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Originally published online as doi:10.1189/jlb.0403160 on July 15, 2003

Published online before print July 15, 2003
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(Journal of Leukocyte Biology. 2004;75:18-26.)
© 2004 by Society for Leukocyte Biology

Biology of Toll receptors: lessons from insects and mammals

Jean-Luc Imler*,1 and Liangbiao Zheng{dagger}

* Institut de Biologie Moléculaire et Cellulaire, CNRS, 67000 Strasbourg, France
{dagger} Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven CT 06520, USA

1Correspondence: UPR9022-CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67000 Strasbourg, France. E-mail: JL.Imler{at}ibmc.u-strasbg.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 CONCLUSIONS
 REFERENCES
 
Toll receptors are type I transmembrane proteins that play important roles in development and immunity in animals. Comparison of the genomes of mouse and human on one side and of the fruitfly Drosophila and the mosquito Anopheles (two dipteran insects) on the other, revealed that the four species possess a similar number of Toll receptors (~10). However, phylogenetic analyses indicate that the families of Toll receptors expanded independently in insects and mammals. We review recent results on these receptors, which point to differences in the activation and signaling between Tolls in insects and Toll-like receptors (TLRs) in mammals. Whereas mammalian TLRs appear to be solely dedicated to host-defense, insect Tolls may be predominantly linked to other functions, probably developmental.

Key Words: Drosophila • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
CONCLUSIONS
 REFERENCES
 
Innate immunity is the first-line host-defense that controls the initial steps of the immune response in multicellular organisms [1 ]. In mammals, it plays a direct role in the activation and orientation of the subsequent adaptive immune response. Rapid and efficient, it relies on nonclonal receptors that recognize conserved molecular patterns on the surface of infectious microorganisms. These so-called pattern recognition receptors (PRRs) detect signature molecules expressed by pathogens such as lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan (PGN) from Gram-positive bacteria, double-stranded (ds)RNA from viruses or ß-glucans from fungi [2 ]. PRRs long remained elusive, until work in the fruitfly Drosophila melanogaster revealed the importance of the receptor Toll in the response to infections by fungi and Gram-positive bacteria [3 , 4 ]. Drosophila is a powerful genetic model that has been useful to decipher the molecular mechanisms controlling development, oncogenesis and, more recently, immunity in animals [5 ]. One defining feature of the Drosophila immune response is the rapid secretion of a cocktail of potent antimicrobial peptides into the blood upon septic injury. These peptides, which are active against various types of infectious agents including Gram-negative and Gram-positive bacteria, as well as fungi, can be used as markers of the immune response. Genetic investigations on the regulation of the expression of the genes encoding antimicrobial peptides have led to the identification of two pathways: the Toll pathway, activated in response to infection by Gram-positive bacteria and fungi, leads to expression of the antifungal peptide Drosomycin, and the Imd pathway, activated by Gram-negative bacterial infections, triggers expression of the genes encoding antibacterial peptides such as Diptericin (reviewed in [6 7 8 ]).

Following the demonstration that Toll carries important immune functions, 10 (or so) homologues, the Toll-like receptors (TLRs) were found in mammals and shown to play a critical role in cellular activation by a variety of microbial molecules such as LPS (TLR4), peptidoglycan (TLR2), flagellin (TLR5), dsRNA (TLR3), and unmethylated CpG DNA motifs (TLR9). The importance of TLRs to host-defense in mammals and humans, in particular, is illustrated by the description of polymorphisms in the genes encoding hTLR4 or hTLR2. These mutations result in inactive or hypoactive receptors and are associated with increased sensitivity to different infectious agents [9 10 11 12 ]. The genomes of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae also contain families of ~10 Toll receptors. We review below the structure of Toll/TLR receptors in insect and mammals, as well as our current understanding of the molecular mechanisms through which they are activated and of the signaling pathways they trigger.

1. Structure and evolution of Toll receptors
Toll-related receptors are characterized by a 150-amino-acid intracytoplasmic domain named TIR, which they share with members of the interleukin-1 receptor (IL-1R) family and plant disease resistance (R) genes. The TIR (Toll/IL-1R/R) domains have been found in many proteins involved in development and innate immunity in both animals and plants (reviewed in [13 , 14 ]). On the basis of phylogenetic analysis of the TIR domain, these proteins fall into three groups: IL-1R, Toll/TLR, and cytosolic TIR proteins (Fig. 1 ). Members of the IL-1R and Toll/TLR groups are transmembrane proteins with an intracellular TIR domain. The hallmark of the IL-1R group, which is specific to vertebrates, is the presence of three extracellular immunoglobulin domains. The cytokine IL-1 interacts with a dimeric receptor composed of two structurally related subunits: the type I IL-1 receptor (IL-1RI) and the IL-1R accessory protein (IL-1RAcP), and initiates intracellular signaling events, eventually leading to inflammation. Similarly, the heterodimeric interleukin-18 receptor (IL-18R/AcPL) is activated by IL-18 [15 ]. The Toll/TLR group, present in vertebrates and invertebrates, is characterized by an extracellular domain composed of amino-terminal leucine-rich repeats (LRRs) flanked by characteristic cysteine clusters on the C-terminal (CF motif) or N-terminal (NF motif) side of LRRs (Fig. 1) . The third group is cytosolic, including molecules from invertebrates and vertebrates, but also from plants. In plants, three types of TIR-containing proteins are known: those with TIR only (TX), those with TIR coupled to a nucleoside binding site (NBS) (TN), and those with TIR coupled to NBS and LRR domains (TNL). The genome of the thale cress Arabidopsis thaliana contains an astounding 91 genes that conform to this general TNL architecture [16 ]. Importantly, several TNL molecules, such as the N protein from tobacco, the L6 protein from flax, and the RPS2 protein from Arabidopsis have been shown to participate in the resistance of plants to a wide spectrum of pathogens, from viruses to fungi [17 ]. In addition 19 TN proteins and 31 TX proteins have been identified [16 ]. These TN and TX proteins may correspond to TIR domain containing cytosolic proteins in animals, such as MyD88, TIRAP (also named Mal) and TRIF/TICAM-1 (see below).



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  		Figure 1. TIR domain receptors in animals and plants. (A) Schematic representation of molecules containing TIR domains in Drosophila melanogaster, Homo sapiens and the plant Arabidopsis thaliana. The characteristic structural domains of these molecules are indicated. TIR: Toll/IL-1R/R domain; LRR: Leucine-rich repeat; CF: COOH-flanking cysteine cluster; NF: NH2-flanking cysteine cluster; Ig: immunoglobulin domain; NBS: nucleotide binding site. (B) Phylogenetic analysis of TIR domains. The TIR domain was identified from each protein sequence from human (h), mouse (m), D. melanogaster (Dm), Anopheles gambiae (Ag), and A. thaliana (At) by SMART [105 ]. Alignment and an unrooted neighbor joining tree was generated with Clustal X [106 ]. The cytosolic TIR domain-containing molecules are shown in green. Only a subset of A. thaliana proteins (one example each from the TNL, TN, and TX types) are shown. A subset of the members of the IL-1R family is shown in blue. The Toll receptors form two subgroups, one containing most insect Tolls shown in purple, and the other containing mammalian TLRs, as well as D. melanogaster and A. gambiae Toll-9, drawn in orange.

 
As mentioned above, Toll receptors are present in both vertebrates and invertebrates. Surprisingly, the genomes of dipteran insects and mammals encode a similar number (around 10) of these receptors. Nine Toll-related genes from Drosophila have been identified [18 19 20 ]. Analysis of the genome sequence of another dipteran, the malaria vector Anopheles gambiae, revealed 10 Toll-related genes. Clear orthologs have been found only for four of them (Toll-6, -7, -8 and -9). In the mosquito, Toll-1 and Toll-5 have been duplicated, while Toll-2 (or 18W) has been lost [21 ]. Similarly, Toll-1 has been duplicated in another mosquito, the yellow fever vector Aedes aegypti [22 ]. In human and mouse, 10 and 11 TLR genes have been found, respectively [23 ]. hTLR10 does not have an ortholog in the mouse genome, whereas mouse TLR11 and TLR12 are absent from the human genome.

In spite of the similarity in the size of the families of Toll/TLR receptors in insect and mammals, and the overall conserved organization of structural domains between these molecules, insect Tolls and mammalian TLRs exhibit a number of structural and functional differences. Whereas mammalian TLRs contain only a single CF cysteine cluster flanking the C-terminal end of the LRRs and juxtaposed to the plasma membrane, most Drosophila Tolls contain two or more such clusters, including the divergent NF motif (Fig. 1A) . Phylogenetic analysis of the better conserved intracytoplasmic TIR domain also reveals two subgroups of Toll receptors, one composed of most insect Tolls and the other of mammalian TLRs (Fig. 1B) . The only exception is the Toll-9 orthologs, which fall in the TLR subfamily for both the structure of the ectodomain and the sequence of the TIR domain. This suggests that the families of Toll-related receptors in insects and mammals result from independent expansion and diversification after the divergence of invertebrate and vertebrates [18 , 23 , 24 ]. Consistent with this view, two TLR genes from the sea urchin, Strongylocentrotus purpuratus, and one from the sea squirt, Ciona intestinalis, cluster with other deuterostome TLRs (S. M. Kanzok, unpublished results).

In keeping with the sequence analysis described above, and as outlined throughout the rest of this article, significant differences are also apparent between the function of insect Tolls and mammalian TLRs. In particular, there are still no indications that, apart from Toll, the other members of the family in Drosophila participate in the control of the immune response. Indeed, Drosophila Tolls do not seem to participate in the Imd pathway-mediated response to Gram-negative bacterial infections [19 , 20 , 25 , 26 ]. Only Toll-5 (also called Tehao) and Toll-9 have been shown to activate the drosomycin promoter (a target of the Toll pathway) in tissue-culture cells, but the in vivo significance of these results is not clear at this stage [19 , 20 , 27 , 28 ]. Furthermore, the expression patterns of Drosophila Tolls also contrast with those of mammalian TLRs. Indeed, several Tolls are most highly expressed at the crucial developmental stages of embryogenesis and metamorphosis [20 ]. In the Drosophila embryo, Toll genes have distinct expression patterns, and their expression is dynamically changing throughout development [29 ]. These complex and tissue-specific patterns of expression suggest a role in embryonic development for most Tolls in Drosophila. Therefore, one intriguing hypothesis is that whereas TLRs in mammals diversified to accommodate a growing set of microbial molecules, Tolls in insects evolved to carry different functions, linked to development. Indeed, Toll, 18w and more recently Tollo/Toll-8 have been shown to carry developmental functions in Drosophila [25 , 30 31 32 33 ]. As a consequence, we must leave open the possibility that the immune function of Toll and of the TLRs does not reflect a common ancestry, but rather results from convergent evolution (see below).

2. Activation of Toll and TLRs
Both Toll in Drosophila and TLRs in mammals are activated upon microbial challenge. However, in agreement with the phylogenetic analysis described above, the molecular mechanisms operating appear to be distinct. The current model points to a direct activation of TLRs by microbial molecules, whereas activation of Toll in Drosophila requires the cysteine-knot growth factor Spaetzle, which functions as a ligand for Toll.

The first indication that TLRs may function as pattern recognition receptors came from the positional cloning of the locus responsible for LPS hyporesponsiveness in C3H/HeJ mice, which revealed an inactivating mutation in the gene encoding TLR4 [34 , 35 ]. The TLR4 gene is deleted in C57BL/10ScCr mice, which also exhibit defective response to LPS. Generation of TLR4 knockout mice confirmed that TLR4 is an essential component of the LPS receptor complex and that inactivation of this receptor enables mice to resist endotoxic shock [36 ]. Other TLRs were shown to be essential for the response to a variety of microbial molecular patterns such as peptidoglycan and lipopeptides (TLR2) [36 37 38 39 ], double-stranded RNA from viruses (TLR3) [40 ], flagellin from bacterial flagella (TLR5) [41 ], or unmethylated CpG motifs found in bacterial DNA (TLR9) [42 ] (Fig. 2A ). Although no microbe-derived agonists have been identified so far for TLR7 and TLR8, these receptors have been shown to mediate activation of antiviral immunity in response to low-molecular mass compounds of the imidazoquinoline family [43 ]. These TLRs may therefore be activated by viral molecular patterns, like TLR3. Consistent with their function at the forefront of the innate immune system, TLRs are expressed in monocytes and dendritic cells [23 ], but also in a variety of other cells, including epithelial cells (e.g., [44 45 46 47 48 ]).



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  		Figure 2. Activation of Toll and Toll-like receptors. (A) TLR3, TLR5 and TLR9 are required for cellular activation by double-stranded (ds) RNA from viruses, flagellin from flagellated bacteria, and unmethylated CpG DNA motifs found in bacterial or viral DNA. TLR7 is required for the response to the antiviral imidazoquinoline compound R-848. (B) Association of TLR2 with TLR1 or TLR6 is required for cellular activation by diacylated or triacylated bacterial lipoproteins (BLP). (C) Activation of TLR4 by lipopolysaccharide (LPS) requires the coreceptors CD14 and MD2. (D) In D. melanogaster, Toll is activated upon binding of a cleaved form of the cytokine Spaetzle (Spz C106). Peptidoglycan from Gram-positive bacteria is recognized by the blood-borne peptidoglycan recognition protein PGRP-SA, which is then thought to activate a proteolytic cascade culminating in the processing of the Spz precursor.

 
Some TLRs have been shown to form heterodimers, thus broadening their recognition spectrum (Fig. 2B) : for instance, TLR2 associates with TLR1 to mediate response to the triacylated bacterial lipopeptides Pam3CSK4 or OspA, and with TLR6 to respond to the diacylated mycobacterial lipopeptide MALP-2. TLR2 is also required for the response to peptidoglycan, although, in this case, neither TLR1 nor TLR6 is required [36 37 38 39 ]. Because TLR2 homodimerization is not sufficient to trigger signaling [49 ], this suggests that TLR2 associates with another member of the family to mediate response to PGN. TLRs can also associate with accessory molecules to mediate efficient recognition and response to microbial molecular patterns. This is best exemplified by TLR4, which requires association of the 25kDa molecule MD-2 with its ectodomain to reach the plasma membrane and interact with LPS [50 ]. Cell activation by LPS has also long been known to require the GPI-anchored coreceptor CD14 [51 ] (Fig. 2C) .

In addition to microbial molecules, TLRs have been reported to be activated by endogenous agonists such as heat-shock proteins, inflammatory mediators or fragments of molecules from the extracellular matrix like proteoglycans, hyaluronic acid, fibronectin, or antimicrobial peptides [52 53 54 55 56 57 58 ]. A common feature of these molecules is that they are generated in response to stress or as a consequence of tissue injury. An important concern about these studies, despite the many precautions of the authors, is the possible contamination of the TLR agonists with LPS or other microbial compounds [59 ]. Nevertheless, these results raise the intriguing possibility that TLRs may be activated, possibly synergistically, by molecules derived from the nonself but also from the damaged self. This may enable the innate immune system to distinguish between harmless commensal and infectious microbes that generate tissue damage.

Importantly, there is still considerable uncertainty about the molecular mechanisms mediating TLR activation. In particular, there is as-yet no strong biochemical evidence that molecules such as LPS, dsRNA or lipopeptides directly interact with TLRs. Although radiolabeled iodinated lipid A has been shown to interact with TLR4, the same labeled ligand was also shown to interact with TLR2 [60 ]. Thus, the molecular events leading to TLR activation remain to be clearly established, even though elegant pharmacological experiments based on species-specific sensitivity to various types of LPS agonists strongly suggest that TLR4 and LPS are in close proximity in the receptor complex [61 , 62 ].

In Drosophila, significant progress has recently been made in our understanding of the activation of the Toll receptor by Gram-positive bacteria and fungi. The Spaetzle gene, which encodes a cysteine-knot growth factor, has long been known to be required upstream of Toll [3 ]. This molecule is synthesized as an inactive precursor that is processed by a serine-protease [63 ]. The released carboxyl-terminal fragment, which contains the cysteine-knot motif, then binds to and activates Toll [64 ] (Fig. 2D) . Interestingly, the Drosophila genome encodes a family of six Spaetzle-related genes, which may encode ligands for the other members of the Toll family [65 ]. Thus, Toll functions like a cytokine receptor in Drosophila and not like a PRR. In fact, pattern recognition occurs upstream of Toll, in the hemolymph, and there is now clear genetic evidence that the pathway branches upstream of Toll, involving distinct PRRs that mediate recognition of Gram-positive bacteria and fungi. Indeed, activation of the Toll pathway by Gram-positive bacteria, but not fungi, is affected in semmelweiss (seml) mutant flies [66 ], and the opposite (normal response to Gram-positive bacteria but no response to fungi) is observed in persephone (psh) mutant flies [67 ]. The gene seml encodes the secreted peptidoglycan recognition protein PGRP-SA, whereas psh encodes a serine-protease. In the current model for activation of the Toll receptor in Drosophila, pattern recognition is mediated by soluble PRRs (e.g., PGRP-SA), which activate a proteolytic cascade involving serine proteases (e.g., Persephone) and leading to processing of Spaetzle and generation of an active Toll ligand. This mechanism of amplification may be best suited for an animal with an open circulatory system like Drosophila, in which all organs bathe in the hemolymph. These results point to interesting functional similarities between Toll and the mammalian IL-1R, rather than TLRs [64 ]. In addition, they reveal a crucial aspect of host-defense shared by Drosophila and mammals, namely the importance of TIR domain-containing cytokine receptors in the control of the inducible synthesis of effector molecules, such as antimicrobial peptides (e.g., [68 , 69 ]).

3. Signaling pathways downstream of TLRs
Upon stimulation, TLRs activate the transcription factors NF-{kappa}B and AP1, leading to production of inflammatory cytokines, such as TNF{alpha} and IL-6, and up-regulation of the costimulatory molecules CD80 and CD86 on dendritic cells (DC) [70 ]. TIR domains play a critical role in TLR signaling, as shown by the fact that a single point mutation in the TIR domain of murine TLR4 (P712H, replacement of proline at position 712 by a histidine) abolishes the host immune response to LPS [34 ]. The cytoplasmic factor MyD88 contains a TIR domain that mediates binding to the cognate domains of IL-1R or TLRs through homophilic interactions (Fig. 3A ). MyD88, which is recruited to the receptors after stimulation, contains an amino-terminal death domain that enables it to bind the death domain-containing serine-threonine kinases of the IRAK family (reviewed in [71 ]). Therefore, MyD88 appears to function as an adaptor between receptors of the TLR or IL-1R families and downstream signaling kinases. Genetic studies using MyD88-deficient mice confirmed that this factor is essential for the NF-{kappa}B-dependent induction of the genes encoding the cytokines TNF{alpha} and IL-6 in response to TLR agonists [72 , 73 ]. Unexpectedly, analysis of MyD88 mutant mice also pointed to the existence of a MyD88-independent pathway downstream of some TLRs. Indeed, NF-{kappa}B (and AP1) induction by LPS (TLR4 agonist) or dsRNA (TLR3 agonist) in MyD88-deficient mice was not abolished but only delayed [40 , 72 ]. Furthermore, DC maturation is not affected when MyD88-deficient cells are stimulated with LPS or dsRNA [40 , 74 ]. By contrast, when MyD88-deficient cells are treated with stimuli that activate TLR2 or TLR9, induction of NF-{kappa}B and DC maturation is completely abolished. Further studies showed that TLR3 and TLR4 can activate the transcription factor IRF3 and induce production of the cytokine IFNß in a MyD88-independent manner [75 , 76 ]. These results reveal the existence of two groups of TLRs, one of which (e.g., TLR3, TLR4) does not entirely depend on MyD88 for signaling. Two additional TIR domain adapters have recently been described. Genetic evidence indicated that the first one, TIRAP (also called MAL), is an essential cofactor of MyD88 to mediate induction of NF-{kappa}B and AP1 in response to stimulation of TLR2 or TLR4 [77 , 78 ] (Fig. 3A) . In this regard, the function of TIRAP is reminiscent of that of the Drosophila death domain-containing protein Tube, which is an essential cofactor of the Drosophila homologue of MyD88 (DmMyD88) [26 , 79 80 81 ] (Fig. 3B) . Other TLRs do not require TIRAP to activate NF-{kappa}B and AP1. The second adaptor, TICAM-1/TRIF, has so far only been characterized in tissue-culture cells. These experiments indicate that TICAM-1/TRIF is involved in the regulation of the IFNß promoter by TLR3 and suggest that it may be involved in the MyD88-independent pathway downstream of TLR3 and TLR4 [82 , 83 ] (Fig. 3C) . Thus, signaling by TLRs is more complex than initially anticipated, and the diversity of extracellular agonists for TLRs could be mirrored by a panel of intracytoplasmic adaptor molecules. The recent discovery that sequences outside the well-defined death and TIR domains are important for the function of MyD88 and DmMyD88 also challenges the initial concept that these molecules function as simple adaptors [84 , 85 ].



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  		Figure 3. Signaling by Toll/TLR receptors. (A-C) The receptor complex. All TLRs can signal to the promoter of the gene encoding TNF{alpha} through MyD88, but some of them also require the related molecule TIRAP (also known as MAL) (A). In D. melanogaster, Toll signaling involves the homologue of MyD88 (DmMyD88) and the death domain adaptor Tube (B). TLR3 and TLR4 can activate a specific genetic program, including induction of the gene-encoding interferon-ß. The adaptor TICAM1/TRIF is probably involved in this pathway (C). TIR domains are shown as shaded ovals and death domains as striped rectangles. (D) The TLR/IL-1R signaling pathway in mammals. P: phosphorylation; Ub: Lys63-mediated polyubiquitination; pm: plasma membrane. The nucleus is shaded. See the text for details and references.

 
Significant progress has recently been made in the understanding of the signaling events downstream of MyD88 (Fig. 3D) . A family of related kinases, the IRAKs, plays a critical role at this stage. The first member of this family, IRAK-1, is recruited to the receptor independently of MyD88. By contrast, IRAK-4 is recruited at the receptor through binding MyD88. Once at the receptor, IRAK-1 becomes phosphorylated by IRAK-4 and subsequently by autophosphorylation (reviewed in [86 ]). As a result, the kinases dissociate from the receptor complex and interact with the downstream component TRAF6. Genetic evidence indicates that IRAK-4 plays an essential role in the activation of the pathway, as responses to TLR agonists (as well as to IL-1) are abolished in IRAK-4 knockout mice [87 ]. By contrast, the responses to TLR agonists are only attenuated in IRAK-1 mutant cells, suggesting that other proteins, possibly IRAK-2, can substitute for IRAK-1 [88 , 89 ]. The last member of the family, IRAK-M, appears to be a negative regulator of the pathway, as responses to TLR agonists are exacerbated in IRAK-M mutant cells [90 ]. Once activated, the RING-finger containing factor TRAF6 activates a heterodimer composed of two ubiquitination proteins called Uev1A and Ubc13 [91 ]. These proteins trigger polyubiquitination of TRAF6 and possibly other factors. Importantly, this polyubiquitination differs from the classical formation of ubiquitin chains extending on Lysine (K) 48 of ubiquitin, which leads to proteasome-mediated degradation of the modified protein. Nonclassical ubiquitin chains are linked through K63, and in the case of TRAF6, this modification has been shown to trigger association with the MAP3 kinase TAK1 [92 ]. Once activated, TAK1 can directly phosphorylate and activate the kinases IKKß and MKK6. IKKß is part of a large complex that also includes the essential regulatory component IKK{gamma}/NEMO. This kinase phosphorylates the NF-{kappa}B cytoplasmic inhibitor I{kappa}B, thus releasing the transcription factor that translocates to the nucleus where it activates target genes. MKK6 phosphorylates the kinase JNK, which leads to activation of AP1 (reviewed in [93 ]) (Fig. 3D) . AP1 and NF-{kappa}B then mediate transcriptional induction of genes encoding molecules involved in inflammation (cytokines, iNOS, E-selectin, ...), but also triggering the onset of the adaptive immune response (CD40, CD80, CD86). In Drosophila, the molecules bridging the IRAK-4 homologue Pelle to the I{kappa}B homologue Cactus in the Toll-signaling pathway are less clearly defined at this stage. Structural homologues of TRAFs exist in flies, but no mutants have been reported as yet. Surprisingly, the homologues of IKKß and IKK{gamma}/NEMO were shown to be essential in the Imd pathway, but not in the Toll pathway [94 , 95 ]. Conversely, a recently published study using tissue culture cells reveal that the Drosophila atypical protein kinase C (PKC) and the homologue of the scaffold protein p62 are involved in the Toll pathway, but not in the Imd pathway [96 ].

TLRs may also activate other signaling pathways, involving tyrosine kinases. This was first shown for TLR2, which can recruit the Rac GTPase and the SH2 adaptor p85, upon phosphorylation of a membrane proximal tyrosine residue by a still unknown kinase [97 ]. P85 then leads to activation of the PI3 kinase, and production of phosphorylated lipids, which act as second messengers to activate the serine/threonine protein kinase Akt. Akt in turn triggers induction of NF-{kappa}B transcriptional activity, independently of I{kappa}B degradation. Tyrosine kinases are also involved in TLR3 signaling. The gene 561, which encodes a regulator of protein synthesis, is one of the most highly induced gene in virus-infected cells. Induction of this gene by viral dsRNA is mediated by TLR3 and is blocked by pharmacological inhibitors of tyrosine kinases. In agreement with these findings, specific tyrosine residues in the TIR domain of TLR3 were shown to be essential for the induction of the gene 561 [98 ]. Thus, TIR domains may have to be considered as platforms allowing the docking of different kinds of adaptor molecules, containing either TIR or SH2 domains.

4. TLRs and the adaptive immune response
As mentioned in the introduction, one function of innate immunity is to initiate and orient the adaptive immune response. Recent results have shown that TLRs participate in the induction of the adaptive immune response, in keeping with their essential role in innate immunity. Indeed, induction of the adaptive immune response is impaired in MyD88-deficient mice: immunization of mice with a protein antigen mixed with complete Freund’s adjuvant generates a strong immune response in wild-type mice, but not in MyD88 knockout animals. Unexpectedly, the humoral response controlled by T helper 2 (TH2) cells is not affected in MyD88-deficient mice, and antibodies of the IgE and IgG1 isotypes are still raised against the injected protein [99 ]. This suggests that TLRs are necessary for priming TH1 responses, but not TH2 responses. Along the same line, it was recently shown that MyD88-deficient mice develop a polarized nonprotective TH2 response when infected by the intracellular parasite Leishmania major, instead of the IL-12 mediated TH1 response that results in protection of genetically resistant wild-type mice [100 ]. However, an elegant independent study using a mouse model of allergic sensitization to inhaled antigen recently reported that LPS signaling through TLR4 is necessary to induce both TH1 and TH2 responses and that the type of adaptive response induced depends on the dose of LPS used: Low doses of LPS trigger a TH2 response in this model, whereas inhalation of high doses of LPS results in TH1 responses [101 ]. These data point to a possible role of TLRs in inflammatory diseases such as allergic asthma.

Activation of the adaptive immunity relies not only on TH cells, but also on T regulatory (TR) cells, a small subset of CD4+ T cells, which express the CD25 marker, and suppress T cell activation. TR cells are involved in the control of a number of immune-related pathologies such as autoimmunity, graft rejection, and inflammatory bowel disease [102 ]. Like TH cells, TR cells are under the influence of cells from the innate immune system, and induction of adaptive immunity involves both activation of TH cells and suppression of TR cells. The group of Medzhitov recently reported that TLRs not only control upregulation of costimulatory molecules on APCs to activate TH cells, but are also required to block TR cells. This new TLR-mediated activity involves the TLR/MyD88-dependent secretion of cytokines including IL-6, which are responsible for the block of suppression [103 ]. This study shows that TLRs act at two different levels to activate T cell responses, thus strengthening their importance in the onset of adaptive immunity.

Many microbial molecules that activate TLRs are found in adjuvants. Recent studies in humans confirm the importance of TLRs in vaccine efficacy. Indeed, Flavell and collaborators identified individuals with very low antibody titers after vaccination with Borrelia burgdorferi (the causative agent of Lyme disease) outer surface lipoprotein OspA and could show that these low-responder individuals expressed significantly lower levels of TLR1 at the cell surface than normal responders. Studies in TLR1-deficient mice confirmed the importance of this receptor in the success of vaccination with OspA [37 ].

Finally, there are also suggestions that TLRs are involved in autoimmunity. For example, it was recently shown that activation of B cells producing a class of self-reactive antibodies known as Rheumatoid Factor is mediated by chromatin-IgG complexes, and requires engagement of both the B cell antigen receptor and a TLR, most likely TLR9 [104 ]. Thus, innate immunity and TLRs, in particular, represent a new investigation field to better understand and develop treatment for autoimmune diseases.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 CONCLUSIONS
REFERENCES
 
In summary, Toll receptors share with members of the IL-1R family a cytoplasmic TIR domain, which is also evident in plant proteins and, as a consequence, was probably already present at the origin of eukaryotic life [23 ]. The fact that TIR domain-containing molecules are involved in host-defense reactions in most taxa today strongly argues that the ancestral function of the domain is related to immune defense. A careful analysis of the structure and function of Toll receptors in insects and mammals nevertheless suggests that two lines of Toll receptors, the insect Tolls and the mammalian TLRs, evolved independently to carry specific functions, possibly as a consequence of the significant physiological differences between these animals. In insects, our current hypothesis is that most Tolls carry developmental functions and that the immune function attributed to Toll may result from convergent evolution. In mammals, it has become apparent that TLRs control multiple aspects of the immune response, both innate and adaptative. As a consequence of their central role, and as illustrated above, these receptors are potentially involved in a wide range of pathologies affecting the immune system, from septic shock to allergic asthma or autoimmune diseases. Drugs that target TLRs or their signaling pathways could therefore offer promising new treatments for these diseases.

Received April 17, 2003; revised June 3, 2003; accepted June 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 CONCLUSIONS
 REFERENCES
 

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D. Schikorski, V. Cuvillier-Hot, M. Leippe, C. Boidin-Wichlacz, C. Slomianny, E. Macagno, M. Salzet, and A. Tasiemski
Microbial Challenge Promotes the Regenerative Process of the Injured Central Nervous System of the Medicinal Leech by Inducing the Synthesis of Antimicrobial Peptides in Neurons and Microglia
J. Immunol., July 15, 2008; 181(2): 1083 - 1095.
[Abstract] [Full Text] [PDF]

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 Sci SignalHome page
T. M. Burch-Smith and S. P. Dinesh-Kumar
The Functions of Plant TIR Domains
Sci. Signal., August 28, 2007; 2007(401): pe46 - pe46.
[Abstract] [Full Text] [PDF]

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 ScienceHome page
R. M. Waterhouse, E. V. Kriventseva, S. Meister, Z. Xi, K. S. Alvarez, L. C. Bartholomay, C. Barillas-Mury, G. Bian, S. Blandin, B. M. Christensen, et al.
Evolutionary Dynamics of Immune-Related Genes and Pathways in Disease-Vector Mosquitoes
Science, June 22, 2007; 316(5832): 1738 - 1743.
[Abstract] [Full Text] [PDF]

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 GENES CELLSHome page
O. Harari-Steinberg, R. Cantera, S. Denti, E. Bianchi, E. Oron, D. Segal, and D. A Chamovitz
COP9 signalosome subunit 5 (CSN5/Jab1) regulates the development of the Drosophila immune system: effects on Cactus, Dorsal and hematopoiesis.
Genes Cells, February 1, 2007; 12(2): 183 - 195.
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 J. Biol. Chem.Home page
S. W. Shin, G. Bian, and A. S. Raikhel
A Toll Receptor and a Cytokine, Toll5A and Spz1C, Are Involved in Toll Antifungal Immune Signaling in the Mosquito Aedes aegypti
J. Biol. Chem., December 22, 2006; 281(51): 39388 - 39395.