Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family

The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.     

In silico approach to inhibition of signaling pathways of Toll-like receptors 2 and 4 by ST2L

Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.     

Contributions of Unique Intracellular Domains to Switchlike Biosensing by Toll-like Receptor 4

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.     

Cutting edge: TNFR-associated factor (TRAF) 6 is essential for MyD88-dependent pathway but not toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent pathway in TLR signaling

Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-kappaB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-kappaB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-kappaB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling.     

Genetic analysis of Toll/Interleukin-1 Receptor (TIR) domain sequences from rhesus macaque Toll-like receptors (TLRs) 1–10 reveals high homology to human TLR/TIR sequences

Toll-like receptors (TLRs) form a major group of pattern recognition receptors of the innate immune system that sense molecular patterns on microbes. The cytoplasmic Toll/Interleukin-1 Receptor (TIR) signaling domain is instrumental in inducing a signaling cascade upon recognition of specific ligands by TLRs. Because nonhuman primates are used as models of infectious and immune processes, we sought to obtain an increased understanding of nonhuman primate TLRs. We obtained the nucleotide sequences of the TIR domains of rhesus macaque TLRs 1–10 and examined their genetic relationships to TLRs from humans and mice. Alignment of the deduced amino acid sequences revealed macaque-specific changes mostly outside the conserved Box regions of the TLR/TIR domain. Assessment of mutational biases among TLRs from multiple species revealed a strong overall bias towards synonymous substitutions, with a few short regions showing evidence for positive selection outside the Box regions. This first presentation of the TLR/TIR domain sequences from nonhuman primates indicates that although there are species-specific differences, a high level of sequence homology exists in the critical signaling Box regions of macaque, human, and murine TLR/TIR domains. These findings suggest that animal models, including nonhuman primates, will be useful in modeling human TLR pathophysiology and therapy.     

Inhibition of Toll-like receptors TLR4 and 7 signaling pathways by SIGIRR: A computational approach

Toll-like receptors (TLRs) belong to the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily which is defined by a common cytoplasmic Toll/interleukin-1 receptor (TIR) domain. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to trigger an immediate defensive response. SIGIRR (single immunoglobulin interleukin-1 receptor-related molecule), another member of the TLR/IL-1R superfamily, acts as a negative regulator of MyD88-dependent TLR signaling. It attenuates the recruitment of MyD88 adaptors to the receptors with its intracellular TIR domain. Thus, SIGIRR is a highly important molecule for the therapy of autoimmune diseases caused by TLRs. So far, the structural mechanism of interactions between SIGIRR, TLRs and adaptor molecules is unclear. To develop a working hypothesis for this interaction, we constructed three-dimensional models for the TIR domains of TLR4, TLR7, MyD88 and SIGIRR based on computational modeling. Through protein–protein docking analysis, we developed models of essential complexes involved in the TLR4 and 7 signaling and the SIGIRR inhibiting processes. We suggest that SIGIRR may exert its inhibitory effect through blocking the molecular interface of TLR4, TLR7 and the MyD88 adaptor mainly via its BB-loop region.     

Investigating the effect of key mutations on the conformational dynamics of toll‐like receptor dimers through molecular dynamics simulations and protein structure networks

The Toll‐like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen‐associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain‐containing proteins. Mutations in the BB loop of the Toll‐like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lps^d mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain‐containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein–protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild‐type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways.     

Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Ks's (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.     

Calcineurin negatively regulates TLR-mediated activation pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-kappaB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-kappaB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IkappaB-alpha degradation, MAPK activation, and TNF-alpha production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.     

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.