TLR4 and TLR8 agonist combination markedly reduces CCL2 mRNA accumulation: role of NF-κB signaling pathway. (A-B) MD-DCs were treated with LPS (100 ng/mL) or R848 (2 μg/mL) alone or in combination. After 6 hours, total RNA was extracted and retrotranscribed. Real-time PCR was performed using primers specific for human CCL2 (A) and IL-12p40 (B) mRNA on cDNA normalized with β-actin. One representative experiment of 3 performed is shown. Results, analyzed by the relative quantification method (2^−ΔΔCt method), are presented as fold increase/reduction of CCL2 and IL-12p40 gene expression. (C) MD-DCs were treated with of 10 μM TPCK or left untreated. At the concentration used, this inhibitor did not affect cell viability (data not shown). After 30 minutes, some cultures were exposed to TLR ligands, as described in the legend to Figure 2. After 18 hours of culture, supernatants were harvested and frozen for CCL2 determination. Data are expressed as mean ± SD of duplicate wells and are representative of 4 independent experiments. P values were calculated by Student t test, and statistical significance is indicated comparing results from TPCK-treated cells versus untreated cells. (D) NF-κB activation was measured in nuclear extracts (5 μg) after a 2-hour stimulation with TLR ligands by a transcription factor ELISA. Data are expressed as mean ± SD of duplicate wells and are representative of 3 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from MD-DCs treated with single agonists versus control cells and from MD-DCs treated with a combination of the agonists versus the single treatments.