Toll/IL-1 Receptor Research Articles

Immunobiology of Toll‐like receptors: Emerging trends

Toll-like receptors (TLR), a family of evolutionarily conserved pathogen recognition receptors, play pivotal role as primary sensors of invading pathogens. TLR identify molecular motifs of infectious agents (pathogen associated molecular patterns) and elicit an effective defensive response against them. Mammalian TLR derive their name from the Drosophila Toll protein, with which they share sequence similarity. So far, expression of 10 TLR is known in humans. The adaptor proteins, myeloid differentiation factor 88 and Toll IL-1 receptor (TIR) domain containing adaptor inducing IFN-beta (TRIF) are the key players in the TLR signalling cascade leading to the activation of nuclear factor (NF)-kappaB and interferon regulatory factor-3, respectively. Targeted manipulation of the TLR signalling pathway has immense therapeutic potential and may eventually prove to be a boon in the development of innovative treatments for diverse disease conditions. There is accumulating evidence that TLR agonists have tremendous potential as novel therapeutic targets. In this review, we have discussed the immunobiology of TLR and emphasize significant advances made within the ever-expanding field of TLR that provide intriguing insights efficacious in unravelling the complexities associated with TLR.

The interleukin-1 families of cytokines and receptors: therapeutic potential for immunomodulation and the treatment of inflammatory disorders

The interleukin (IL)-1 cytokine family includes 11 distinct members that are related in their genomic and protein structures. The initial members of this family, IL-1α and IL-1β, were discovered over two decades ago and are now considered as prototypic pro-inflammatory cytokines. Their activity is downregulated by a third family member, termed IL-1-receptor antagonist (IL-1ra). Another IL-1 family cytokine that was also identified early on is IL-18, an agonist cytokine possessing potent immunostimulatory activities. Subsequently, sequence homology searches in DNA databases led to the discovery of genes that encode additional family members. IL-1 cytokines exert their function through structurally related cell surface receptors that have a Toll/IL-1 receptor (TIR) module in their intracellular region and one to three copies of immunoglobulin (Ig)-like domains in their extracellular region. At present, 11 members of the IL-1 receptor family are known. A hallmark of IL-1 receptor signalling, which is mediated via the TIR domain, is the activation of the transcription factor NF-κB and the mitogen-activated protein kinases (MAPK). Owing to their possible roles in immune disease mechanisms, IL-1 and its relatives have long been considered as potential targets of therapeutic intervention in inflammatory and autoimmune conditions. Strong evidence for the importance of IL-1 in pathogenic inflammation was recently provided by the clinical use of a recombinant form of human IL-1ra for the treatment of rheumatoid arthritis and certain autoinflammatory syndromes. The discovery and characterisation of the IL-1 families of cytokines and receptors, as well as the exploration of their therapeutic potential, has generated a large body of patent literature over the past few years. Here, this literature is reviewed and discussed in the context of current research findings.

Binding specificity of Toll-like receptor cytoplasmic domains.

MyD88 participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated Toll-like receptors (TLR). Yeast two-hybrid experiments reveal that the TIR domains of human TLR differ in their ability to associate with MyD88: The TIR of TLR2 binds to MyD88 but the TIR of the closely related TLR1, 6, or 10 do not. Using chimeric TIR domains, we define the critical region responsible for differential MyD88 binding, and use a computational analysis of the critical region to reveal the amino acids that differ between MyD88 binders and non-binders. Remarkably, a single missense mutation created in TLR1 (N672D) confers on it the ability to bind MyD88, without affecting its association with other proteins. Mutations identified as critical for MyD88 binding also affect signaling of TLR pairs in mammalian cells. To investigate the difference between MyD88 binders and non-binders, we identify novel interacting proteins for each cytoplasmic domain of TLR1, 2, 6, and 10. For example, heat shock protein (HSP)60 binds to TLR1 but not to TLR2, and HSP60 and MyD88 appear to bind the same region of the TIR domain. In summary, interactions between the TLR, MyD88, and novel associated proteins have been characterized.

MyD88-dependent and -independent signaling by IL-1 in neurons probed by bifunctional Toll/IL-1 receptor domain/BB-loop mimetics

Interleukin (IL)-1beta is a pluripotent proinflammatory cytokine that signals through the type-I IL-1 receptor (IL-1RI), a member of the Toll-like receptor family. In hypothalamic neurons, binding of IL-1beta to IL-1RI mediates transcription-dependent changes that depend on the recruitment of the cytosolic adaptor protein myeloid differentiation primary-response protein 88 (MyD88) to the IL-1RI/IL-1 receptor accessory protein (IL-1RAcP) complex through homomeric Toll/IL-1 receptor (TIR)-TIR interactions. Through design and synthesis of bifunctional TIR mimetics that disrupt the interaction of MyD88 with the IL-1RI/IL-1RAcP complex, we analyzed the involvement of MyD88 in the signaling of IL-1beta in anterior hypothalamic neurons. We show here that IL-1beta-mediated activation of the protein tyrosine kinase Src depended on a MyD88 interaction with the IL-1RI/IL-1RAcP complex. The activation of the protein kinase Akt/PKB depended on the recruitment of the p85 subunit of PI3K to IL-1RI and independent of MyD88 association with the IL-1RI/IL-1RAcP complex. These bifunctional TIR-TIR mimetics represent a class of low-molecular-weight compounds with both an antiinflammatory and neuroprotective potential. These compounds have the potential to inhibit the MyD88-dependent proinflammatory actions of IL-1beta, while permitting the potential neuronal survival supporting actions mediated by the MyD88-independent activation of the protein kinase Akt.

TNF receptor‐associated factor‐1 (TRAF1) negatively regulates Toll/IL‐1 receptor domain‐containing adaptor inducing IFN‐β (TRIF)‐mediated signaling

Toll-like receptor 3 (TLR3) plays an important role in antiviral responses through recognizing viral double-stranded RNA produced during viral infection and mediating induction of type I IFN. TRIF is a Toll/IL-1 receptor (TIR) domain-containing adaptor protein that is associated with TLR3 and critically involved in TLR3-mediated signaling. In yeast two-hybrid screens, we identified TNF receptor-associated factor (TRAF)1 as a TRIF-interacting protein. The TRAF-C domain of TRAF1 and the TIR domain of TRIF were responsible for their interaction. Overexpression of TRAF1 inhibited TRIF- and TLR3-mediated activation of NF-kappaB, IFN-stimulated response element and the IFN-beta promoter. Overexpression of TRIF caused caspase-dependent cleavage of TRAF1. The cleaved N-terminal but not C-terminal fragment of TRAF1 was responsible for inhibiting TRIF signaling. Mutation of the caspase cleavage site of TRAF1 or addition of the caspase inhibitor crmA inhibited TRAF1 cleavage and abolished the ability of TRAF1 to inhibit TRIF signaling, suggesting that TRIF-induced cleavage of TRAF1 is required for its inhibition of TRIF signaling. Our findings provide a novel mechanism for negative regulation of TRIF-mediated signaling.

A Novel Splice Variant of Interleukin-1 Receptor (IL-1R)-Associated Kinase 1 Plays a Negative Regulatory Role in Toll/IL-1R-Induced Inflammatory Signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.

TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF

Toll-like receptors (TLR) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an essential role in intracellular eradication of engulfed pathogens. Here, we demonstrate the physical and functional association between components of the cytosolic NADPH oxidase and TLR-mediated signaling molecules. Cytosolic components of NADPH oxidase suppressed TLR-mediated NF-kappaB activation as well as IFN-beta promoter activation. We demonstrate that TNF-associated factor (TRAF) 4 associates with p47(phox), a component of cytosolic NADPH oxidase, and physically interacts and functionally counteracts with TRAF6 and Toll-IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF) molecules that critically regulate TLR-mediated signaling. TRAF4 mRNA expression was elicited in RPMI 8226 cells following LPS or CpG DNA treatment. These results suggest that TRAF4 participates in the molecular mechanism underlying silencing of TLR-mediated signaling through the interaction with molecules harboring phagosome/endosome membrane.

Interactive Sites in the MyD88 Toll/Interleukin (IL) 1 Receptor Domain Responsible for Coupling to the IL1β Signaling Pathway

Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1beta, a key inflammatory cytokine. IL1beta receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1beta-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1beta signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1beta-induced and NFkappaB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix alphaA, 195-197 in Box 2, and 275 in betaE-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro200 by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a three-dimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed.

Molecular Determinants of TRIF Proteolysis Mediated by the Hepatitis C Virus NS3/4A Protease

Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses. HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its proline-rich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-beta (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P(6) position in lieu of the acidic residue present in viral substrates. Circular dichroism (CD) spectroscopy confirmed that a substantial fraction of TRIF exists as polyproline II helices, and inclusion of the polyproline track increased affinity of P side TRIF peptides for the HCV-BK protease. A polyproline II peptide representing an SH3 binding motif (PPPVPPRRR, Sos) bound NS3 with moderate affinity, resulting in inhibition of proteolytic activity. Chemical shift perturbations in NMR spectra indicated that Sos binds a 3(10) helix close to the protease active site. Thus, a polyproline II interaction with the 3(10) helix likely facilitates NS3/4A recognition of TRIF, indicating a significant difference from NS3/4A recognition of viral substrates. Because SH3 binding motifs are also present in NS5A, a viral protein that interacts with NS3, we speculate that the NS3 3(10) helix may be a site of interaction with other viral proteins.

Peptide-mediated Interference of TIR Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-κB

Myeloid differentiation factor 88 (MyD88) plays a crucial role in the signaling pathways triggered by interleukin (IL)-1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the Toll/IL-1 receptor (TIR) domain in MyD88 homodimerization in cell-free and in vitro experimental settings by using epta-peptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified glutathione S-transferase-MyD88 TIR or co-immunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 epta-peptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-kappaB. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signaling in vivo.

Human TLR10 Is a Functional Receptor, Expressed by B Cells and Plasmacytoid Dendritic Cells, Which Activates Gene Transcription through MyD88

Human TLR10 is an orphan member of the TLR family. Genomic studies indicate that TLR10 is in a locus that also contains TLR1 and TLR6, two receptors known to function as coreceptors for TLR2. We have shown that TLR10 was not only able to homodimerize but also heterodimerized with TLRs 1 and 2. In addition, unlike TLR1 and TLR6, TLR10 was expressed in a highly restricted fashion as a highly N-glycosylated protein, which we detected in B cell lines, B cells from peripheral blood, and plasmacytoid dendritic cells from tonsil. We were also able to detect TLR10 in a CD1a(+) DC subset derived from CD34(+) progenitor cells which resemble Langerhans cells in the epidermis. Although we were unable to identify a specific ligand for TLR10, by using a recombinant CD4TLR10 molecule we also demonstrated that TLR10 directly associates with MyD88, the common Toll IL-1 receptor domain adapter. Additionally, we have characterized regions in the Toll IL-1 receptor domain of TLR10 that are essential in the activation of promoters from certain inflammatory cytokines. Even though TLR10 expression has not been detected in mice, we have identified a partial genomic sequence of the TLR10 gene that was present but nonfunctional and disrupted by a retroviral insertion in all mouse strains tested. However, a complete TLR10 sequence could be detected in the rat genome, indicating that a functional copy may be preserved in this species.

Evolution and integration of innate immune recognition systems: the Toll-like receptors

Toll-like receptors (TLRs) recognize specific components of micro-organisms and trigger the activation of innate immunity. TLR-mediated signaling pathways are now rapidly being elucidated. TLR signaling originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain, which is conserved among all TLRs. Furthermore, TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP and TRAM, play essential roles in TLR signaling. MyD88 is essential for inflammatory cytokine production via all TLRs, whereas TRIF is involved in TLR3- and TLR4-mediated MyD88-independent induction of IFN-β. Thus, innate immunity represents a skilful system that senses microbial invasion and initiates appropriate immune responses.

Evolution and integration of innate immune recognition systems: the Toll-like receptors

Toll-like receptors (TLRs) recognize specific components of micro-organisms and trigger the activation of innate immunity. TLR-mediated signaling pathways are now rapidly being elucidated. TLR signaling originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain, which is conserved among all TLRs. Furthermore, TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP and TRAM, play essential roles in TLR signaling. MyD88 is essential for inflammatory cytokine production via all TLRs, whereas TRIF is involved in TLR3- and TLR4-mediated MyD88-independent induction of IFN-beta. Thus, innate immunity represents a skillful system that senses microbial invasion and initiates appropriate immune responses.

Modulation of Toll?interleukin 1 receptor mediated signaling

Toll-like receptors (TLRs) belong to the Toll-interleukin 1 receptor superfamily, which is defined by a common intracellular Toll-IL-1 receptor (TIR) domain. A group of TIR domain containing adaptors (MyD88, TIRAP, TRIF and TRAM), are differentially recruited to the Toll-IL-1 receptors, contributing to the specificity of signaling. The IL-1 mediated signaling pathway serves as a "prototype" for other family members. Genetic and biochemical studies reveal that IL-1R uses adaptor molecule MyD88 to mediate a very complex pathway, involving a cascade of kinases organized by multiple adapter molecules into signaling complexes, leading to activation of the transcription factor NFkappaB. Several Toll-like receptors utilize variations of the "prototype" pathway by employing different adaptor molecules. Double-stranded RNA triggered, TLR3-mediated signaling is independent of MyD88, IRAK4, and IRAK. The adapter molecule TRIF is utilized by TLR3 to mediate the activation of NFkappaB and IRF3. LPS-induced, TLR4-mediated signaling employs multiple TIR-domain containing adaptors, MyD88/TIRAP to mediate NFkappaB activation, TRIF/TRAM for IRF3 activation. Recent studies have also begun to unravel how these pathways are negatively regulated. SIGIRR (also known as TIR8), a member of TIR superfamily that does not activate the transcription factors NFkappaB and IRF3, instead negatively modulates responses. Cells from SIGIRR-null mice show enhanced activation in response to either IL-1 or certain Toll ligands. In addition to SIGIRR, several other negative regulators have been shown to inhibit the TIR signaling, including ST2, IRAKM, MyD88s, SOCS1, and Triad3A. The coordinated positive and negative regulation of the TIR signaling ensures the appropriate modulation of the innate and inflammatory responses.

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha.

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.

Toll-like receptor 3-mediated activation of NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-beta.

We have previously shown that double-stranded RNA-triggered, Toll-like receptor 3 (TLR3)-mediated signaling is independent of MyD88, IRAK4, and IRAK. Instead, TRAF6, TAK1, and TAB2 are recruited to TLR3 on poly(I.C) stimulation. TRAF6-TAK1-TAB2 are then translocated to the cytosol where TAK1 is phosphorylated and activated, leading to the activation of IkappaB kinase and NFkappaB. The present study addressed two important questions: (i) How are TRAF6, TAK1, and TAB2 recruited to TLR3? (ii) Are TRAF6, TAK1, and TAB2 also required for TLR3-mediated IRF3 activation? Recently, a novel Toll-IL-1 receptor (TIR)-containing adapter, TIR domain-containing adapter inducing IFN-beta (TRIF), was shown to play a critical role in TLR3-mediated activation of NF-kappaB and IRF3. We found that TLR3 recruits TRAF6 via adapter TRIF through a TRAF6-binding sequence in TRIF (PEEMSW, amino acids 250-255). Mutation of this TRAF6-binding sequence abolished the interaction of TRIF with TRAF6, but not with TLR3. Interestingly, mutation of the TRAF6-binding site of TRIF only abolished its ability to activate NF-kappaB but not IRF3, suggesting that TLR3-mediated activation of NF-kappaB and IRF3 might bifurcate at TRIF. In support of this finding, we showed that DN-TRAF6 and DN-TAK1 blocked poly(I.C)-induced NF-kappaB but not IRF3 activation. Furthermore, whereas poly(I.C)-induced NF-kappaB activation is completely abolished inTRAF6-/- MEFs, the signal-induced activation of IRF3 is TRAF6 independent. In conclusion, TRIF recruits TRAF6-TAK1-TAB2 to TLR3 through its TRAF6-binding site, which is required for NF-kappaB but not IRF3 activation. Therefore, double-stranded RNA-induced TLR3/TRIF-mediated NF-kappaB and IRF3 activation diverge at TRIF.

Identification of a TLR4- and TRIF-dependent activation program of dendritic cells.

Dendritic cell activation by Toll-like receptors (TLR) is crucial for the generation of protective immune responses. In addition to the common myeloid differentiation factor 88 (MyD88)-dependent signaling pathway, TLR4 engages the adaptor protein Toll/IL-1 receptor (TIR)-domain-containing adaptor inducing IFN-beta (TRIF), leading to interferon regulatory factor 3 (IRF-3) activation and type I interferon production. Using microarray expression profiling we now identify TRIF as a major regulator of the TLR4-triggered activation program of dendritic cells. We show that the expression of 47% of the genes that are responsive to TLR4 stimulation in wild-type dendritic cells is significantly altered in cells carrying a loss-of-function mutation of TRIF. Specifically, expression of IL-12, IL-18, and IL-23 was impaired in the absence of functional TRIF, suggesting that TLR4-promoted Th1 responses are TRIF-dependent. Furthermore, we provide evidence that TRIF regulates TLR4-mediated gene expression both by type I IFN-dependent and -independent mechanisms. Whereas dendritic cell production of CXCL10 and CCL12 was dependent on both TRIF and the type I interferon receptor, expression of IL-6 required TRIF but not type I interferon activity. Functional TRIF was also required for the normal induction of numerous genes considered important for host defense against diverse pathogens.Together, these data therefore identify TRIF as a crucial regulator of TLR4-dependent dendritic cell responses.

Innate immunity and Toll-like receptors

A recently described family of receptors, called the Toll-like receptors (TLRs), may help explain what triggers the production of inflammatory mediators such as tumour necrosis factor and chemokines in diseases such as rheumatoid arthritis. TLRs recognize microbial products and trigger signalling pathways, which culminate in enhanced expression of immune and inflammatory genes. They are emerging as key players in the pathogenesis of infectious and inflammatory diseases. TLRs are defined by the presence of leucine-rich repeats and a signalling domain, which contains the Toll/IL-1 receptor (TIR) domain. An important finding is that the cystolic domain of the receptor for the cytokine IL-1 is similar to TLRs and that they signal in a similar way. Ten TLRs exist in humans and the best characterized are TLR4, which senses lipopolysaccharide from Gram-negative bacteria, and TLR3, which senses viral RNA. Signalling by each TLR is initiated by the receptor TIR domain and recruits via homotypic adapter proteins that contain TIR domains. Five such adapters have been discovered to date including MyD88, Mal (also known as TIRAP), Trif, Tram, and SARM. Signals activated include the transcription factor NF-κB and mitogen-activated protein kinases, which lead to induction of gene expression. Differences are emerging between TLRs in terms of which adapter is recruited by which TLR. This may lead to specificities in TLR signalling, with pathways being triggered that are specific for the elimination of the invading microbe. Manipulation of proteins with TIR domains presents a number of opportunities for enhancing host defence or disease resistance, or strategies to limit inflammation. In the context of chronic inflammatory diseases, roles for TLRs in the pathogenesis of inflammation are emerging in conditions such as rheumatoid arthritis and systemic lupus erythematosus. Examples include the expression and action of TLRs in synoviocytes and a role for TLR9 in the production of rheumatoid factor from autore-active B cells. Since TLRs are likely to be key drivers of TNF production, limiting their actions may have clinical benefit in inflammatory diseases. Additionally, since signals like NF-κB and p38 mitogen-activated protein kinase are activated by TLRs, inhibiting these processes will limit the effect of TLRs. Recent evidence, that certain receptor signals activated by TLRs may have an inflammatory rather than an immune role, raises the possibility of limiting the proinflammatory effects of TLRs without affecting roles in host defence. Furthermore, certain members of the TLR family (SIGIRR and ST2) may in fact be inhibitory receptors, and several negative signals generated during TLR action have been identified. As information on TLRs accumulates, more opportunities will present themselves for drug development.

Yamamoto et al. Role of adapter TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 2003;301:640-3

Toll-like receptors (TLRs) function by activating MyD88-dependent and -independent signaling pathways. In this study, mice were generated with a genetic deficiency in a Toll/IL-1 receptor (TIR) domain–containing adapter protein (TRIF) and were analyzed for their response to Toll stimuli. Notably, TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-β. In addition, inflammatory cytokine production triggered by TLR-4 ligand (but not other TLR ligands) was severely impaired in cells derived from TRIF-deficient mice. Only mice deficient in both MyD88 and TRIF had complete loss of NF-κβ activation in response to TLR-4 stimulation. Collectively, these findings indicate a central role for TRIF as an adapter molecule in stimulating MyD88-independent TLR signaling pathways.