The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of adapter proteins MyD88 and TRIF (180.5)

Abstract The Asp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram negative sepsis and infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of these polymorphisms on TLR4 expression, TLR4- MD2 interactions, LPS binding, and activation of MyD88- and TRIF-dependent pathways. Complementation of HEK293/CD14/MD2 transfectants with WT or mutant YFP-TLR4s revealed similar TLR4 expression levels, TLR4-MD2 interactions, and LPS binding. The presence of the D299G polymorphism led to impaired LPS-induced phosphorylation of p38 and TBK-1, activation of NF-κB and IRF-3, and induction of IL-8 and IFN-β mRNA. The T399I TLR4 species elicited responses that showed no statistical difference with those caused by WT receptor. In contrast to WT TLR4, expression of the D299G mutant in TLR4-/- mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β genes. Co-immunoprecipitation revealed deficient LPS-elicited interaction of MyD88 and TRIF with D299G TLR4, in contrast to robust adapter recruitment to WT TLR4. Our data indicate the D299G polymorphism compromises TLR4 signaling by impairing recruitment of MyD88 and TRIF, suggesting that its interference with TLR4 dimerization and assembly of docking platforms in the TIR domains that enable adapter recruitment