Fig. 5.

Functional activation of mφs by MPs isolated from chagasic patients. MPs were harvested from the plasma of seropositive CA and CS subjects. MPs from seronegative NH individuals were used as controls. a–d THP-1 mφs were incubated in triplicate with medium only (none) or with NH-MPs, CA-MPs, and CS-MPs for 1 h. Cell-free supernatants were utilized to measure ROS levels by amplex red assay (a) and measure nitrate/nitrite levels by Griess reagent assay (b). c, d THP-1 mφs were incubated for 1 h with MPs as above. Cells were loaded with JC-1 or MitoSOX Red probes, and analyzed by fluorimetry. Changes in mitochondrial membrane potential measured as J aggregates (red)/J monomers (green) ratio (c) and MitoSOX Red fluorescence as a measure of mitochondrial ROS production (d) are shown. e Plasma-derived MPs from NH, CA, and CS subjects were added in triplicate to THP-1 mφs. Cells were incubated for 24 h, and supernatants were utilized in a Bioplex assay for a panel of 17 human cytokines and chemokines. f, g ELISA was performed to measure the IL-1β and IFN-γ levels in cell-free supernatants collected 48 h after incubation of THP-1 mφs with MPs. In all bar graphs, data are plotted as mean value ± SEM, and significance is presented as * p < 0.05, ** p < 0.01, *** p < 0.001 (media only or NH-MPs [controls] vs. CA-MPs or CS-MPs).