Fig. 2.

Targeting inhibition of nucleic acid-sensing TLRs by E6446. (A) (Upper) HEK293 cells stably transfected with plasmids carrying TLR4/MD2, TLR7, or TLR9 genes and the NF-κB reporter gene ELAM-1–luciferase were stimulated with the appropriate ligand (LPS with soluble CD14, R848, or oligo 2006) overnight. Next, Steady-Glo reagent (Promega, Inc.) was added to the wells, and the amount of luciferase activity in each sample was quantified in a Wallac Envision counter. (Lower) Ficoll-separated mononuclear cells were isolated from healthy volunteer donors, washed, and plated with stimulatory oligonucleotide CpG ODN 2216, R848, or ssRNA in the presence of the lyposomal trasfection reagent DOTAP in complete RPMI for 72 h. IL-6 levels in supernatant were quantified by ELISA (R&D Systems) and expressed as the percentage of levels found in nonstimulated cell supernatants. (B) (Upper) BALB/c mice were predosed with 60 mg/kg of E6446 1.5 h before challenge with the indicated dose of Oligo 1668 (Oligos, Etc.). At 2 h postchallenge, serum was collected and assayed for IL-6 by ELISA. (Lower) Spleen cells from mice were incubated with CpG ODN 1401 (1 μg/mL) or R818 (1 μM) for 72 h in the presence of increasing concentrations of E6446. (C) Mice were orally treated with 20 mg·kg⁻¹·d⁻¹ of E6446 during 5 d. Two hours after the last dose, spleen cells were harvested and challenged with the indicated stimuli. Cytokine levels in culture supernatants were analyzed by Searchlight multiplex. *P < 0.05.