5572. 14. Muzio M, Ni J, Feng P, Dixit VM: (Pelle) family member -2 and as proximal mediators of IL-1 signaling. Science 1997, 278:1612-1615. 15. Wesche H, Gao X, Li X, Kirschning CJ, Stark GR, Cao Z: -M is a novel member of the Pelle/interleukin-1 receptor-associated kinase () family. J Biol Chem 1999, 274:19403-19410. 16. Kobayashi K, Hernandez LD, Galan JE, Janeway CA Jr, Medzhitov R, Flavell RA: -M is a negative regulator of Tolllike receptor signaling. Cell 2002, 110:191-202. -M is a kinase-inactive family member expressed in monocytes and macrophages, and is induced upon TLR stimulation. Surprisingly, this inhibits cytokine production resulting from TLR signaling. -Mdeficient mice have enhanced inflammatory responses to bacterial infection, indicating that -M dampens this response. 17. Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C, Takada H, Wakeham A, Itie A, Li S et al.: Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking -4. Nature 2002, 416:750-756. In this paper, the authors generate and analyze -4 knockout mice, revealing an essential role for -4 in TLR signaling. These mice have striking impairments in the ability to produce cytokines (IL-1, TNF and IL- 6) both in vitro and in vivo as a result of IL-1 or TLR stimulation. The animals are also resistant to lethal septic shock (a TLR4 response) and are dramatically more susceptible to bacterial and viral infections than wildtype animals. 18. Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S: Targeted disruption of the gene results in loss of IL-1- and IL-18-mediated function. Immunity 1998, 9:143-150. 19. Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S: Endotoxininduced maturation of -deficient dendritic cells. J Immunol 2001, 166:5688-5694. 20. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S: Unresponsiveness of -deficient mice to endotoxin. Immunity 1999, 11:115-122. 21. Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K, Morr M, Muhlradt PF, Akira S: Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophageactivating lipopeptide-2 activates immune cells through a tolllike receptor 2- and -dependent signaling pathway. J Immunol 2000, 164:554-557. 22. Schnare M, Holt AC, Takeda K, Akira S, Medzhitov R: Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein. Curr Biol 2000, 10:1139-1142. 23. Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt PF, Sato S, Hoshino K, Akira S: Lipopolysaccharide stimulates the - independent pathway and results in activation of IFNregulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001, 167:5887-5894. 24. Horng T, Barton GM, Medzhitov R: TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2001, 2:835-841. 25. Fitzgerald KA, Palsson-McDermott EM, Bowie AG, Jefferies CA, Mansell AS, Brady G, Brint E, Dunne A, Gray P, Harte MT et al.: Mal (-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 2001, 413:78-83. 26. Yamamoto M, Sato S, Hemmi H, Sanjo H, Uematsu S, Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T et al.: Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 2002, 420:324-329. See annotation to [27 ]. 27. Horng T, Barton GM, Flavell RA, Medzhitov R: The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 2002, 420:329-333. References [26,27 ] describe the phenotype of TIRAP deficient mice. These mice have impaired B-cell proliferation and cytokine production in www.current-opinion.com

Recognition of microbial infection by TLRs Kopp and Medzhitov 401 response to stimulation with TLR2 and TLR4 ligands but not with other TLR ligands, indicating differences in the signal transduction pathways among TLRs. 28. Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T: TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol 2003, 4:161-167. See annotation to [29 ]. 29. Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, Takeda K, Akira S: Cutting edge: a novel Toll/IL-1 receptor domaincontaining adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol 2002, 169:6668-6672. References [28,29 ] describe a novel adaptor protein that links TLR activation with the induction of IRF3. Because IRF3 is involved in IFN-b production, TLRs utilizing this adapter could potentially stimulate an antiviral response. TRIF/TICAM appears to operate downstream of a only a subset of TLRS. Both papers demonstrate that TRIF/TICAM can interact directly with TLR3 and can activate the IFN-b promoter. 30. Edelson BT, Unanue ER: -dependent but Toll-like receptor 2-independent innate immunity to Listeria: no role for either in macrophage listericidal activity. J Immunol 2002, 169:3869-3875. 31. Seki E, Tsutsui H, Tsuji NM, Hayashi N, Adachi K, Nakano H, Futatsugi-Yumikura S, Takeuchi O, Hoshino K, Akira S et al.: Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice. J Immunol 2002, 169:3863-3868. 32. Takeuchi O, Hoshino K, Akira S: Cutting edge: TLR2-deficient and -deficient mice are highly susceptible to Staphylococcus aureus infection. J Immunol 2000, 165:5392-5396. 33. Scanga CA, Aliberti J, Jankovic D, Tilloy F, Bennouna S, Denkers EY, Medzhitov R, Sher A: Cutting edge: is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J Immunol 2002, 168:5997-6001. 34. Echchannaoui H, Frei K, Schnell C, Leib SL, Zimmerli W, Landmann R: Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation. J Infect Dis 2002, 186:798-806. 35. Koedel U, Angele B, Rupprecht T, Wagner H, Roggenkamp A, Pfister HW, Kirschning CJ: Toll-like receptor 2 participates in mediation of immune response in experimental pneumococcal meningitis. J Immunol 2003, 170:438-444. 36. Reiling N, Holscher C, Fehrenbach A, Kroger S, Kirschning CJ, Goyert S, Ehlers S: Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. J Immunol 2002, 169:3480-3484. 37. Kang TJ, Chae GT: Detection of Toll-like receptor 2 (TLR2) mutation in the lepromatous leprosy patients. FEMS Immunol Med Microbiol 2001, 31:53-58. 38. Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J, Schoen RT, Medzhitov R, Fikrig E, Flavell RA: Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 2002, 8:878-884. 39. Wooten RM, Ma Y, Yoder RA, Brown JP, Weis JH, Zachary JF, Kirschning CJ, Weis JJ: Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi. J Immunol 2002, 168:348-355. 40. Wang X, Moser C, Louboutin J-P, Lysenko ES, Weiner DJ, Weiser JN, Wilson JM: Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung. J Immunol 2002, 168:810-815. 41. Bernheiden M, Heinrich JM, Minigo G, Schutt C, Stelter F, Freeman M, Golenbock D, Jack RS: LBP, CD14, TLR4 and the murine innate immune response to a peritoneal Salmonella infection. J Endotoxin Res 2001, 7:447-450. 42. Abel B, Thieblemont N, Quesniaux VJ, Brown N, Mpagi J, Miyake K, Bihl F, Ryffel B: Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J Immunol 2002, 169:3155-3162. 43. Netea MG, Van Der Graaf CA, Vonk AG, Verschueren I, Van Der Meer JW, Kullberg BJ: The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J Infect Dis 2002, 185:1483-1489. 44. 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. 45. 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. 46. Haeberle HA, Takizawa R, Casola A, Brasier AR, Dieterich HJ, Van Rooijen N, Gatalica Z, Garofalo RP: Respiratory syncytial virusinduced activation of nuclear factor-kappab in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways. J Infect Dis 2002, 186:1199-1206. 47. 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 - dependent signaling pathway. Nat Immunol 2002, 3:196-200. 48. 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. 49. 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. See annotation to [50 ]. 50. 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. References [49,50 ] identified that antiviral genes are induced by TLR3 and TLR4, but not by TLR2 or TLR9. By utilizing gene chip analysis, the authors discovered a broad set of genes dependent on NF-kB that are induced by apparently all four of these TLRs, and a subset of genes that are dependent on IRF3 that are induced by only TLR3 and TLR4. 51. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, Soudais C, Dupuis S, Feinberg J, Fieschi C et al.: Pyogenic bacterial infections in humans with -4 deficiency. Science 2003, 299:2076-2079. www.current-opinion.com