Analysis of NETosis and ROS generation by neutrophils of HR and LR TB patients. Neutrophils from HR and LR TB patients were incubated at 5 × 106 cells/mL during 4 h with Mtb-Ag (10 µg/ml). After treatment with DNase I, (A) DNA release was measured in the supernatants by using an specific stain (Invitrogen, S34860) and (B) myeloperoxidase (MPO) activity was determined in the supernatants by employing TMB. Bars represent the mean values of (A) the ratio of the released DNA Mtb-Ag/Medium ± SEM and (B) the OD450nm Mtb-Ag/OD450nm Medium ± SEM. (C) Representative images of NETs (DAPI) acquired by confocal microscopy (60X oil objective). Upper panel: HR TB patient; lower panel: LR TB patient. (D) Neutrophils (1.5 × 106 cells/ml) from HR and LR TB patients were incubated with 2′,7′ –dichlorofluorescin diacetate (DCFDA, 50 µM) during 15 min and then stimulated with or without Mtb-Ag (10 µg/ml) during 60 minutes. Finally, DCFDA fluorescence was evaluated to monitor ROS production by flow cytometry. Bars represent the mean of the ratio ROS Mtb-Ag/Medium ± SEM. (E) A representative example for ROS production in HR TB (left panel) or LR TB (right panel). Statistical differences were calculated using the Wilcoxon signed rank test for paired samples (***P < 0.001) or the Mann-Whitney nonparametric test for unpaired samples (#P < 0.05; ####P < 0.0001). N, HR= (A: 4; B: 6; C: 13); N, LR = (A: 6; B: 10; C: 19).
