SUMMARY Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll-IL-1R (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling. Cell-permeable peptides derived from the D helix and the segment immediately N terminal to TLR2 TIR domain potently inhibited TLR2-mediated cytokine production. The D helix peptide, 2R9, also potently inhibited TLR4, TLR7, and TLR9, but not TLR3 or TNF-α signaling. Cell imaging, co-immunoprecipitation, and in vitro studies demonstrated that 2R9 preferentially targets TIRAP. 2R9 diminished systemic cytokine responses elicited in vivo by synthetic TLR2 and TLR7 agonists; it inhibited activation of macrophages infected with influenza strain A/PR/8/34 (PR8) and significantly improved survival of PR8-infected mice. Thus, 2R9 represents a TLR-targeting agent that blocks protein interactions downstream of activated TLRs.

Background: Assembly of TLR signaling complexes is mediated by a cooperative interaction of TIR domains present in TLRs and TLR adapters.Results: This work identifies several TIRAP/Mal-derived peptides that inhibit TLR4 and TLR2 signaling in vitro and in vivo.Conclusion: TIRAP/Mal-derived inhibitory peptides block TLR signaling by interfering with signaling complex assembly.Significance: Inhibitory peptides indicate TIRAP TIR interfaces and provide leads for development of TLR-targeting drugs.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

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.signaling complex assembly | TLR4 targeting | TIR domain recognition site | decoy peptides T oll-like receptors (TLRs) initiate innate immune responses by recognizing specific pathogen-associated molecules; for example, TLR4 recognizes lipopolysaccharides (LPSs) of Gramnegative 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-adapterlike (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 mitogenactivated 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...

TLR2 heterodimers with TLR1 or TLR6 recognize distinct pathogen-associated molecules such as tri- and di-acylated lipopeptides. The activated TLR2 heterodimers recruit Toll-IL-1R domain- (TIR-) containing adapter proteins, TIRAP and MyD88, through the receptor TIR domains. Molecular recognition mechanisms responsible for agonist-driven, TIR domain-mediated receptor-adapter interactions as well as the structure of resultant signaling complexes remain unknown. We previously reported that the cell-permeable peptide derived from helix D of TLR2 TIR (2R9) specifically binds TIRAP in vitro and in cells and thereby inhibits TIRAP-dependent TLR signaling. This study demonstrates that cell-permeable peptides from D helix of TLR1 or TLR6, peptides 1R9 and 6R9 respectively, inhibit signaling mediated by cognate TLR2 co-receptors. Interestingly, 1R9 and 6R9 bind different TLR2 adapters, as they selectively bind MyD88 and TIRAP TIR, respectively. Both peptides block the agonist-induced co-immunoprecipitation (co-IP) of TLR2 with TIRAP or MyD88, but not TLR2 co-IP with co-receptors. Our data suggest that D helices of TLR1 and TLR6 TIR domains are adapter recruitment sites in both co-receptors; yet the sites recruit different adapters. The D helix in TLR1 is the MyD88 docking site, whereas in TLR6 this site recruits TIRAP.