Effect of single or combined treatment with TLR3, TLR4, and TLR8 agonists on the secretion of CCL2 in MD-DCs. (A) MD-DCs at day 5 of culture were exposed to different concentrations of p(I:C), LPS, and R848. After 18 hours, supernatants were collected and CCL2 content was measured by ELISA. Data are expressed as mean ± SD of culture duplicates and are representative of 5 independent experiments. P values were calculated by ANOVA comparing results from TLR ligand-stimulated versus unstimulated cells. (B-C) MD-DCs were treated with p(I:C) (20 μg/mL), LPS (100 ng/mL), or R848 (2 μg/mL), either alone or in combination. Eighteen hours later, supernatants were collected for CCL2 (B) and IL-12p70 (C) determination. Results are presented as means ± SD of duplicate wells from one representative of 6 independent experiments. P values were calculated by ANOVA among the single and combined TLR ligand-stimulated cells. (D) MD-DCs were treated with unpurified LPS (100 ng/mL), ultrapure LPS (100 ng/mL), R837 (2 μg/mL), or R848 (2 μg/mL), either alone or in combination. Eighteen hours later, supernatants were collected for CCL2 determination. Results are presented as means ± SD of duplicated wells from one representative of 5 independent experiments. P values were calculated by ANOVA and indicate significant reduction of CCL2 content in cultures simultaneously treated with ultrapure LPS and R848 versus cultures treated with R848 alone. Conversely, no significant modulation in CCL2 content was observed in cultures treated with R837 alone with respect to control cultures.