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.

No abstract

Neuroinflammation is instrumental in neuronal damage observed in various neuropathologies. Thus, understanding pro-inflammatory pathways of human glial cells is important in identifying potential pharmacological strategies to modulate inflammatory signaling in these cells. Microglia-derived cytokines and chemokines are instrumental in neuroinflammation and several proinflammatory mediators activate these cells to include bacterial lipopolysaccharide (LPS) that signals through toll-like receptor 4 (TLR4). Microglial TLR4 signaling is not well studied; therefore, our goal is to investigate the molecular mechanisms of TLR4 microglial signaling, which entailed treatment of human microglial cells (CHME-5) with LPS. LPS induced NF-κB activation, demonstrated by increased NF-κB p65 binding activity and phosphorylated p65 expression in CHME-5 cells. TLR4 expression also increased alongside NF-κB activation. On the other hand, co-exposure to the μ-opioid receptor antagonist, β-funaltrexamine (β-FNA), down-regulated LPS-induced NF-κB activation. To date, our data suggest that the inflammatory actions of microglial TLR4 signaling may be partly due to modulation of NF-κB activation. Thus, we have also initiated studies to elucidate the effects of LPS and β-FNA on other key TLR4 signal transduction proteins. Together, these ongoing studies are expected to further elucidate the mechanisms of LPS-induced TLR4 signaling in human microglia.

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.