Figure 4.

Decreased Trypanosoma cruzi replication by mammalian target of rapamycin inhibition was independent of cytoplasmic ROS and IDO. (A) Bone marrow-derived macrophages (BMDM) from C57BL/6 mice were pretreated with DMSO as control or with rapamycin (100 nM) during 90 min. After pretreatment cells were washed and uninfected (UI) or T. cruzi-infected (T. cruzi) (1:5, cell:parasite ratio) were cultured at different times and then processed for the experiments. Besides, BMDM were stimulated with LPS + IFNγ (1 µg/mL + 100 ng/mL), during 24 h as positive control. BMDM at 6 and 24 h postinfection were stained with anti-F480 (PE) and anti-CD11b (APC) mAbs. Then, cells were incubated with 20 µM H2DCFDA probe, 20 min at 37°C for intracellular reactive oxygen species (ROS) detection. Bars represent mean ± SD of from three independent experiments (ns, no significant difference vs. DMSO). (B–C) Parasite replication in BMDM from C57BL6 mice pretreated with DMSO as control, or with Rapamycin (RAPA: 100 nM) during 90 min, or with 1-methyl tryptophan (1-MT: 100 µM; 24 h) or with 1-MT (24 h) + RAPA (90 min). After pretreatment BMDM were washed and then infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) during 24 h. Besides, BMDM without inhibitors pretreatment, were stimulated with LPS + IFNγ (1 µg/mL + 100 ng/mL), during 24 h as positive control. After that, non-internalized parasites were removed and 72 h later intracellular amastigotes were counted by indirect immunofluorescence. (B) 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 and **p < 0.005, vs. DMSO). (C) A representative image shows cell nucleus stained with DAPI and parasites in green. Inserts show an area from the image (arrowhead) at higher magnification, indicating infected macrophages.