The Impact of the Asp299Gly and Thr399Ile Polymorphisms on Toll-like Receptor 4 Signal Transduction

Abstract

Toll-like receptors (TLRs) sense microbial pathogens to activate innate immune cells and prime adaptive immunity. Asp299Gly (D299G) and Thr399Ile (T399I) TLR4 polymorphisms underlie an increased risk of certain inflammatory and infectious diseases, but the mechanisms by which they affect TLR4 signaling are poorly understood. In this study, I determined the impact of the D299G and T399I polymorphisms on TLR4 expression and interactions with myeloid differentiation factor 2 (MD2), LPS binding and activation of the myeloid differentiation primary response gene (MyD) 88- and TIR domain-containing adapter inducing IFN-β (TRIF)-dependent pathways. Complementation of human embryonic kidney (HEK) 293 cells with transfected wild-type (WT) or mutant TLR4 variants expressing yellow fluorescent protein (YFP) revealed comparable TLR4 expression, TLR4-MD2 interactions, and LPS binding. 293/CD14/MD2 cells expressing D299G or T399I YFP-TLR4s had deficient LPS-induced MyD88 recruitment to D299G YFP-TLR4, phosphorylation of TBK1, IRF3 and p38, activation of NF-κB- and IRF3-driven reporters, and induction of IL-8 and IFN-β mRNA. In contrast to WT YFP-TLR4, expression of the D299G YFP-TLR4 in immortalized TLR4-/- mouse macrophages failed to elicit LPS- mediated induction of TNF-α and IFN-β genes. Thus, the D299G and T399I polymorphisms do not affect TLR4 expression, TLR4-MD2 interaction and LPS binding, but impair LPS-induced activation of the MyD88- and TRIFdependent pathways, suggesting their interference with TLR4 dimerization and assembly of docking platforms within the TIR domains.