Fig. 3
T7-EA is a TLR7-selective agonist that induces local cytokine and chemokine expression. a, b T7-EA, T7 and R848 were incubated with HEK293 cells expressing either human TLR7 or TLR8 at concentrations ranging from 0.01 to 10 μM. The HEK-Blue 293 reporter system is based on HEK293 cells that are stably transfected with hTLR7 or hTLR8 and a SEAP reporter. SEAP activity can be used to quantify NFκB activation. c Human PBMCs (2 × 106/mL) were incubated with T7-EA and T7 at concentrations ranging from 0.01 to 10 μM for 18 h. The levels of IFNα in the culture supernatants were determined by ELISA. d BMDMs (0.5 × 106/mL) derived from C57BL/6 mice were incubated with T7-EA, T7 and R848 at concentrations ranging from 0.01 to 10 μM for 22 h. IL-6 levels in the supernatants were measured by ELISA. e, f Balb/c mice (n = 3) were injected with 35 nmol T7, T7-EA, R848 or vehicle (10% DMSO in saline) in a 50-μL volume in the gastrocnemius muscles. Then, 1, 3 and 7 days after injection, the muscles were harvested. RNA was isolated from muscles, and TNF-α and CCL2 expression levels at the site of injection were determined by real-time-PCR. A student’s t test was used for data analysis. The data represent the mean ± SD of triplicates and are representative of three independent experiments. ** P < 0.01. T7, SZU-101; BMDMs, bone marrow derived macrophages