Misako Matsumoto

TIR-containing Adapter Molecule (TICAM)-2, a Bridging Adapter Recruiting to Toll-like Receptor 4 TICAM-1 That Induces Interferon-β

Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-κB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-β genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-β induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-κB and the IFN-β promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-β, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.

Cutting Edge: NF-κB-Activating Kinase-Associated Protein 1 Participates in TLR3/Toll-IL-1 Homology Domain-Containing Adapter Molecule-1-Mediated IFN Regulatory Factor 3 Activation

TLRs signal the presence of microbial patterns and activate transcription factors. In TLR3 and TLR4, the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM-1) (also called Toll/IL-1R domain-containing adapter inducing IFN-β (TRIF)) mediates IFN regulatory factor 3 (IRF3) phosphorylation followed by IFN-β production. The regulatory subunit TNFR-associated factor family member-associated NF-κB activator (TANK) couples with the kinase complex IκB kinase-related kinase ε/NF-κB-activating kinase (NAK) (TANK-binding kinase 1 (TBK1)) that involveTICAM-1-dependent IFN-β induction. There are several TANK-homologous proteins. We tested whether TICAM-1 binds and coprecipitates with TANK or its family proteins. The results are: 1) the TANK family protein NAK-associated protein 1 (NAP1), but not TANK, coprecipitates withTICAM-1; 2) NAP1 overexpression markedly enhances TBK1-mediated IFN-β promoter activation; 3) a dominant-negative form, NAP (158–270), suppresses IRF3 activation in response to poly(I:C) or LPS; 4) RNA interference targeting of the NAP1 message results in a failure of poly(I:C)-mediated IRF3 polymerization and IFN-β production. Thus, NAP1 is the kinase subunit responsible for TLR3/4-mediated IFN-β induction in the TICAM-1 pathway.

NAK-Associated Protein 1 Participates in Both the TLR3 and the Cytoplasmic Pathways in Type I IFN Induction

TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-β promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-β promoter activation >24 h after poly(I:C) or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-1R domain-containing adapter-inducing IFN-β) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-β promoter activation by RSV infection. Virus-mediated activation of the IFN-β promoter was largely abrogated by the gene silencing of IFN-β promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, IκB kinase-related kinase ε and TANK-binding kinase 1 participated in IFN-β induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-β promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-β promoter.

Surface-Expressed TLR6 Participates in the Recognition of Diacylated Lipopeptide and Peptidoglycan in Human Cells

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-κB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys30 and Cys36 in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-κB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.