Insect Toll-like receptors

Insects defend themselves against infectious microorganisms by synthesizing potent antimicrobial peptides. Drosophila has appeared in recent years as a favorable model to study this innate host defense. A genetic analysis of the regulation of the antifungal peptide drosomycin has demonstrated a key role for the transmembrane receptor Toll, which prompted the search for mammalian homologs. Two of these, Toll-like receptor (TLR)2 and TLR4, recently have been shown to play a critical role in innate immunity against bacteria. Six additional Drosophila Toll-related genes (Toll-3 to Toll-8) are descibed in addition to 18-wheeler. Two of these genes, Toll-3 and Toll-4, are expressed at a low level. In contrast, Toll-6, -7, and -8 are expressed at high levels during embryogenesis and molting, suggesting that, like Toll and 18w, they perform developmental functions. Finally, Toll-5 is expressed only in larvae and adults. By using chimeric constructs, the capacity of the signaling Toll/IL-1R homology domains of these receptors to activate antimicrobial peptide promoters has been tested and it has been found that only Toll and Toll-5 can activate the drosomycin promoter in transfected cells, thus demonstrating specificity at the level of the Toll/IL-1R homology domain. In contrast, none of these constructs activate antibacterial peptide promoters, suggesting that Toll-related receptors are not involved in the regulation of antibacterial peptide expression. This result was independently confirmed by the demonstration that a dominant-negative version of the kinase Pelle can block induction of drosomycin by the cytokine Spaetzle, but does not affect induction of the antibacterial peptide attacin by lipopolysaccharide (Tauszig, 2000).

Because the (Toll/IL-1R homology) TIR domain is the most conserved region between Toll and its mammalian homologs, and it is shared by other molecules critical for the control of innate immunity such as MyD88 and the IL-1 and IL-18 receptors, gene sequences encoding TIR domains were sought in the Drosophila genome by using the BLAST program. This search revealed the existence of six genes encoding receptors containing a TIR domain, in addition to Toll and 18w. Alignment of the TIR domains of the Drosophila molecules with the mammalian type I IL-1R, the human Toll homolog TLR4 and MyD88 shows a high degree of conservation throughout a stretch of 150 aa, with many identical residues in all sequences. The eight Drosophila Toll-related proteins share higher similarity between them than with any mammalian TLR, suggesting that these two groups of proteins have evolved independently. The Drosophila Tolls fall into several subsets. With regard to the TIR domains, Toll-3 and Toll-4 share 79% identity and Toll and Toll-5 have 60% identity, as is also the case for 18W and Toll-7. Toll-6 is less related to the other seven Drosophila Tolls (Tauszig, 2000).

Examination of the full amino acid sequence of the mammalian and insect TIR-containing proteins reveals that they all contain a putative transmembrane domain and an extracellular domain composed of leucine-rich repeats (LRRs), flanked by characteristic cysteine-rich motifs. However, the arrangement of these Cys motifs differs between mammalian and Drosophila Tolls. Whereas the mammalian TLRs described to date only contain a membrane-proximal cysteine-rich flanking motif at the C-terminal end of the LRRs, Drosophila Tolls contain C- and N-flanking cysteine-rich motifs. The extracellular regions of Drosophila Toll-related molecules are less conserved than the TIR domains, and the arrangement of the C- and N-flanking motifs is different, suggesting that some of these receptors may activate common targets in response to different signals. Finally, the TIRs of Toll, 18W, and Toll-6, -7, -8 are followed by a C-terminal extension. These extensions do not present any obvious motif that could hint to a function, with the exception of polyglutamine stretches in 18W, Toll-7, and Toll-8. In the other Tolls (Toll-3 to Toll-5), a stop codon is present a few residues after the TIR domain, as in mammalian TLRs and members of the IL-1R family (Tauszig, 2000).

The Toll protein's extracellular domain contains leucine-rich repeats (LRR), implicated in intermolecular interactions, and its large intracellular domain transduces a signal that eventually reaches the nucleus. Toll genes of D. pseudoobscura and D. virilis, and two distinct Toll-like genes of the grasshopper, Schistocerca americana, show a Toll-specific subfamily of LRR, and a strikingly high degree of conservation in the cytoplasmic domain. Many aa residues conserved among the insect Toll-like cytoplasmic domains are also conserved in mammalian and avian type-I interleukin-1 receptors and the hypothetical product of a transcript, MyD88, found in murine myeloid cells (Yamagata, 1994).

18 wheeler (18w) is expressed in a segmental pattern in Drosophila embryos and also in cells that migrate extensively. 18 wheeler transcripts accumulate in embryos in a pattern reminiscent of segment polarity genes. Mutations in 18w cause death during larval development and early adulthood. Escaping mutant adults often display leg, antenna, and wing deformities, presumably resulting from improper eversion of imaginal discs. Sequence analysis indicates that 18w encodes a transmembrane protein with an extracellular moiety containing many leucine rich repeats and cysteine motifs; an intracellular domain bearing homology to the cytoplasmic portion of the interleukin-1-receptor. Expression of 18W protein in non-adhesive Schneider 2 cells promotes rapid and robust aggregation of cells. Analysis of the expression of 18w in different mutant backgrounds shows that it is under control of segment polarity and homeotic genes. The data suggest that the 18W protein participates in the developmental program specified by segmentation and homeotic genes as a cell adhesion or receptor molecule that facilitates cell movements (Eldon, 1994).

Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. Several enhancer trap lines have been isolated, expressing beta-galactosidase in small subsets of muscle fibers prior to innervation. Two of these are inserts in connectin and Toll, members of the LRR gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears (Nose, 1992).

The gene Toll (Tl) encodes a maternally supplied interleukin 1 receptor-related transmembrane protein, a key component required to establish dorsoventral polarity in the Drosophila embryo. Tl homologs of a primitive dipteran, Clogmia albipunctata, and of the beetle Tribolium castaneum have been isolated. Tribolium Tl protein (Tl) lacks sequences in the C-terminal portion of the cytoplasmic domains that are conserved in the dipteran homologs. The Tl homolog of Tribolium mediates the ventralizing activity when expressed as a gain-of-function variant in transgenic Drosophila, indicating that the sequences conserved in the Diptera are not essential for Tl signaling. In contrast to Drosophila where Tl gene expression occurs maternally and supplies uniformly distributed Tl in the egg membrane, Tl transcripts form a ventral-to-dorsal gradient in the Tribolium blastoderm stage embryo. This localized expression pattern of Tl transcripts, as compared with the strong maternal and ubiquitous expression in Drosophila and Clogmia embryos, suggests that dorsoventral patterning in long-germ band and short-germ band insects (see Tribolium early embryonic development) involves the same components but different modes of their action. Nuclear accumulation of Dorsal protein seems to parallel the local expression of Tl in the Tribolium embryo, suggesting that local Tl expression is signaling dependent. The localized Tl expression in Tribolium embryos is consistent with the proposal of an inducible and self-amplifying activation system that causes molecular asymmetry in embryos of short germ band insects. Thus the mode of initating dorsoventral polarity in Tribolium is different from Drosophila. The localized zygotic expression of Tl in a short-germ band embryo provides an entry point towards the analysis of the mechanisms underlying DV patterning in such embryos (Maxton-Kuchenmeister, 1999).

Dorsoventral polarity of the Nasonia embryo primarily relies on a BMP gradient formed without input from Toll

In Drosophila, Toll signaling leads to a gradient of nuclear uptake of Dorsal with a peak at the ventral egg pole and is the source for dorsoventral (DV) patterning and polarity of the embryo. In contrast, Toll signaling plays no role in embryonic patterning in most animals, while BMP signaling plays the major role. In order to understand the origin of the novelty of the Drosophila system, DV patterning was examined in Nasonia vitripennis (Nv), a representative of the Hymenoptera and thus the most ancient branch points within the Holometabola. Previous studies have shown that while the expression of several conserved DV patterning genes is almost identical in Nasonia and Drosophila embryos at the onset of gastrulation, the ways these patterns evolve in early embryogenesis are very different from what is seen in Drosophila or the beetle Tribolium. In contrast to Drosophila or Tribolium, wasp Toll was found to have a very limited ventral role, whereas BMP is required for almost all DV polarity of the embryo, and these two signaling systems act independently of each other to generate DV polarity. This result gives insights into how the Toll pathway could have usurped a BMP-based DV patterning system in insects. In addition, this work strongly suggests that a novel system for BMP activity gradient formation must be employed in the wasp, since orthologs of crucial components of the fly system are either missing entirely or lack function in the embryo (Ozuak, 2014).

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta

Proteolytic activation of phenoloxidase (PO) and the cytokine Spatzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. This study presents biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta. Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. This study found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR(176)*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH(112)*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpatzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, this study has elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar (Wang, 2020).

Plant Toll-like receptors

The products of plant disease resistance genes are postulated to recognize invading pathogens and rapidly trigger host defense responses. The resistance gene N of tobacco mediates resistance to the viral pathogen tobacco mosaic virus (TMV). Sequence analysis of the N gene shows that it encodes a protein of 131.4 kDa with an amino-terminal domain similar to that of the cytoplasmic domain of the Drosophila Toll protein and the interleukin-1 receptor (IL-1R) in mammals, a nucleotide-binding site (NBS), and 4 imperfect leucine-rich repeats (LRR). The sequence similarity of N, Toll, and IL-1R suggests that N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway (Whitham, 1994).

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. This gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos form from cells expressing SERK. During somatic embryogenesis, SERK expression ceases after the globular stage. In plants, SERK mRNA can only be detected transiently in the zygotic embryo up to the early globular stage, but not in unpollinated flowers or in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo. Clues about the function of SERK might be found in the homology between SERK and two proteins of Drosophila that are required for the establishment of the dorsoventral polarity in the embryo. The kinase domain of SERK shows homology to the Pelle protein. Pelle itself is activated by the Toll transmembrane receptor. The ligand-binding domain of Toll, as in SERK, consists of leucine rich repeats (LRRs). In the plant embryo sac a situation may exist whereby an unknown inducer is present uniformly, but embryo formation awaits the presence of the SERK protein for SERK mediated embryo induction (Schmidt, 1997).

Conservation of the Toll signaling pathway in C. elegans

Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. Deletion mutants for four genes supposed to function in a nematode Toll signaling pathway have been generated and characterized. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. However, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. It is concluded that in C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter role, it functions in the context of a behavioral mechanism that keeps worms away from potential danger. When compared to the family of Drosophila TLR proteins, TOL-1 is most similar overall to Toll-8, closely followed by Toll-6. TOL-1, however, lacks a C-terminal extension after the TIR domain. Such an extension is found in certain Drosophila Toll-family proteins (Pujol, 2001).

Conservation of the Spatzle/Toll signaling pathway function in Xenopus

The Spatzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose patterning functions are conserved between insects and vertebrates. Although there is no experimental evidence that upstream events are conserved as well, the following question was posed: can a vertebrate embryo respond to maternal components of the fly Dorsal pathway? A dorsalizing activity is demonstrated for the heterologous Easter, Spatzle and Toll proteins in UV-ventralized Xenopus embryos; dorsalization is inhibited by a co-injected dominant Cactus variant. Thus the epistatic relationships between upstream and downstream components of the Drosophila dorsoventral (d/v) pathway are maintained in the frog, as is evident from the inhibtion of Spz and Easter activity by the dominant Cactus mutation. It is concluded that the Dorsal signaling pathway is a component of the conserved d/v patterning system in bilateria (Armstrong, 1998).

Mammalian Toll-like receptors

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. The molecular cloning of a class of putative human receptors is reported, with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors (TLRs 1-5) are probably the direct homologs of the fly molecule; as such, they could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role (akin to Toll in the dorsoventralization of the Drosophila embryo) as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, it is shown that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors, MyD88 factors, and plant disease-resistance proteins recognizes a parallel/fold with an acidic active site. A similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria (Rock, 1998).

The 22- and 31-LRR ectodomains of Toll and 18 Wheeler, respectively, are most closely related to the comparable 18-, 19-, 24- and 22-LRR arrays of TLRs 1-4. However, a striking difference in the human TLR chains is the common loss of an approximately 90-residue cysteine-rich region that is variably embedded in the ectodomains of Toll and 18 Wheeler. These cysteine clusters are bipartite, with distinct top (ending in an LRR) and bottom (stacked atop an LRR) halves; the top module recurs in Drosophila and human TLRs as a conserved juxtamembrane spacer. It is suggested that the flexibly located cysteine clusters in Drosophila receptors (when mated top to bottom) form a compact module with paired termini that can be inserted between any pair of LRRs without altering the overall fold of TLR ectodomains (Rock, 1998)

Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. The cloning and characterization of a human homolog of the Drosophila toll protein (Toll) is reported. Toll has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. A constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells (Medzhitov, 1997).

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. A 50-amino acid region (residues 477-527) is critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein (Heguy, 1992).

The type-I interleukin-1 receptor (IL-1R) of chick embryo fibroblast's intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila. The alignment between chicken and fly proteins shows the presence of four aa blocks with more than 80% homology. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain (Guida, 1992).

A new human gene, GARP, is localized in the 11q14 chromosomal region. GARP comprises two coding exons and is expressed as two major transcripts of 4.4 and 2.8 kilobases, respectively. It encodes a putative transmembrane protein of 662 amino acids, the extracellular portion of which is almost entirely made of leucine-rich repeats. The GARP protein has structural similarities with the human GP Ib alpha and GP V platelet proteins, and with the Chaoptin, Toll, and Connectin adhesion molecules of Drosophila (Ollendorff, 1994).

The open reading frame of MyD88, a gene induced in myeloid differentiation, is related to the cytoplasmic domains of the interleukin-1 receptor and the Toll gene product (Hultmark, 1994).

The interleukin-1 receptor (IL-1R) signaling pathway leads to nuclear factor kappa B (NF-kappaB) activation in mammals and is similar to the Toll pathway in Drosophila: the IL-1R-associated kinase (IRAK) is homologous to Pelle. Two additional proximal mediators have been identified that are required for IL-1R-induced NF-kappaB activation: IRAK-2, a Pelle family member, and MyD88, a death domain-containing adapter molecule. MyD88 and the cytoplasmic domain of IL-1R accessory protein (IL-1RAcp) possess sequence similarity with Drosophila Toll. Both IRAK-2 and MyD88 associate with the IL-1R signaling complex. Dominant negative forms of either attenuate IL-1R-mediated NF-kappaB activation. MyD88-induced NF-kappaB activity is specifically inhibited by expression of constructs of TRAF6, suggests that TRAF6 functions downstream of MyD88. Therefore, IRAK-2 and MyD88 may provide additional therapeutic targets for inhibiting IL-1-induced inflammation (Muzion 1997).

Using data base searches, four new proteins have been identified that share homology with the signaling domain of the type I interleukin-1 receptor (IL-1RI): human "randomly sequenced cDNA 786" (rsc786), murine MyD88, and two partial Drosophila open reading frames, MstProx and STSDm2245. Comparisons between these new proteins and known IL-1RI homologous proteins such as Toll, 18-Wheeler, and T1/ST2 reveal six clusters of amino acid similarity. Does sequence similarity between the signaling domain of IL-1RI and the three mammalian family members indicate functional similarity? Chimeric IL-1RI receptors expressing the putative signaling domains of T1/ST2, MyD88, and rsc786 were assayed by three separate IL-1 responsive assays (NF-kappaB, phosphorylation of an epidermal growth factor receptor peptide, and an interleukin 8 promoter-controlled reporter construct) for their ability to transduce an IL-1-stimulated signal. All three assays were positive in response to the T1/ST2 chimera, while the MyD88 and rsc786 chimeras failed to respond. These data indicate that the sequence homology between IL-1RI and T1/ST2 indicates a functional homology as well (Mitcham, 1996).

Toll-like receptors - protein interactions

IL-1 is a proinflammatory cytokine that signals through a receptor complex of two different transmembrane chains to generate multiple cellular responses, including activation of the transcription factor NF-kappaB. MyD88, a previously described protein of unknown function, is recruited to the IL-1 receptor complex following IL-1 stimulation. MyD88 binds to both IRAK (IL-1 receptor-associated kinase) and the heterocomplex (the signaling complex) of the two receptor chains and thereby mediates the association of IRAK with the receptor. Ectopic expression of MyD88 or its death domain-containing N-terminus activates NF-kappaB. The C-terminus of MyD88 interacts with the IL-1 receptor and blocks NF-kappaB activation induced by IL-1, but not by TNF. Thus, MyD88 plays the same role in IL-1 signaling as TRADD and Tube do in TNF and Toll pathways, respectively: it couples a serine/threonine protein kinase to the receptor complex (Wesche, 1997).

Signal transduction through Toll-like receptors (TLRs) originates from their intracellular Toll/interleukin-1 receptor (TIR) domain, which binds to MyD88, a common adaptor protein containing a TIR domain. Although cytokine production is completely abolished in MyD88-deficient mice, some responses to lipopolysaccharide (LPS), including the induction of interferon-inducible genes and the maturation of dendritic cells, are still observed. Another adaptor, TIRAP (also known as Mal), has been cloned as a molecule that specifically associates with TLR4 and thus may be responsible for the MyD88-independent response. LPS-induced splenocyte proliferation and cytokine production are abolished in mice lacking TIRAP. As in MyD88-deficient mice, LPS activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases is induced with delayed kinetics in TIRAP-deficient mice. Expression of interferon-inducible genes and the maturation of dendritic cells is observed in these mice; they also show defective response to TLR2 ligands, but not to stimuli that activate TLR3, TLR7 or TLR9. In contrast to previous suggestions, these results show that TIRAP is not specific to TLR4 signalling and does not participate in the MyD88-independent pathway. Instead, TIRAP has a crucial role in the MyD88-dependent signalling pathway shared by TLR2 and TLR4 (Yamamoto, 2002).

Mammalian Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defence responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88. Although members of the TLR and the interleukin-1 (IL-1) receptor families all signal through MyD88, the signalling pathways induced by individual receptors differ. TIRAP, an adaptor protein in the TLR signalling pathway, has been identified and shown to function downstream of TLR4. Mice deficient in the Tirap gene have been generated. TIRAP-deficient mice respond normally to the TLR5, TLR7 and TLR9 ligands, as well as to IL-1 and IL-18, but have defects in cytokine production and in activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases in response to lipopolysaccharide, a ligand for TLR4. In addition, TIRAP-deficient mice are also impaired in their responses to ligands for TLR2, TLR1 and TLR6. Thus, TIRAP is differentially involved in signalling by members of the TLR family and may account for specificity in the downstream signalling of individual TLRs (Horng, 2002).

Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Proteins associated with the cytoplasmic domain of mouse TLR4 have been investigated by yeast two-hybrid screening: JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, was identified as a TLR4-associated protein. The homolog of JIP3 has been reported in Drosophila and Caenorhabiditis elegans (Bowman, 2000; Byrd, 2001), indicating JIP3 is evolutionally well conserved. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as evidenced by the observation that the two other JIPs, JIP1 and JIP2, fail to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increases the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impairs LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibits LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, these data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs (Byrd, 2001).

Signaling downstream of Toll-like receptors

The atypical protein kinase C (aPKC)-interacting protein, p62, interacts with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). The inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this, p62 is shown to selectively interact with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6 (Sanz, 2000).

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-kappaB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE - glycation is an uncontrolled, non-enzymatic reaction of sugars with proteins) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, was investigated in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produce activation of NF-kappaB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increase the activity of IKKbeta, HMGB1 exposure results in activation of both IKKalpha and IKKbeta. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-kappaB-dependent transcription by HMGB1. Transfections with dominant negative constructs have demonstrated that TLR 2 and TLR 4 are both involved in HMGB1-induced activation of NF-kappaB. In contrast, RAGE plays only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS (Park, 2004)

Toll-like receptors and axis formation

The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo. A homologous pathway exists in Xenopus, but its role during normal frog development has not yet been established. Xenopus MyD88 (XMyD88), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK kinases, has been cloned. Overexpression of a dominant-negative form of XMyD88 in the frog embryo blocks Toll receptor activity, specifically inhibits axis formation and reduces expression of pivotal organizer genes. The observed stage-dependency of interference suggests a function for maternal XMyD88 soon after fertilization. It is concluded that XMyD88 activity is required for normal Spemann organizer formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation (Prothmann, 2000).

In order to analyze the effect of dominant-negative XMyD88 on early development, RNA in situ hybridizations were performed for genes involved in embryonic patterning. Ventroposterior markers such as bmp4, vent1, vent2, or Xwnt8 are not upregulated on the dorsal side of the embryo, and expression of the dorso-anterior markers Xnot2, XFD-1, Xnr4, Xotx2 and chordin were not significantly reduced in their expression domains. In addition, the pan-mesodermal marker Xbra is expressed at normal levels, indicating that TGF-beta dependent mesoderm induction occurs in dominant negative XMyD88-injected embryos. However, the mRNA levels of a subset of organizer genes are strongly reduced or ablated. These genes encode the multifunctional head-inducer Cerberus (Cer); the TGF-beta family member and neural inducer Xenopus Nodal-related 3 (Xnr3); the homeobox protein Goosecoid (Gsc), and the BMP-antagonist Noggin. Whether these four genes are direct targets of a XMyD88-dependent signaling pathway is currently not known, and dominant negative XMyD88 may also affect the expression of additional genes. Nevertheless, based on what is known about the functions of these pivotal regulators in vertebrate development, their transcriptional inhibition fits to the observed morphological and histological phenotype (Prothmann, 2000).

Mammalian Toll-like receptors and natural immunity

The human homolog of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll occurs through sequential recruitment of the adapter molecule MyD88 and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated factor 6 (TRAF6) and the nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) are both involved in subsequent steps of NF-kappaB activation. Conversely, a dominant negative version of TRAF6 fails to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways (Muzio, 1998).

Interleukin-1 (IL-1) is a proinflammatory cytokine that has several effects in the inflammation process. When it binds to its cell-surface receptor, IL-1 initiates a signaling cascade that leads to activation of the transcription factor NF-kappaB and is relayed through the protein TRAF6 and a succession of kinase enzymes, including NF-kappaB-inducing kinase (NIK) and I kappaB kinases (IKKs). However, the molecular mechanism by which NIK is activated is not understood. The MAPKK kinase TAK1 (Drosophila homolog: TGF-ß activated kinase 1) acts upstream of NIK in the IL-1-activated signaling pathway and TAK1 associates with TRAF6 during IL-1 signaling. Stimulation of TAK1 causes activation of NF-kappaB, which is blocked by dominant-negative mutants of NIK, and an inactive TAK1 mutant prevents activation of NF-kappaB that is mediated by IL-1 but not by NIK. Activated TAK1 phosphorylates NIK, which stimulates IKK-alpha activity. These results indicate that TAK1 links TRAF6 to the NIK-IKK cascade in the IL-1 signaling pathway (Ninomiya-Tsuji, 1998).

Remarkable structural and functional similarities exist between the Drosophila Toll/Cactus/Dorsal signaling pathway and the mammalian cytokine-mediated interleukin-1 receptor (IL-1R)/I-kappaB/NF-kappaB activation cascade. In addition to a role regulating dorsal-ventral polarity in the developing Drosophila embryo, signaling through Drosophila Toll (dToll) activates the nonclonal, or innate, immune response in the adult fly. Recent evidence indicates that a human homolog of the dToll protein participates in the regulation of both innate and adaptive human immunity through the activation of NF-kappaB and the expression of the NF-kappaB-controlled genes IL-1, IL-6, and IL-8, thus affirming the evolutionary conservation of this host defense pathway. The cloning is reported of two novel human genes, TIL3 and TIL4 (Toll/IL-1R-like-3, -4) that exhibit homology to both the leucine-rich repeat extracellular domains and the IL-1R-like intracellular domains of human and Drosophila Toll. Northern analysis shows distinctly different tissue distribution patterns, with TIL3 expressed predominantly in ovary, peripheral blood leukocytes, and prostate, and TIL4 expressed primarily in peripheral blood leukocytes and spleen. Chromosomal mapping by fluorescence in situ hybridization localizes the TIL3 gene to chromosome 1q41-42 and TIL4 to chromosome 4q31.3-32. Functional studies shows that both TIL3 and TIL4 are able to activate NF-kappaB, though in a cell type-dependent fashion. Together with human Toll, TIL3 and TIL4 encode a family of genes with conserved structural and functional features involved in immune modulation (Chaudhary, 1998).

The Toll-mediated signaling cascade using the NF-kappaB pathway has been shown to be essential for immune responses in adult Drosophila. A human homolog of the Drosophila Toll protein induces various immune response genes via this pathway. Signaling by the human Toll receptor employs an adaptor protein, MyD88, and induces activation of NF-kappaB via the Pelle-like kinase IRAK and the TRAF6 protein, similar to IL-1R-mediated NF-kappaB activation. However, Toll and IL-1R signaling pathways are not identical with respect to AP-1 activation. These findings implicate MyD88 as a general adaptor/regulator molecule for the Toll/IL-1R family of receptors for innate immunity (Medzhitov, 1998).

MyD88 has a modular organization, an N-terminal death domain (DD) related to the cytoplasmic signaling domains found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, and a C-terminal Toll domain similar to that found in the expanding family of Toll/interleukin-1-like receptors (IL-1R). This dual domain structure, together with the following observations, supports a role for MyD88 as an adapter in IL-1 signal transduction; MyD88 forms homodimers in vivo through DD-DD and Toll-Toll interactions. Overexpression of MyD88 induces activation of the c-Jun N-terminal kinase (JNK) and the transcription factor NF-kappaB through its DD. A point mutation in MyD88, MyD88-lpr (F56N), which prevents dimerization of the DD, also blocks induction of these activities. MyD88-induced NF-kappaB activation is inhibited by the dominant negative versions of TRAF6 and IRAK, which also inhibit IL-1-induced NF-kappaB activation. Overexpression of MyD88-lpr or MyD88-Toll (expressing only the Toll domain) acts to inhibit IL-1-induced NF-kappaB and JNK activation in a 293 cell line overexpressing the IL-1RI. MyD88 coimmunoprecipitates with the IL-1R signaling complex in an IL-1-dependent manner (Burns, 1998).

Bacterial lipopolysaccharides stimulate Toll-like receptors

Bacterial lipopolysaccharide (LPS) induces activation of the transcription factor nuclear factor kappaB (NF-kappaB) in host cells upon infection. LPS binds to the glycosylphosphatidylinositol (GPI)- anchored membrane protein CD14, which lacks an intracellular signaling domain. The role of mammalian Toll-like receptors (TLRs) as signal transducers for LPS was investigated. Overexpression of TLR2, but not TLR1, TLR4, or CD14 confers LPS inducibility of NF-kappaB activation in mammalian 293 cells. Mutational analysis demonstrates that this LPS response requires the intracellular domain of TLR2. LPS signaling through TLR2 is dependent on serum that contains soluble CD14 (sCD14). Coexpression of CD14 synergistically enhances LPS signal transmission through TLR2. In addition, purified recombinant sCD14 can substitute for serum to support LPS-induced TLR2 activation. LPS stimulation of TLR2 initiates an interleukin 1 receptor-like NF-kappaB signaling cascade. These findings suggest that TLR2 may be a signaling component of a cellular receptor for LPS (Kirschning, 1998).

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown. Toll-like receptor 2 (TLR2) is a signaling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1-receptor-associated kinase is required for this response. These results indicate that TLR2 is a direct mediator of signaling by LPS (Yang, 1998).

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-kappaB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-kappaB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocks TLR4-mediated transgene activation in a dose-dependent manner. These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-kappaB activation and subsequent cellular events (Chow, 1999).

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiate apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulates nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activate the respiratory burst through hTLR2. The respiratory burst of phagocytes generates reactive oxygen species (ROS) that contribute to antimicrobial defense. A monoclonal antibody specific for hTLR2 (mAb 2392) was isolated. This antibody completely abrogates the generation of ROS by peripheral blood leukocytes (PBLs) after exposure to sBLP, indicating that mAb 2392 is a blocking antibody. Although a role for other TLRs cannot be excluded, these data indicate that hTLR2 is critical for BLP responses and link Toll receptors to a transcription-independent, antimicrobial defense mechanism. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms. Although many bacterial pathogens induce apoptosis in host cells, the implications of this phenomenon remain elusive. BLP-induced apoptosis could be important for (1) the initiation of inflammation, (2) the resolution of inflammation, and (3) generating the proper signals necessary for adaptive immune responses. The observation that sBLPs are excellent adjuvants supports the third hypothesis. The BLP-hTLR2 apoptotic pathway emerges as a mechanism potentially fulfilling multiple roles in the genesis and progression of innate and adaptive immune responses to bacteria (Aliprantis, 1999).

Toll: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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