The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors

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 (refs 1-3). 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 (refs 4, 5). Here we report the generation of mice deficient in the Tirap gene. 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.     

Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4

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. Here we report that 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, our 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.     

RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.     

Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.     

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.     

Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3

Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, and induce antimicrobial immune responses. Double-stranded RNA (dsRNA) is a molecular pattern associated with viral infection, because it is produced by most viruses at some point during their replication. Here we show that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-kappaB and the production of type I interferons (IFNs). TLR3-deficient (TLR3-/-) mice showed reduced responses to polyinosine-polycytidylic acid (poly(I:C)), resistance to the lethal effect of poly(I:C) when sensitized with d-galactosamine (d-GalN), and reduced production of inflammatory cytokines. MyD88 is an adaptor protein that is shared by all the known TLRs. When activated by poly(I:C), TLR3 induces cytokine production through a signalling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kappaB and mitogen-activated protein (MAP) kinases independently of MyD88, and cause dendritic cells to mature.     

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.     

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.     

UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes

Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs.     

Programming the magnitude and persistence of antibody responses with innate immunity

Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.     

CD36 is a sensor of diacylglycerides

Toll-like receptor 2 (TLR2) is required for the recognition of numerous molecular components of bacteria, fungi and protozoa. The breadth of the ligand repertoire seems unusual, even if one considers that TLR2 may form heteromers with TLRs 1 and 6 (ref. 12), and it is likely that additional proteins serve as adapters for TLR2 activation. Here we show that an N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2 (a diacylated bacterial lipopeptide) and to lipoteichoic acid. Homozygous mice are hypersusceptible to Staphylococcus aureus infection. Cd36(obl) macrophages readily detect S-MALP-2, PAM(2)CSK(4), PAM(3)CSK(4) and zymosan, revealing that some--but not all--TLR2 ligands are dependent on CD36. Already known as a receptor for endogenous molecules, CD36 is also a selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer.     

Distinct molecular mechanism for initiating TRAF6 signalling

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.