Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo (P1232)

Abstract Toll/IL-1R domain- (TIR-) containing adapter inducing IFN-β- (TRIF-) related adapter molecule (TRAM) enables recruitment of TRIF to activated TLR4 and thereby mediates the TLR4-driven induction of TRIF-dependent cytokines. A library of cell-permeating decoy peptides derived from TRAM TIR domain has been screened for the ability of individual peptides to inhibit TLR4 signaling in primary macrophages. Peptides derived from TRAM TIR BB loop (TM4) and C helix (TM6) inhibited the LPS-induced activation of MyD88- and TRIF-dependent cytokines, as well as MAPK activation. TM4 and TM6 did not block macrophage activation induced by TLR2, TLR9, or RIG-I-like receptor agonists. Both TM4 and TM6 blocked co-immunoprecipitation of TRAM and TLR4 ectopically expressed in HEK293T cells. Both peptides also blocked the LPS-induced recruitment of MyD88 to TLR4 in primary murine macrophages. In vivo examination of TRAM-derived peptides demonstrated that all peptides that were inhibitory in vitro, profoundly suppressed systemic inflammatory response elicited in mice by a sublethal LPS dose, and protected against a lethal LPS challenge. Both intraperitoneal and intravenous administration of a select peptide effectively mitigated the cytokine production induced by an intraperitoneal LPS injection. This research identifies novel TLR inhibitors effective in vitro and in vivo and validates the approach taken in this study as a rational way for development of signaling inhibitors and therapeutics leads.