Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain

Toll/IL-1R resistance (TIR) domain–containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow–derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF–TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF–TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF–TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.Toll-like receptors (TLRs) initiate innate immune responses by recognizing specific pathogen-associated molecules; for example, TLR4 recognizes lipopolysaccharides (LPSs) of Gram-negative bacteria (1, 2). Ligand recognition induces dimerization of cytoplasmic Toll/IL-1R resistance (TIR) domains of two receptor molecules and causes recruitment of intracellular TIR domain-containing adapters. Four adapter proteins participate in TLR4 signaling: myeloid differentiation factor 88 (MyD88) (3), TIR domain-containing adapter protein, also known as MyD88-adapter-like (TIRAP–Mal) (4, 5), TIR domain–containing adapter-inducing IFN-β, also known as TLR adaptor molecule 1 (TRIF–TICAM-1) (6, 7), and TRIF-related adaptor molecule also known as TLR adaptor molecule 2 (TRAM–TICAM-2) (8, 9). TIRAP–Mal is important for MyD88 recruitment to the signaling complex located at the plasma membrane to initiate early NF-κB and mitogen-activated protein kinase (MAPK) activation and induce “MyD88-dependent” proinflammatory cytokines, such as TNF-α and IL-1β (4, 5, 10). TRAM is important for TRIF recruitment to the endosomally located TLR4 signaling complexes to activate IFN regulatory factor 3 (IRF3) and induce IRF3-dependent cytokines, such as IFN-β and RANTES (regulated upon activation normal T-cell expressed and secreted) (8, 9, 11).A typical TIR domain consists of the central five stranded parallel β sheets (the strands are designated as βA–βE) surrounded by 5 α-helices (i.e., αA–αE) (12, 13). The TIRAP–Mal TIR domain has an atypical fold compared with other resolved mammalian TIR structures in that the position of its β-strand B is shifted by 12–18 amino acids toward the C terminus, so that TIRAP TIR does not have a helix B but has an unusually long AB loop (14, 15). Structures of the TIR domains of TLR4, TRIF, and TRAM have not been yet resolved. The TIR domain is a key structural feature present in all TLRs and TLR adapter proteins. TIR domains mediate transient homotypic or heterotypic protein interactions required for agonist-driven assembly of TLR signaling complexes (13, 16, 17). Multiple interactions of TIR domains of TLRs and TLR adapters are required to mediate adapter recruitment and stabilize initial complex (18–20). It has been proposed that TLR4 activation leads to formation of several compositionally distinct complexes. Kagan et al. proposed that TLR4 engages TIRAP–MyD88 and TRAM–TRIF sequentially at distinct cellular locations (11), thus implying that the two sets of adapters may compete for the same binding site at the TLR4 homodimer. However, it remains unclear how exactly the four adapters interact with each other and TLR4 to orchestrate TLR4 signaling.The presumed mechanism of signaling inhibition by a decoy peptide is that the peptide competes with its prototype protein for the prototype’s docking site and thereby prevents a protein–protein interaction required for signaling (19). In this study, we have examined cell-permeable decoy peptides derived from the TIR domain of TRIF. Two peptides, TF4 and TF5, from the second loop (BB loop) and the second helical region (helix B) of the TRIF TIR, respectively, potently inhibited LPS-induced activation of MAPKs and induction of MyD88-dependent and TRIF-dependent cytokines in wild-type macrophages. TF5 did not inhibit TLR4 signaling in TRIF^−/− immortalized bone-marrow–derived macrophages (iBMDMs) but did exhibit full activity in the MyD88^−/− cells. TF5 inhibits TLR4-driven macrophage signaling at a lower dose in vitro compared with TF4 and binds to both TRAM and TLR4, whereas TF4 targets TLR4 but not the TRAM TIR. In a mouse model of TLR4-driven inflammation, TF5 potently decreased the systemic cytokine levels induced in mice by a sublethal LPS dose, and dramatically improved survival of mice challenged with a lethal LPS dose.