Asp299Gly polymorphism decreases TLR4-elicited activation of MyD88- and TRIF-dependent signaling pathways (116.17)

Abstract

Abstract Innate immune cells detect Gram-negative bacterial lipopolysaccharide (LPS) via Toll-like receptor (TLR) 4 that employs myeloid differentiation primary response gene (MyD) 88 and TIR-domain containing adapter-inducing IFN-β (TRIF) pathways to activate NF-κB, IRF3, and induce pro-inflammatory cytokines and type I IFNs, respectively. Asp299Gly and Thr399Ile TLR4 polymorphisms have been associated with an increased risk of Gram-negative sepsis and certain bacterial infections. The mechanisms by which these mutations impact TLR4 signal transduction are incompletely understood. We used transfection-based complementation of TLR4-/- HEK293 cells with CD14, MD2, wild-type (WT) or polymorphic YFP-TLR4s to determine the impact of the Asp299Gly and Thr399Ile polymorphisms on LPS binding and TLR4-elicited activation of MyD88 and TRIF pathways. In contrast to strong LPS responses induced by WT TLR4, the Asp299Gly variant elicited decreased phosphorylation of p38 and IRF3, activation of NF-κB- and IFN-β-driven reporters, and expression of IL-8 mRNA. 293/MD2 cells expressing WT or polymorphic TLR4s showed comparable LPS binding, and had similar total levels of transfected YFP-TLR4s and Flag-MD2, as well as similar expression of total p38 and β-tubulin. Thus, the D299G polymorphism compromises TLR4-mediated activation of MyD88 and TRIF-dependent pathways, and these signaling deficiencies are not due to lower expression levels of the TLR4 receptor complex.