Figure 1.

MIM-3 acts as an immune-modulator of macrophages’ cytokine secretion without affecting the phagocytosis capabilities of granulocytes. (A). Representative scheme of the CD14⁺-derived macrophages’ cytokine secretion assessment protocol. CD14⁺ cells were obtained from peripheral blood mononuclear cells (PBMCs) isolated from one healthy donor and cultivated for 6 days in complete medium supplemented with M-CSF 50 ng/mL alone (M0), IFN-γ at 20 ng/mL (M1), IL-4 at 5 ng/mL (M2a), or IL-10 at 20 ng/mL (M2c), as background polarization cytokines and in the presence of either MIM-3 or vehicle (Veh.). An additional 24 h of LPS treatment (100 ng/mL) were applied as an inducer of an inflammatory status during the last day of the MIM-3/Veh. treatment. AA: amino acids. Cytokine secretion was evaluated by ELISA. (B–I) Effect of MIM-3 on the secretion of the cytokines: IL12p70, TNF-α, IL-1β, IL12p40, IL-6, IL-23, arginase and TARC. The results are expressed in percentage of the cytokine secretion in the Veh. -treated conditions, the Veh. being set at 100% for each one of the differentiation backgrounds. The M1-macrophages induced by IFN-γ and treated with MIM-3 are represented with the green- and blue-squared histograms, the M2a macrophages induced by IL-4 and treated with MIM-3 are represented with the green- and pink-squared histograms, and the M2c macrophages induced by IL-10 and treated with MIM-3 are represented with the green- and yellow-squared histograms. Each condition has been performed in triplicate. (J) Human granulocytes were preincubated for 10 min with MIM-3, or vehicle (Veh.), or 10 µM N-formyl-methionyl-leucyl-phenylalanine (fMLP) as a positive phagocytosis inducer. Fluorescent beads were then added to the culture medium for an additional 45 min. Each condition was performed in triplicate and each histogram represents the mean ± SD of the fluorescence as a percentage of the Veh.-treated conditions, set at 100%.