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. 2018 Feb 20;9:313. doi: 10.3389/fimmu.2018.00313

Figure 7.

Figure 7

Mitochondrial ROS (mtROS) production is important to control parasite replication during mammalian target of rapamycin (mTOR) inhibition in Trypanosoma cruzi-infected macrophages. (A) Bone marrow-derived macrophage (BMDM) from C57BL/6 mice pretreated with DMSO as control or with rapamycin (100 nM) during 90 min. After pretreatment cells were washed and uninfected or infected T. cruzi BMDM (1:5, cell:parasite ratio) were cultured at 3 and 6 h. Besides, BMDM without rapamycin pretreatment were stimulated with LPS + ATP (1 µg/mL and 5 mM) during 3 and 6 h. At indicates times postinfection (p.i.), BMDM were stained with anti-F480 (FITC) and anti-CD11b (APC) mAbs. Then, cells were incubated with 5 µM MitoSOX probe (PE) 15 min at 37°C and analyzed by Flow cytometry. Bars display mean fluorescence intensity (MFI) of mtROS on F4/80+ CD11b+ gated populations. Experiments were repeated three times with similar results being obtained and are expressed as mean ± SD (*p < 0.05 and **p < 0.005 vs. DMSO). A representative histogram of at least three independent experiments is shown. (B) Parasite replication in BMDM from C57BL6 mice pretreated with DMSO as control (24 h) rapamycin (100 nM, 90 min), with DPI (20 µm, 3 h) or with DPI + rapamycin. After pretreatment, cells were washed and infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) during 24 h. Besides, BMDM without inhibitors pretreatment, were stimulated with LPS (1 µg/mL) + IFNγ (100 ng/mL) or with IL-4 (80 ng/mL) and infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) during 24 h as controls. After that, non-internalized parasite were removed and 72 h later intracellular amastigotes were counted by indirect immunofluorescence. Intracellular replication of T. cruzi is expressed as number of parasites per 100 cells, quantified by ImageJ software and represent mean ± SD from three independent experiments (*p < 0.05; vs. rapamycin). (C) A representative image from DMSO, rapamycin, DPI, rapamycin + DPI pretreated BMDM, show cell nucleus stained with DAPI and parasites in green. Inserts show an area from the image (arrowhead) at higher magnification, indicating infected macrophages.