Toll‐like receptors: molecular mechanisms of the mammalian immune response

The unrelenting bombardment by microbial pathogens from the environment and their increasing resistance to medical treatments are a constant threat to the survival of all organisms on earth. Microbes are covered by molecular patterns that are common among a broad range of pathogens. These include the lipopolysaccharides (LPS) of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria, lipoproteins of bacteria and parasites, glycolipids of mycobacteria, mannans of yeast and double-stranded RNAs of viruses.1,2 Recognition of and responses to these molecules are controlled by a wide variety of cellular receptors. The best characterized receptors are the T-cell receptor and B-cell antibody receptor of the adaptive immune response, the specificity of which is randomly generated and clonally selected during the development of T and B lymphocytes. 3 Unlike the receptors of the adaptive immune system, the pattern recognition receptors of the innate immune system have predetermined specificity generated early on in evolution and play an essential role in the determination of self versus non-self during the initial rapid responses to infection.1,2 As described in this review, a family of cell surface receptors, termed Toll-like receptors, are emerging as key regulators of host responses to infection. The origins of the mammalian innate immune response are clearly seen with the involvement of the Toll protein in Drosophila development and host defence. Prior to the discovery of its involvement in Drosophila host defence, Toll was identified by a genetic screen for a defect in the establishment of dorsal–ventral polarity. This toll mutant was a dominant-ventralizing mutation in contrast to the recessive-dorsalizing mutants dorsal and gastrulation defective. Further mutant screens and double-mutant strategies characterized the order of other genes involved in this developmental pathway. 4 The best characterized being the Rel signalling pathway involving the activation of family members dorsal, dif and relish. 5–8 The factors involved in the embryonic Toll signalling pathway have been identified and characterized. 9 Activation of Toll requires the endogenous ligand Spaetzle, which is proteolytically cleaved to its active form by a series of serine proteases genetically upstream of spaetzle and toll. Stimulation of Toll leads to the activation of Rel proteins, such as Dorsal, through signalling factors Tube and Pelle, resulting in the degradation of Cactus and release of Dorsal. Cactus is the Drosophila homologue of the mammalian I-κB family that sequesters and inhibits Rel family members in the cytoplasm prior to activation. Once released, Rel proteins travel to the nucleus where they positively or negatively regulate target genes involved in many cellular events. In fact, recent studies have placed Toll signalling pathways at the centre of mediating the up-regulation of Drosophila host responses to infection.4,10,11 The Drosophila immune response relies on the production of antimicrobial peptides, and each peptide has inherent specificity for its particular class of pathogen. 12 In an analysis of the regulation of these peptides it was found that they were all highly regulated by κB sites in their promoters. In fact, the Rel family members Dorsal, Dif and Relish are all activated in response to infection. 5–8 Genetic analysis identified the Spaetzle–Toll–Pelle–Tube–Cactus signalling cascade as an important regulator not only of Rel protein activation, but also in the induction of antimicrobial peptides. As a result, differential responses to yeast and bacterial infection have been observed between Toll family members. Toll and Spaetzle mutant flies suffer severe fungal infection and are unable to induce the expression of the antifungal peptide, Drosomycin. Although many of the same factors involved in the embryonic pathway are utilized, the Toll–antifungal pathway leads to the activation of Dif, but not Dorsal, in the regulation of the drosomycin gene. 10 In addition, there are mechanisms in place to respond to bacterial infection. The Toll family member, 18-wheeler (18w), has been linked to the production of Attacin, an antibacterial peptide.13,14 Mutants for ird and imd have also been linked to antibacterial responses. 6 In contrast to mutants biased to specific pathogens (toll, spaetzle, 18w, ird and imd), Cactus is required for both antifungal and antibacterial responses. This suggests that Cactus is a common player in both kinds of responses and may therefore involve separate Cactus/Rel complexes activated by independent upstream pathways. 8 This is a potential mechanism for the differential regulation of Rel family target genes. Strikingly, similar receptors and signalling pathways have been observed recently in other species, ranging from plants to mammals, a fact which emphasizes the conservation of molecules throughout evolution. The link between Drosophila Toll (dToll) and mammalian Toll-like receptors exemplifies the evolutionary connection between Drosophila host defence and the mammalian innate immune response, which has been considered for many years to be the ancient arm of the mammalian immune system. Mammalian Toll homologues have been termed Toll-like receptors. Nine have been published. 15 The first functional analysis of mammalian Toll-like receptor was the study of hToll4 by Medzhitov et al. 16 The hToll4 mRNA was isolated from dendritic cells, γδ T cells, T helper type 1 (Th1) and 2 (Th2) αβ T cells and B cells. A dominant active form of the receptor induced cytokine expression and NF-κB activation when expressed in monocyte cell lines. 16 These findings and the homology with dToll suggest that Toll-like receptors may play an important role in mammalian host responses to infection. Toll-like receptors have moderate homology with dToll, but more importantly share a common structure. Toll-like receptor structure includes leucine-rich repeats in the extracellular domain15,17,18 and a cytoplasmic domain which shares significant homology with the interleukin-1 receptor (IL-1R) signalling domain, termed the Toll/IL-1 receptor homologous region. 19 The leucine-rich repeats of the extracellular domain mediate the response to conserved pathogen-associated molecular patterns commonly found among microbial organisms. A number of pattern recognition receptors recognize these molecules, including mannose and scavenger receptors, Toll-like receptors and CD14.1,2 There is considerably more diversity found in the extracellular domains of Toll-like receptors, when compared to the intracellular signalling domain. 20 This has also been observed in the Toll-like receptors of plant species. 21 Analysis of the leucine-rich repeats of plants suggests that the extracellular domain plays an important role in the specificity of host responses to infection. 22 Although leucine-rich repeats play a role in the recognition of microbial ligands, their involvement in Toll-like receptor function has yet to be fully addressed. Responses to LPS can cause septic shock in extreme Gram-negative bacterial infections, causing a severe health risk to the population every year. The search for the ‘LPS receptor’ has a long history. Three strains of mice, C3H/HeJ, C57BL/10ScCr and C57BL/10ScN, are naturally hyporesponsive to high doses of LPS. These mutations are referred to as the lpsd gene.23,24 These strains of mice showed inherent specificity by their increased susceptibility to Gram-negative infection, and normal responses to Gram-positive bacteria. Furthermore, LPS hyporesponsive macrophages from lpsd mutant mouse strains respond normally to microbial lipoproteins. 23–25 This suggested that different receptors were involved in responding to different pathogens. Following the identification of the mouse tlr4 gene and considerable mapping of the lpsd gene locus, lpsd was identified as the lps gene. 26–28 In a 1999 review, Samuel D. Wright described Toll-like receptors as, ‘the missing piece of the puzzle’. 29 In fact, the mutations that occurred in all three mouse strains occurred in the tlr4 gene. The C3H/HeJ hyporesponsiveness is caused by a dominant negative mutation 30 owing to a proline to histidine substitution mutation at amino acid 712. C57BL/10ScCr and C57BL/10ScN strains carry a recessive tlr4 null mutation. 26–28 Another Toll-like receptor, TLR2, was also implicated in responses to LPS. TLR2 conferred LPS responsiveness to a normally non-responsive cell line, HEK293. TLR2 activated NF-κB in response to LPS and an anti-TLR2 antibody inhibited the production of tumour necrosis factor-α (TNF-α) and IL-12 p40 from monocytes stimulated with LPS. 31–33 (Please see NOTE ADDED IN PROOFat end of Reference list.) Hamster macrophages and the Chinese hamster ovary CHO cell line carry a TLR2 null mutation, but are still responsive to LPS. 34 While TLR2 may be LPS responsive in stably transfected cell lines, genetic evidence supports TLR4 as the LPS receptor. Toll-like receptor-deficient (TLR–/–) mice support this model. TLR4–/– mice are hyporesponsive to LPS, while TLR2–/– mice still respond normally. The results from the Toll-like receptor knockouts also suggest that there is clear specificity for microbial ligands between Toll-like receptor family members.35,36 Although the evidence supports TLR4 as a key component of the LPS receptor, TLR4 mutant mice have been observed to be responsive to LPS under some conditions. Responsiveness has been observed with LPS from different species of Gram-negative bacteria 37 as well as with extremely high doses of LPS. 38 In addition, activation can be restored to normal levels by treatment with interferon-γ (IFN-γ). 39 Although there appear to be clear differences in specificity between TLR4 and TLR2 in mouse, the ability of human TLR2 to be activated by LPS and the inhibition of LPS-induced cytokine activation by an anti-human TLR2 blocking antibody suggest that there may either be some factors in common between TLR4 and TLR2 specificities or species differences between human and mouse Toll-like receptors. 31–33 Clearly, the mechanisms underlying responses to microbial ligands are very complex and may involve multiple factors that determine the specificity of Toll-like receptor responsiveness. Much like the specificity of Drosophila Toll family members, mammalian Toll-like receptor family members are emerging as having their own specificity for different classes of microbial molecules. Microbial molecules which elicit strong responses from the innate immune system are quite common among a broad range of pathogens. These pathogen-associated molecular patterns are generally repetitive in nature and are distinct from the molecules found in the host. In addition, pathogen-associated molecular patterns are not subject to antigenic variation, but are essential for the survival and pathogenicity of the organism.1,2 Microbial carbohydrates are a good example of this, which are a common structure that decorate the cell walls of many species of bacteria. 40–42 Lipid modifications are also common among microbial molecules. 43–48 It is thought that pathogen-associated molecular patterns are the target of pattern recognition receptors, such as Toll-like receptors; however, the exact mechanism of this interaction is under much debate. While much of the focus of Toll-like receptor biology has centred on its role in mediating LPS responsiveness, there has been much interest in identifying other pathogens and molecules that activate TLRs. Although a growing number of Toll-like receptors have been identified, TLR2 and TLR4 are the only Toll-like receptors that have been shown to be responsive to microbial ligands. TLR4 is clearly the main LPS receptor, but what role do TLR4 and TLR2 play in the responses to other antigens? Microbial lipoproteins are very common among bacteria including mycobacteria, Gram-positive and Gram-negative bacteria, such as the spirochaetes Borrelia burgdorferi and Treponema pallidum. These microbial lipoproteins share a common triacyl motif at their N-termini that is responsible for their stimulatory properties. 43–47 Much like many of these bacterial ligands, microbial lipoproteins are strong stimulators of macrophage activation, including the production of cytokines and the up-regulation of inflammatory events. 48–52 TLR2 was found to mediate responsiveness to lipoproteins from mycobacteria, and species. TLR2 was found to be the of activation, while were found not to be anti-TLR2 antibody inhibited of cytokine production from macrophages stimulated with lipoproteins. as well as the Toll-like receptor-deficient mice, that TLR2 is the receptor for microbial While TLR4 has been shown to also mediate responses to some lipoteichoic acids and LPS, TLR2 to a broad range of molecules from a wide variety of pathogens. TLR2 has been shown to be involved in the of as well as in responses to to the 19 and from TLR2 to be the regulator of Gram-positive responses. lpsd mice respond normally to Gram-positive infection while being more to Gram-negative infection. Gram-positive bacterial ligands that through TLR2 include cell and lipoteichoic responsiveness to pathogens by multiple receptors is of Drosophila host defence. Drosophila Toll receptors mediate responses to a specific class of resulting in the production of target genes that that infection. It that this has been conserved at some with mammalian Toll-like receptors which have an growing of ligands. This to the activation of Toll-like specific target genes. the range of microbial ligands, these innate receptors a mechanism by which the immune system can respond to distinct classes of molecules. Further studies with Toll-like receptor-deficient mice be essential to TLR2 in to the specificity of responses and the susceptibility to infection. The between Drosophila host defence and mammalian innate are Although the suggest that TLR2 and TLR4 have clear specificity for different microbial ligands, the mechanism of activation is still there a interaction between Toll-like receptors and their ligands, or are there endogenous ligands, similar to the Drosophila Spaetzle, that activate the receptors in response to Furthermore, what role do Toll-like receptor such as and play in this protein which may play a role in Toll-like receptor function is was the first protein to be identified as an LPS receptor. many molecules, including LPS, microbial cell walls and Once from the surface of the LPS is to or of by protein protein The of a complex at the results in cellular activation of cell expression with increased of many cell to LPS and other microbial molecules in their ability to activate signalling and cytokine is to the of the by a not the the it mediate LPS signalling but may a to activate intracellular signalling pathways. this to mice are hyporesponsive to LPS, that plays a role in this to this for a expression not LPS responsiveness to cell lines. when is with TLR2 or activation of NF-κB by LPS, microbial or is These are with the role of in cellular activation and cytokine may be involved in responsiveness. studies of TLR4 suggested that it was not responsive to LPS activation when stably expressed in cells. studies identified a which was shown to with TLR4 and its responsiveness to LPS. Furthermore, may be required for TLR4 the do other Toll-like receptors similar to other Toll-like receptor family members have not been shown to be responsive to microbial they may or with other Toll-like receptor family members to there interaction between Toll-like receptors and their microbial The specificity in Toll-like cell lines, as well as the mice, suggests that interaction may be the of this interaction is the molecular Unlike the strong of with ligands LPS and microbial the of with LPS is much be for this of other proteins, such as and may this 20 studies suggest that there is a interaction in a of responsiveness to different of A by genetic and mouse TLR4 were to between different of A when in C3H/HeJ there have yet to be studies a strong between Toll-like receptors and their ligands. The activation of Drosophila Toll during infection requires the endogenous ligand Much like the of Spaetzle in the developmental Toll is proteolytically cleaved to its active Spaetzle, by fungal 10 The serine proteases and involved in the developmental however, are not involved in the response to fungal infection. 10 of Spaetzle is required for induced expression of antimicrobial peptides Toll in but the involved in the immune response has yet to be Spaetzle and Toll are required for antifungal responses in endogenous ligand has been identified for antimicrobial responses upstream of the Toll family member, 10 have been observed in other species and are thought to play a role in mammalian immune response, as serine proteases have been observed in the These proteases share with the Drosophila serine The proteases B and which are homologues of and similar to found in The activation of the mammalian cascade a pathway of proteases activated during infection. These mechanisms clearly factors in common between organisms. there a cascade and endogenous ligand upstream of Toll-like receptor have that Toll-like receptors are activated by an endogenous studies of endogenous ligands for Toll-like receptors have only recently to A study by et al. implicated the protein as an endogenous TLR4 that it as a to the innate immune In fact, is normally in the cell but is to the in response to cell Responses to LPS and are very C3H/HeJ mice are not responsive to that like LPS, TLR4 stimulatory of Although this is the first example of an endogenous protein Toll-like receptor signalling a interaction of TLR4 with has yet to be A role for in the activation of responses to LPS is not et al. identified of serine from cells. of LPS-induced activation of and the production of and macrophage cell hyporesponsiveness to LPS. there are between organisms. Drosophila of is regulated by the serine that there may be multiple levels of regulation in LPS response expression is inhibited by and LPS responsiveness. This responsiveness is with of LPS and may to activation by LPS. Further of an endogenous Toll-like receptor ligand more and genetic to molecules involved in Toll-like receptor activation. The immune responses of Drosophila and not only share common receptors, but also share conserved signalling pathways involved in the activation of host The Toll-like receptors share significant homology with the intracellular signalling domain. 19 In fact, they share common signalling molecules of the Activation of NF-κB of a number of receptors including receptors, the receptor, dToll and Toll-like receptors. Activation of the receptor domain results in of the through its own receptor domain. Furthermore, mice do not respond to either or LPS, its role in these signalling receptor protein through domain of both proteins resulting in the of is to the activation of such as The and which are involved in the of members of the NF-κB I-κB family members are resulting in I-κB NF-κB to the nucleus where the active can gene expression of such cytokines as IL-12 and molecules and A number of signalling pathways have been implicated of microbial ligands, such as LPS and lipoproteins. In fact, the regulation of many genes requires more signalling pathway. the IL-12 p40 gene is regulated by NF-κB and protein family In to the NF-κB signalling other signalling pathways have been linked to Toll-like receptor signalling pathways. of the dominant active form of TLR4 not only NF-κB but also and In fact and are involved in the regulation of many and The protein pathway is also activated in response to microbial ligands, of Toll-like receptor activation as The to be involved in LPS-induced NF-κB activation of TLR2 and TLR4 in transfected cell and in the macrophage cell line A negative mutant NF-κB activation in both of these cell and is in response to LPS. The specific to the activation of of Toll-like receptors are however, the suggests that while is essential for responses to LPS, is not required for the activation of NF-κB or pathways. The Toll-like receptor, protein and pathways. There is growing evidence that Toll-like receptors to the activation of a number of different signalling pathways that to the specificity of target gene activation and cellular responses. Toll-like receptors appear to be involved in the activation of a growing of mammalian host responses to infection. The signalling pathways and target genes of Toll-like receptors have on the host responses are The innate immune response is for the early of microbial pathogens and the of infection. In the adaptive immune response is and only do innate and adaptive immune responses each they are The innate immune response the type of adaptive immune response through the production of which are regulated in by Toll-like receptor Activation of is not but on activation generated by cells of the innate immune of the innate immune system, cells that have been in with the can responses through the production of cytokines and Toll-like receptors are clearly involved in the activation of cytokines by microbial ligands. of a dominant active TLR4 in macrophage cell cytokine production and the expression of molecules. 16 IL-12 is a regulator of immune responses the induction of responses from T and B lymphocytes. Microbial lipoproteins activate the IL-12 p40 through a A negative mutant inhibits the induction of the IL-12 p40 and by microbial lipoproteins. Furthermore, an anti-TLR2 antibody the production of IL-12 and other such as from Activation of Toll-like receptors may also to the induction of such as As a result, the of (Th1) and (Th2) immune responses can be by Toll-like receptor target genes. studies have suggested that and cytokines are regulated in response to the same microbial ligands. In fact, the differential expression of IL-12 versus has been observed in such as where the of these cytokines can have on the of The involvement of in this differential regulation of genes be an to in Toll-like receptors are clearly activated by are strong stimulators of IL-12 production and immune The 19 can activate the and production from macrophages in a is for the production of from a antimicrobial pathway. In is required for host defence mice are highly to infection. activation of mammalian Toll-like receptors, by induction of can to an antimicrobial a main function of Drosophila Toll is the induction of antimicrobial peptides, the function of Toll in host defence has been conserved throughout In to macrophage activation and responses to Toll-like receptor activation, the activation of mechanisms has also been et al. that microbial lipoproteins induced of in human including cell and by a of was by and cell was to microbial lipoproteins have the ability to induce both activation of host defence and This signalling pathway is similar to receptor and which can induce both NF-κB activation and In this it is for the immune system to activate host defence by the response from causing such as in by to activation of may also to during the of In a recent review, suggested that the Drosophila immune response is to respond to infection. Toll receptors have clear specificity for the class of antimicrobial peptides that they Toll the antifungal peptide and 18-wheeler the antibacterial peptide, Rel family members have been found to the specificity of target gene activation as for dif mutants production of drosomycin and mutants to A and in to having production of other antimicrobial peptides. there a similar genetic mechanism in the mammalian immune The of the mammalian Toll-like receptor responses multiple levels of Although target genes for each Toll-like receptor have not been identified the Toll-like receptors clearly have specificity for classes of microbial ligands Toll-like receptors may be to increasing the diversity of ligands that these receptors respond as well as, the diversity of target gene activation mice have been generated and are being characterized for NF-κB family involvement in the activation of specific target genes. expression of NF-κB family members has on the production of cytokines and immune responses to infection. as more studies the of NF-κB family members on host responses more A wide variety of cell are responsive to microbial ligands and a of Toll-like receptors. The of these receptors may be to a number of host responses ranging from the production of macrophage cytokines to B-cell and antibody The of the Toll-like receptor to be with many to Toll-like receptors and host LPS, lipoteichoic Toll-like receptors; interleukin-1 receptor protein tumour necrosis The of Toll in Drosophila that Toll proteins a host defence mechanism that has been conserved of of years of evolution. In mammals, Toll-like receptors the innate immune system with the ability to recognize and to a wide of microbial pathogens lipoproteins and Toll-like receptors can activate innate immune responses including a and which as a T-cell responses to pathogens. microbial ligand activation of Toll-like receptors is an early for the induction of IL-12 and the of cytokine responses. It be important to determine by induction of or other activation of leads to a as is in the Toll-like receptor signalling pathway can to the activation of including of the target cells resulting in of the immune response or to the host It be however, to strategies to Toll-like receptors in order to activate host defence mechanisms and to immune responses to a wide variety of in A recent study suggests that LPS a of microbial ligands with distinct of through both human and receptor