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

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1 396 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 Ruslan.Medzhitov@yale.edu This review comes from a themed issue on Host pathogen interactions Edited by Robert L Modlin and Peter Doherty /$ see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI /S (03) 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

2 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

3 398 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

4 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,

5 400 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. 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