Figure 2
Figure 2. Concentration, temporal requirements, and specificity of TLR4 and TLR8 agonist combination for CCL2 inhibition. MD-DCs were treated with a range of concentrations of either LPS or R848. (A) Cultures received fixed amount of R848 (2 μg/mL) and variable concentrations of LPS ranging from 0.1 up to 1000 ng/mL. Data are expressed as mean ± SD of culture duplicates and are representative of 6 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures simultaneously treated with different amounts of LPS and a fixed concentration of R848 with respect to cultures treated with R848 alone. (B) Cultures received a fixed amount of LPS (100 ng/mL) and variable concentrations of R848 ranging from 0.25 up to 5 μg/mL. Eighteen hours later, supernatants were collected for CCL2 content determination. Data are expressed as mean ± SD of culture duplicates and are representative of 6 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures treated with different concentrations of R848 in the presence or in the absence of a fixed amount of LPS. (C) MD-DCs were stimulated with LPS (100 ng/mL) or R848 (2 μg/mL) alone or in combination. Stimuli were added either simultaneously at time 0 or sequentially, with LPS being added 3 hours after R848 or vice versa, R848 3 hours after LPS. CCL2 secretion was measured 18 hours later. Data are expressed as mean ± SD of culture duplicates and are representative of 4 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures simultaneously treated with LPS and R848 with respect to cultures treated with R848 alone.(D) MD-DCs were treated with LPS (100 ng/mL) and R848 (2 μg/mL) alone or in combination. Eighteen hours later, supernatants were collected for CCL1 and CCL4 content determination. Data are expressed as mean ± SD of culture duplicates and are representative of 3 independent experiments. P values were calculated by ANOVA and indicated no significant modulation in CCL1 or CCL4 content in cultures simultaneously treated with LPS and R848 with respect to cultures treated with a single agonist.

Concentration, temporal requirements, and specificity of TLR4 and TLR8 agonist combination for CCL2 inhibition. MD-DCs were treated with a range of concentrations of either LPS or R848. (A) Cultures received fixed amount of R848 (2 μg/mL) and variable concentrations of LPS ranging from 0.1 up to 1000 ng/mL. Data are expressed as mean ± SD of culture duplicates and are representative of 6 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures simultaneously treated with different amounts of LPS and a fixed concentration of R848 with respect to cultures treated with R848 alone. (B) Cultures received a fixed amount of LPS (100 ng/mL) and variable concentrations of R848 ranging from 0.25 up to 5 μg/mL. Eighteen hours later, supernatants were collected for CCL2 content determination. Data are expressed as mean ± SD of culture duplicates and are representative of 6 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures treated with different concentrations of R848 in the presence or in the absence of a fixed amount of LPS. (C) MD-DCs were stimulated with LPS (100 ng/mL) or R848 (2 μg/mL) alone or in combination. Stimuli were added either simultaneously at time 0 or sequentially, with LPS being added 3 hours after R848 or vice versa, R848 3 hours after LPS. CCL2 secretion was measured 18 hours later. Data are expressed as mean ± SD of culture duplicates and are representative of 4 independent experiments. P values were calculated by ANOVA, and statistical significance is indicated comparing results from cultures simultaneously treated with LPS and R848 with respect to cultures treated with R848 alone.(D) MD-DCs were treated with LPS (100 ng/mL) and R848 (2 μg/mL) alone or in combination. Eighteen hours later, supernatants were collected for CCL1 and CCL4 content determination. Data are expressed as mean ± SD of culture duplicates and are representative of 3 independent experiments. P values were calculated by ANOVA and indicated no significant modulation in CCL1 or CCL4 content in cultures simultaneously treated with LPS and R848 with respect to cultures treated with a single agonist.

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