The Asp299Gly Polymorphism Alters TLR4 Signaling by Interfering with Recruitment of MyD88 and TRIF

Abstract

Asp299Gly (D299G) and, to a lesser extent, Thr399Ile (T399I) TLR4 polymorphisms have been associated with Gram negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and TIR domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein (YFP)-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD-2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK binding kinase-1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, while T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4−/− mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Co-immunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD-2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.

High-mobility group box 1 promotes extracellular matrix synthesis and wound repair in human bronchial epithelial cells

“…Further evidence in support of our observations demonstrated that a truncated intracellular domain of RAGE altered cell migration and adhesion and impaired RAGE-induced phosphorylation of ERK1/2, p38 and JNK (27). Canonical TLR4 activation recruits intracellular adaptor proteins MYD88 and TIRAP inducing interferon-␤ (TRIF) downstream of the TLR4 complex (15). In our studies we used CLI-095 to block the intracellular binding site for these adaptor proteins to blunt TLR4-mediated signaling (41).…”

R753Q Polymorphism Inhibits Toll-like Receptor (TLR) 2 Tyrosine Phosphorylation, Dimerization with TLR6, and Recruitment of Myeloid Differentiation Primary Response Protein 88

“…Confocal Microscopy-Confocal microscopy was performed as described previously (43). In brief, cells were seeded (4 ϫ 10 5 cells) on poly-L-lysine-coated coverslips (Fischer Scientific) in phenol-free complete DMEM and cultured for 20 h. Transfection was performed using Lipofectamine 2000 (Invitrogen) transfection reagent followed by gentle washing and recovery for 48 h. Cells were fixed with 2% paraformaldehyde and mounted onto glass slides using a 1,4-diazabicyclo[2.2.2]-octane-based antifade fluorescent mounting medium.…”

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

“…Studies using mouse strains deficient in TLR4 signaling (10)(11)(12), TLR4 expression (10,(13)(14)(15)(16), or using inhibitors of TLR function in wild type (WT) mice (1,17) confirmed that TLR4 contributes to bacterial clearance and the host inflammatory response in the setting of Gram-negative bacterial infection. Furthermore, the TLR4 Asp 299 Gly polymorphism, which results in depressed LPS-TLR4 signaling (18), is associated with increased susceptibility to Gramnegative infections in humans (19). Therefore, the TLR4-dependent response of individual cell types in the setting of bacterial sepsis is likely to be determined by the functional role of each cell type in the overall host response.…”