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Toll signaling pathways in the innate immune response Kathryn V Anderson The Toll signaling pathway, which is required for the establishment of the dorsal–ventral axis in Drosophila embryos, plays an important role in the response of larval and adult Drosophila to microbial infections. Recent genetic evidence has shown that a mammalian Toll-like receptor, mouse Tlr4, is the signal transducing receptor activated by bacterial lipopolysaccharide. Thus, Toll-like receptors appear to detect a variety of microbial components and to trigger a defensive reaction in both Drosophila and mammals. Genetic data from both Drosophila and mice have defined components required for activation of Toll-like receptors and for the downstream pathways activated by the Toll-like receptors. Addresses Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA; e-mail: [email protected] Current Opinion in Immunology 2000, 12:13–19 0952-7915/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations IKK IκB kinase IRAK IL-1-receptor-associated kinase LPS lipopolysaccharide LRR leucine-rich repeat LTA lipoteichoic acid MAPK mitogen-activated protein kinase NIK NF-κB-inducing kinase PAMP pathogen-associated molecular pattern TIR Toll/IL-1-receptor homologous region TLR Toll-like receptor α TNF-α tumor necrosis factor α TRAF6 TNF-receptor-associated factor 6

females hatched. When Wieschaus showed the cuticle pattern of the unhatched embryos to Nüsslein-Volhard, she exclaimed, “Toll!” (a German slang term comparable to crazy or ‘far-out’), and the gene became known by that name. The phenotype was extraordinary because it was dominant and ventralized, clearly the opposite of recessive dorsalizing mutations in two genes that Nüsslein-Volhard knew very well, dorsal and gastrulation defective. With two opposing mutant phenotypes, it was possible to start building genetic pathways, and soon it was clear that dorsal acted downstream of Toll, whereas gastrulation defective acted upstream. Another nine genes with related phenotypes were discovered in systematic genetic screens; double mutant analysis ordered the genes into a linear pathway of action [1]. All 12 of the Drosophila genes in this embryonic signaling pathway have now been characterized on a molecular level [2]. In the Drosophila embryo, a proteolytically processed product of the spätzle gene activates Toll. Spätzle is a novel protein, with a pattern of cysteine residues that suggest it would form a cysteine-knot structure [3]. Spätzle is secreted as an inactive precursor that is activated by proteolytic cleavage. The activation of Spätzle is regulated by a protease cascade that includes at least four serine proteases of the trypsin family. Activation of Toll leads, through steps requiring Tube and Pelle, to the degradation of Cactus, a Drosophila IκB protein, which is complexed with Dorsal, a Drosophila Rel/NF-κB protein. Dorsal, once released from the complex with Cactus, moves into embryonic nuclei where it activates a set of specific target genes and represses another set.

Introduction All animals can mount rapid defense responses that kill invading microbes. As described in this review, a remarkable convergence of Drosophila developmental genetics, mouse genetics, large-scale sequencing projects and DNA transfection experiments has revealed that Toll signaling pathways are key mediators of the response to bacteria and fungi in both Drosophila and mammals.

The Drosophila Toll pathway: embryonic patterning Toll was discovered as an essential component of the pathway that establishes the dorsal–ventral axis of the early Drosophila embryo. If any component in that genetic pathway is missing, no ventral or lateral cell types develop; these embryos lack all mesoderm and the entire nervous system. The first mutants in Toll were discovered in the genetic screens performed by Nüsslein-Volhard and Wieschaus. While searching for zygotic lethal mutations that affected embryonic patterning, Wieschaus discovered a line in which none of the embryos laid by heterozygous

The Drosophila Toll pathway: response to microbial infection The role of the Toll pathway in the Drosophila immune response could not have been predicted from its role in early development and came instead from the studies of promoters of genes induced in response to infection. One of the principal responses of insects to microbial infection is the transcriptional induction of a battery of genes encoding antimicrobial peptides. All the antimicrobial peptide genes include κB elements in their upstream regions [4], suggesting that NF-κB/Rel proteins might be activators of insect immune responses. Dorsal was the first insect protein known that would regulate transcription through κB sites. Indeed, nuclear localization of Dorsal, and two more recently described Drosophila Rel proteins, Dif and Relish, are all rapidly activated in response to infection ([5–8]; LP Wu, KV Anderson, unpublished data). Subsequent genetic experiments showed that Spätzle, Toll, Pelle, Tube and Cactus are all required for the rapid transcriptional induction of the gene encoding an antifungal peptide, Drosomycin, in

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genes related to Toll have been reported [13–15]; more are certain to appear as human genome sequencing accelerates. As described below, all of these Toll genes are candidates to mediate innate immune responses.

Figure 1

TLR MyD88 IRAK

TRAF6

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Signalsome

IKKα/β PO4

SCF–ubiquitin ligase complex

IκB

NF-κB

Ubiquitin PO4

IκB

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Slimb Current Opinion in Immunolgy

Signaling downstream of Toll receptors is mediated by several protein complexes. The first complex includes the activated TLR, an adaptor molecule such as MyD88 (or Tube in Drosophila), and a SIIK (serinethreonine innate immunity) kinase such as IRAK (or Pelle in Drosophila). The activated TLR complex in turn activates the IKK/signalsome complex in a step mediated by TRAF6. The IKK complex includes NIK, which phosphorylates and activates IKKβ; activated IKKβ phosphorylates target serines in the amino-terminal domain of IκB. Once phosphorylated, the IκB–NF-κB complex becomes associated with a ubiquitin ligase (in which the recognition subunit is called βTrCP in mammals and Slimb in Drosophila). This complex ubiquitinates IκB, targeting it for degradation by the proteasome. Once IκB is degraded, the nuclear localization signal of NF-κB is revealed and NF-κB moves to the nucleus, where it can activate target genes.

response to infection [9]. In addition, another Toll family member, 18-wheeler, acts in a parallel signaling pathway to mediate specific responses to infection [10].

Families of Toll-like receptors and pathways in flies and mammals Large-scale sequencing projects identified families of Toll genes in both Drosophila and humans. Currently, seven Drosophila genes related to Toll have appeared in the partial genome sequence [11,12]; because approximately half the Drosophila genome sequence is available at this time, it is likely that there will be still more fly genes. Six human

All members of the Toll family are membrane proteins that cross the membrane once and share similar extracellular domains, which include 18–31 leucine-rich repeats (LRRs), and similar cytoplasmic domains of approximately 200 amino acids, which are also similar to the cytoplasmic domain of the IL-1 receptor (the TIR [Toll/IL-1-receptor homologous region]). The cytoplasmic domains of the Toll family define a subclass of TIR domains. The TIR domain of human Toll-like receptor (TLR)4, for example, is 32% identical to Drosophila Toll, and less similar (20% identical) to the TIR domain of the IL-1 receptor. The extracellular domains of the Toll family proteins are large (550–980 amino acids) and could possibly contain more than one ligand-binding domain. Genetic evidence from Drosophila suggests that the aminoterminal half of the extracellular domain of Toll can bind ligand [16,17]. Deletion of most of the LRR region or mutation of one of the four cysteine residues just outside the transmembrane domain leads to constitutive activity of the receptors [13,16,18]. The extracellular domains of the Toll family are quite divergent (the extracellular domains of TLR2 and TLR4 are only 24% identical), making it likely that different ligands activate different receptors. The divergence of extracellular domains is striking even between homologous genes in mice and humans; for example, the extracellular domains of human TLR4 and mouse Tlr4 are only 53% identical, whereas their cytoplasmic domains are 83% identical. The relatively rapid divergence of extracellular domains is reminiscent of the rapid divergence of the LRR sequences found in many of the plant disease genes. In plants, as in animals, LRR-TIR proteins are important in the host response to infection [19]. Comparison between strains has revealed that the LRRs are responsible for specificity of host–pathogen interaction [20•] and suggest that polymorphism between individuals generates sequence diversity that is important at the populationlevel in the ‘arms race’ between host and pathogen [21]. Sequence comparisons of the LRRs of Toll family members within a species will help determine whether rapid sequence changes in the LRRs in animals could aid in the recognition of rapidly evolving pathogens.

Microbial cell wall components as activators of Tlrs Specific components of microbial cell walls are strong activators of innate immune responses. These molecules, the pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) of Gram-negative bacteria, peptidoglycan and lipoteichoic acid (LTA) of Gram-positive bacteria and mannans of fungi, are recognized as foreign by all animals. In mammals, the PAMPs trigger the production

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Figure 2 LPS, LTA? 18-wheeler

Mannans?

Toll

Rel dimer B

LPS Other Toll-like receptor?

Rel dimer C (Relish–Relish?)

Rel dimer A (Dif–Relish?)

Attacin Drosomycin Diptericin Current Opinion in Immunology

A model for the action of multiple Toll receptors. This model is based on data on activation of antimicrobial peptide gene expression in the Drosophila fat body cells (see text for references). In the model, different PAMPs activate different Toll family receptors: a yeast PAMP, for example, activates Toll, while LPS activates another TLR. Activation of each Toll triggers a specific downstream signaling complex that controls the activation of a particular Rel dimer. Toll regulates activation of a Relish–Dif dimer. Each activated Rel dimer then binds with greatest affinity to the regulatory regions of particular target genes; a Relish–Dif dimer activates Drosomycin transcription and a Relish–Relish homodimer activates target genes including Diptericin.

of cytokines including IL-1 and TNF-α. These cytokines are important in the response to infection, but excessive activation of these inflammatory cytokines leads to septic shock, a leading cause of death in patients with bacterial infections [22]. Despite their medical importance, the signal-transducing receptors that mediate responses to PAMPs had not previously been identified. The first experiments to implicate mammalian Toll receptors in responses to PAMPs came from transfection of the cloned receptors into cell lines that could not respond to LPS. In these experiments, human TLR2 (but not TLR4) was sufficient to confer responsiveness of an embryonic kidney cell line to LPS [23•,24•]. In the transfected cells, LPS induced rapid activation of a κB reporter gene and transcription of the inflammatory cytokine IL-8. A strong response required both LPS-binding protein (LBP) and CD14, which binds LPS with high affinity. However, direct binding of LPS to TLR2 was of low affinity, with a dissociation constant (Kd) of 500–700 nM. In contrast, LPS is active in picomolar concentrations, suggesting that accessory proteins are required for LPS activation of TLR2 in vivo.

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affected by the lps mutation is the macrophage. Mutant macrophages do not secrete inflammatory cytokines, fail to phagocytose opsonized particles, and fail to produce reactive oxygen species or nitric oxide in response to LPS stimulation [25,26]. Two mutant alleles of the lps gene have been identified: a semidominant allele (lpsd) arose in the C3H/HeJ strain and a second, recessive allele is present in the C57BL/10ScN and C57BL/10ScCr strains. Mice that are homozygous for mutant alleles of lps are sensitive to infection by Gram-negative bacteria and are resistant to LPS-induced septic shock. Two groups cloned the lps locus by map-based methods [27••,28,29••] and demonstrated that the mutations alter the mouse Tlr4 gene in both strains. The recessive allele is a deletion of the region including the Tlr4 gene, and the semidominant allele in C3H/HeJ is associated with a proline to histidine missense mutation in the cytoplasmic TIR domain of Tlr4. The lps phenotype was recapitulated by a targeted mutation in the Tlr4 gene [30]. The apparent conflict between the biochemical experiments, in which TLR4 failed to confer responsiveness to LPS, and the genetic data, which indicated that the gene is required for LPS responsiveness, points out the dangers of overinterpretion of transfection data. Subsequent experiments showed that TLR4 can mediate activation of a κB reporter in response to LPS in the appropriate cell line [31]. The role of TLR2 in vivo remains unclear. When transfected into appropriate cell lines, TLR2 can be activated by a variety of microbial components. In addition to LPS, the activating components of Gram-positive bacteria (e.g. peptidoglycan and lipoteichoic acid) can activate NF-κB in cells transfected with TLR2 [32–36]. Analysis of the phenotypes produced by targeted mutations in the mouse Tlr2 gene, as well as the other mouse Tlr genes, will be essential in determining which molecules activate particular receptors.

Do pathogen-associated molecular patterns directly activate Toll receptors? Do microbial components such as LPS and LTA act as ligands for Toll receptors? Or do they act indirectly to promote the production of classical ligands? The binding affinities of the Toll receptors for LPS–CD14–LBP complexes are lower than would be expected if LPS acts as a direct ligand for TLRs on their own. A novel secreted protein, MD-2, was found to increase the response of TLR4 to LPS [37]. Such accessory proteins might increase the affinity TLRs for ligands such as LPS.

Classical mouse genetics steps in The importance of TLRs in the response to LPS was confirmed by results of the positional cloning of the classical mouse lps mutant, which demonstrated that the Tlr4 gene, rather than Tlr2, is required for response to LPS. Mutations in the lps gene prevent most of the normal responses to LPS: for example, lps mutant B cells fail to proliferate in response to LPS. However, the most significant cell type

An alternative model for activation by PAMPs is suggested by the genetic pathway worked out in the Drosophila embryo, where active Spätzle, the apparent Toll ligand, is produced by localized proteolytic processing of an inactive secreted precursor protein, triggered by a locally modified peptidoglycan [2,38]. In the immune response, LPS and other PAMPs might trigger protease cascades that activate

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classical protein ligands. The proteases that are required for activation of Spätzle in the embryo are not required in the immune response [10]. However, unidentified proteases do seem to be required for processing of Spätzle in response to infection: a mutation in a Drosophila serine protease inhibitor causes constitutive processing of Spätzle and activation of the Toll pathway even in the absence of infection [39••]. Thus, there are at least two modes of TLR activation, which are not mutually exclusive. LPS might bind directly to a specific TLR, or LPS might activate a protease cascade that leads to the rapid production of a classical ligand for a specific TLR. In either case, it is clear that there must be additional components required for TLR activation beyond those identified to date.

Protein complexes mediate activation of Rel proteins by signals from Toll receptors The genetic analysis of the Drosophila embryo and the biochemical experiments in mammalian cells have defined a sequence of events that occur after activation of Toll receptors that lead to nuclear localization of NF-κB/Rel proteins. Signaling by Toll receptors in both Drosophila and in mammalian cells is thought to be mediated by three protein complexes: a complex on the receptor, a complex that phosphorylates the inhibitor protein IκB, and a complex that degrades IκB (Figure 1). In response to signaling, a complex assembles on the receptor itself. This complex includes an adaptor protein (MyD88 in mammals and Tube in Drosophila) and a kinase of the SIIK (serine-threonine innate immunity kinase) family (IRAK [IL-1-receptor-associated kinase] in mammals and Pelle in Drosophila). Genetic evidence in Drosophila shows that Tube and Pelle are required for activation of Dorsal in the embryo and for induction of Drosomycin in response to infection [10,40]. MyD88, a cytoplasmic protein that contains a TIR domain, is part of a protein complex with the cytoplasmic domain of the activated IL-1 receptor [41,42] and has been implicated in TLR signaling by expression of dominant-negative forms of MyD88 [43]. In addition, a targeted mutation in the mouse MyD88 gene prevents the initial response to LPS [44•]. The similarity of the Tlr4 and MyD88 mutant phenotypes argues that they act in the same biochemical process. The role for IRAK in Toll signaling is less straightforward. There are three mouse IRAK genes [41,45], and mice that lack IRAK1 have only subtle deficiencies in responses to IL-1 [46,47]. IRAK1 mutants have not been tested for their responses to LPS or other PAMPs. In mammalian cells, TRAF6 (TNF-receptor-associated factor 6) is thought to link the receptor-associated complex to a second protein complex, the IKK (IκB kinase) complex or signalsome, which phosphorylates IκB. Such a role is confirmed by mouse mutants: cells from Traf6 mutant mice do not activate NF-κB in response to LPS [48•]. A Drosophila Traf6 homologue has been identified [49], but identification of its function in immunity awaits the isolation of mutants.

The signalsome includes the NF-κB-inducing kinase (NIK), the IκB kinase subunits (IKKα, β and γ), associated proteins including IKAP [50], and the inactive Rel–IκB complex. Remarkably, no mutations in Drosophila NIK or IKK subunits have been reported. Null mutations in mouse NIK have not been described, although the classical mouse mutation alymphoplasia is a missense mutation in NIK [51], demonstrating the role for this protein in immune system development, but not implicating it directly in these signaling pathways. Lymph nodes also fail to develop in mice that lack Traf6 [52], suggesting a common developmental role for Traf6 and NIK. The phenotypes of targeted mouse mutations in the two major Ikk subunits demonstrate that Ikkβ is required for cytokine-dependent NF-κB activation. Biochemical experiments had suggested that IKKα and IKKβ were components of an obligate heterodimer and were both required for phosphorylation of the key amino-terminal serine residues of IκB that must be phosphorylated prior to ubiquitination and degradation. The mutant phenotypes, however, make it clear that the two proteins have different functions. Mice that lack Ikkα die at the end of embryogenesis with defects in skin and skeleton; homozygous mutant fibroblasts from these animals activate NF-κB normally in response to IL-1 and TNF-α [53–55]. Mice that lack Ikkβ, in contrast, die at mid-gestation with a phenotype very similar to that of mice that lack the p65 subunit of NF-κB [56•,57•]. Ikkβ-null fibroblasts fail to activate NF-κB in response to IL-1 and TNF-α, demonstrating the essential role of this protein in these pathways. No experiments have yet been reported to determine whether these cells can respond to LPS or LTA, which would confirm the role for Ikkβ in Toll signaling pathways predicted by biochemical experiments [58]. Another Ikk gene has been described recently, Ikk-i, which could also participate in the regulation of IκB degradation [59]. A third protein complex ubiquitinates phosphorylated IκB. This complex includes Drosophila Slimb (or its mammalian homologue βTrCP), which acts as the substrate recognition subunit of a ubiquitin ligase. Genetic experiments in Drosophila have shown that Slimb is required for degradation of Cactus in the Drosophila embryo [60], confirming its role in the Toll pathway. It is likely that there are additional downstream components in Toll signaling pathways that have not yet been identified. Yeast two-hybrid screens have identified novel proteins that interact with TRAF6 and with Pelle [61,62]. Genetic screens in Drosophila have identified a large set of mutants that prevent normal responses to infection [8]. Some of these mutated genes are very likely to encode components of Toll family signaling pathways.

Other signaling pathways downstream of Toll? In addition to activation of NF-κB, overexpression of TLR4 activates the Jun amino-terminal kinase (JNK)

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pathway [42,63] and LPS activates the JNK and p38 mitogen-activated protein kinase (MAPK) pathways [64]. The role of the activation of these pathways by Toll family receptors has not yet been confirmed in vivo. In fact, overexpression of a Drosophila homologue of p38 MAPK inhibited the expression of antimicrobial peptide genes after infection, suggesting that this MAPK could be involved in attenuating the immune response at late times after infection [65]. In Drosophila, GATA transcription factors act together with Rel proteins in gene activation in response to infection. The insect antimicrobial peptide genes, which are transcriptionally activated in response to infection, have both GATA factor and κB sites binding sites in their upstream regulatory regions [66]. Mutations in the GATA site of the cecropin A1 upstream region prevent expression in larva [67]. Serpent, one of the three Drosophila GATA factors, is constitutively present in nuclei, and appears to be required for full induction of the antimicrobial peptide genes in response to infection [67].

Conclusions: recognition of diverse stimuli by a hard-wired immune system Why are large families of Toll receptors involved in the responses to microbial infection? Does each receptor respond to a different lipoprotein or glycolipid to elicit a specific response? Work from Drosophila provides some perspectives on this issue. Infection by different classes of microorganisms activates different sets of antimicrobial peptides [68]. For example, infection with yeast induces the antifungal peptides strongly and antibacterial peptides more weakly. This suggests that different PAMPs could activate different Toll family members; each Toll family member would then be required for the activation of particular target genes (Figure 2). There are mutants in two of the Drosophila Toll genes and their phenotypes are consistent with the hypothesis that different Tolls activate distinct target genes. Toll itself is required for the induction of the antifungal peptide Drosomycin, but not for induction of the antibacterial peptides [9]. 18-wheeler, a Toll family member, is specifically required for the normal induction of the antibacterial peptide gene attacin [10]. Rel protein mutants also affect specific target genes: mutants that lack the Rel protein Dif are impaired in the induction of drosomycin and defensin [69•,70], and mutants that lack Relish completely fail to induce Cecropin A and diptericin and decrease the induction of all the other antimicrobial peptides [71•]. Thus, the data suggest that activation of a specific Toll family receptor leads to nuclear localization of a particular Rel dimer; that dimer could then bind with greatest affinity to the κB sites in the promoters of particular antimicrobial peptide genes and with lower affinity to others. With three known fly Rel proteins activated by the immune response, there are six possible dimers and it is easy to imagine how activation of

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different ratios of the dimers could produce the observed spectra of antimicrobial peptides that result from contact with specific microbial components. The mouse Tlr4 mutant demonstrates that LPS is a specific activator of this receptor, but there is not yet any direct genetic data showing that the other TLRs are activated by specific PAMPs. A family of Toll receptors could, in theory, recognize many different PAMPs. Half a dozen Toll receptors in heteromeric complexes would have the potential to recognize dozens of different microbial components. Future genetic and biochemical studies will reveal whether the Toll family provides an immune response system that responds specifically to a diversity of microbe-specific determinants.

Acknowledgements The author thanks Tim Bestor for help with the figures and for helpful comments on the manuscript. Work was supported by a grant from the National Institutes of Health.

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