Asp299Gly TLR4 polymorphism impairs sensing of Escherichia coli, Rickettsia akari and LPS-mediated activation of MyD88- and TRIF-dependent signaling pathways (37.17)

Abstract

Abstract TLR4 is a key sensor of Gram negative bacteria and LPS. The Asp299Gly (D299G) and Thr399Ile (T399I) SNPs of TLR4 have been associated with increased risks or severity of certain bacterial infections and Gram negative sepsis. This study employed transfection-based complementation of TLR4-negative HEK293T cells to elucidate the role of TLR4 SNPs in sensing Gram-negative bacteria and LPS, and to determine their effect on LPS-elicited activation of MyD88- and TRIF-dependent signaling pathways. Overexpression of wild-type YFP-TLR4 led to marked activation of the NF-κB-dependent pELAM-luciferase reporter in HEK293/CD14/MD2 transfectants exposed to heat-killed E. coli, R. akari and LPS, whereas the D299G (but not T399I) TLR4 exhibited signaling deficiency. Likewise, LPS-mediated activation of TRIF-dependent pRANTES-luciferase and p125 (IFN-beta)-luciferase reporters was significantly lower in HEK293/CD14/MD-2 cells expressing D299G YFP-TLR4. Immunoprecipitation and immunoblot analyses revealed similar total expression levels of wild-type and mutant YFP-TLR4 proteins, and similar LPS binding was observed in HEK293/CD14/MD-2 cells expressing WT or mutant TLR4 species, as detected by FACS. Our data indicate that the D299G SNP impairs TLR4-elicited activation of MyD88- and TRIF-dependent signaling pathways in response to heat-inactivated Gram negative bacteria and LPS not due to lower expression levels of mutant TLR4 or impaired LPS recognition by D299G TLR4/MD2/CD14 complexes.