Signalling adaptors used by Toll-like receptors: an update

Research into the five Toll/IL1 receptor (TIR) adaptor proteins involved in innate immunity continues to advance. Here we outline some of the more recent findings. MyD88 has a key role in signalling by the IL1 receptor complex and TLRs. However, a MyD88-independent pathway of IL1beta signalling in neurons has been described which involves the protein kinase Akt, and which has an anti-apoptotic effect. This pathway may also be important for the mechanism whereby Alum exerts its adjuvant effect since this depends on IL1beta but is MyD88-independent. MyD88 is also involved in tumourigenesis in models of hepatocarcinoma and familial associated polyposis (FAP); negative regulation of TLR3 signalling and in PKCepsilon activation. The adaptor Mal is regulated by phosphorylation and caspase-1 cleavage. A variant form of Mal in humans termed S180L confers protection in multiple infectious diseases. TRAM is controlled by myristoylation and phosphorylation and the localisation of TRAM with TLR4 to endosomes is required for activation of IRF3 and induction of IFNbeta. Finally SARM has been shown to regulate TRIF and also appears to be involved in neuronal injury mediated by oxidative stress in mouse neurons. These advances confirm the importance for the TIR domain-containing adapters in host defence and inflammation.     

A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins

The Toll-like receptor 4 (TLR4) is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR) signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3) and nuclear factor kappaB (NFkappaB) respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.     

Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex.     

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.     

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.     

Evolution of the TIR domain-containing adaptors in humans: swinging between constraint and adaptation

Natural selection is expected to act strongly on immune system genes as hosts adapt to novel, diverse, and coevolving pathogens. Population genetic studies of host defense genes with parallel functions in model organisms have revealed distinct evolutionary histories among the different components-receptors, adaptors, and effectors-of the innate immune system. In humans, however, detailed evolutionary studies have been mainly confined to the receptors and in particular to Toll-like receptors (TLRs). By virtue of a toll/interleukin-1 receptor (TIR) domain, TLRs activate distinct signaling pathways, which are mediated by the five TIR-containing adaptors: myeloid differentiation factor-88 (MyD88), myeloid differentiation factor-88 adaptor-like protein (MAL), toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon (IFN)β (TRIF), toll/interleukin-1 receptor domain-containing adaptor protein inducing IFNβ-related adaptor molecule (TRAM), and sterile α- and armadillo motif-containing protein (SARM). Here, we have examined the extent to which natural selection has affected immune adaptors in humans, using as a paradigm the TIR-containing adaptors. To do so, we characterized their levels of naturally occurring genetic variation in various human populations. We found that MyD88 and TRIF have mainly evolved under purifying selection, suggesting that their role in the early stages of signal transduction is essential and nonredundant for host survival. In addition, the adaptors have been targeted by multiple episodes of positive selection, differing in timing and spatial location. MyD88 and SARM display signatures of a selective sweep that has occurred in all humans, whereas for the other three adaptors, we detected signatures of adaptive evolution that are restricted to specific populations. Our study provides evidence that the contemporary diversity of the five TIR-containing adaptors results from the intermingling of different selective events, swinging between constraint and adaptation.     

Toll receptors and pathogen resistance

Toll receptors in insects, mammals and plants are key players that sense the invasion of pathogens. Toll-like receptors (TLRs) in mammals have been established to detect specific components of bacterial and fungal pathogens. Furthermore, recent evidence indicates that TLRs are involved in the recognition of viral invasion. Signalling pathways via TLRs originate from the conserved Toll/IL-1 receptor (TIR) domain. The TIR domain-containing MyD88 acts as a common adaptor that induces inflammatory cytokines; however, there exists a MyD88-independent pathway that induces type I IFNs in TLR4 and TLR3 signalling. Another TIR domain-containing adaptor, TIRAP/Mal has recently been shown to mediate the MyD88-dependent activation in the TLR4 and TLR2 signalling pathway. Thus, individual TLRs may have their own signalling systems that characterize their specific activities.     

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 (TLR4) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A‐positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.      

1H, 13C, and 15N resonance assignment of the TIR domain of human MyD88

Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like receptor (TLR) signaling pathway. The TIR domain of MyD88 serves as a protein–protein interaction module and interacts with other TIR-containing proteins such as Mal (MyD88 adaptor-like) and Toll-like receptor 4 to form signal initiation complexes. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the TIR domain of MyD88. The resonance assignments obtained in this work will contribute to the study of heteromeric TIR–TIR interactions between MyD88 and TIR-containing receptors or adaptors.     

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.     

Roles for LPS-dependent interaction and relocation of TLR4 and TRAM in TRIF-signaling

Toll-like receptor 4 (TLR4) activates two distinct signaling pathways inducing production of proinflammatory cytokines or type I interferons (IFNs), respectively. MyD88 and TIRAP/Mal are essential adaptor molecules for the former but not for the latter pathway. In contrast, TRIF/TICAM-1 and TRAM/TICAM-2 are essential for both. TIRAP is a sorting adaptor molecule recruiting MyD88 to activated TLR4 in the plasma membrane. TRAM is thought to bridge between TLR4 and TRIF by physical association. Little is known, however, how TRAM interacts with TLR4 or with TRIF during LPS response. Here, we show that TRAM recruits TRIF to the plasma membrane. Moreover, LPS induces upregulation of TLR4-association with TRAM and their subsequent translocation into endosome/lysosome. The internalized signaling complex consisting of TLR4 and TRAM colocalizes with TRAF3, a signaling molecule downstream of TRIF, in endosome/lysosome. These results suggest that TLR4 activates TRIF-signaling in endosome/lysosome after relocation from the cell surface.     

Implication of Toll/IL-1 receptor domain containing adapters in Porphyromonas gingivalis-induced inflammation

Periodontitis is induced by periodontal dysbiosis characterized by the predominance of anaerobic species. TLRs constitute the classical pathway for cell activation by infection. Interestingly, the Toll/IL-1 receptor homology domain adapters initiate signaling events, leading to the activation of the expression of the genes involved in the host immune response. The aim of this study was to evaluate the effects of Porphyromonas gingivalis on the expression and protein-protein interactions among five TIR adapters (MAL, MyD88, TRIF, TRAM and SARM) in gingival epithelial cells and endothelial cells. It was observed that P. gingivalis is able to modulate the signaling cascades activated through its recognition by TLR4/2 in gingival epithelial cells and endothelial cells. Indeed, MAL-MyD88 protein-protein interactions associated with TLR4 was the main pathway activated by P. gingivalis infection. When transient siRNA inhibition was performed, cell viability, inflammation, and cell death induced by infection decreased and such deleterious effects were almost absent when MAL or TRAM were targeted. This study emphasizes the role of such TIR adapter proteins in P. gingivalis elicited inflammation and the precise evaluation of TIR adapter protein interactions may pave the way for future therapeutics in both periodontitis and systemic disease with a P. gingivalis involvement, such as atherothrombosis.     

Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates

The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network.     

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.     

Selective use of TRAM in lipopolysaccharide (LPS) and lipoteichoic acid (LTA) induced NF-kappaB activation and cytokine production in primary human cells: TRAM is an adaptor for LPS and LTA signaling

TLR signal via Toll-IL-1R (TIR) homology domain-containing adaptor proteins. One of these adaptors, Toll-IL-1R domain-containing adaptor inducing IFN-beta-related adaptor molecule (TRAM), has been shown to be essential for TLR4 signaling in TRAM(-/-) mice and cell lines. Previously, we showed that MyD88 or Mal dominant-negative constructs did not inhibit LPS induction of cytokines in primary human M-CSF-derived macrophages. A possible explanation was redundancy of the adaptors during LPS signaling. TRAM is a suitable candidate to compensate for these adaptors. To investigate a potential role for TRAM in LPS signaling in human M-CSF-derived macrophages, we engineered an adenoviral construct expressing dominant-negative TRAM-C117H (AdTRAMdn). Synovial fibroblasts (SF) and human umbilical endothelial cells (HUVECs) were used as a nonmyeloid comparison. AdTRAMdn inhibited LPS-induced signaling in SFs and HUVECs, reducing NF-kappaB activation and cytokine production, but did not inhibit LPS signaling in M-CSF-derived human macrophages. Further investigation of other TLR ligands showed that AdTRAMdn was also able to inhibit signaling initiated by lipoteichoic acid, a TLR2 ligand, in SFs and HUVECs and lipoteichoic acid and macrophage-activating lipopeptide 2 signaling was also inhibited in TRAM(-/-) murine embryonic fibroblasts. We conclude that TRAM is an adaptor protein for both TLR4 and TLR2/6 signaling in SFs, HUVECs, and murine embryonic fibroblasts, but cannot demonstrate a role in human macrophages.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

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.     

Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.