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

Figure 2.

Figure 2

Mammalian target of rapamycin (mTOR) activation in macrophages is essential for parasite replication. (A) Bone marrow-derived macrophage (BMDM) and (B) peritoneal macrophages (PM) from Balb/c mice were pretreated with DMSO as control or with different mTOR inhibitors: rapamycin (50 or 100 nM), PP242 (40 or 80 nM), and LY294002 (10 or 50 nM) during 90 min. Cells were washed and then were infected with Trypanosoma cruzi trypomastigotes (1:5, cell:parasite ratio) during 24 h. Besides, BMDM (A) or PM (B) without inhibitors pretreatment were stimulated with IL-4 (80 ng/mL) and infected with T. cruzi trypomastigotes (1:5, cell:parasite ratio) during 24 h as positive control of infection. After that, non-internalized parasites were removed and 72 h later intracellular amastigotes were counted by indirect immunofluorescence. Parasite replication was expressed as number of parasites per 100 cells, quantified by ImageJ software. Bars represent mean ± SD from three independent experiments (***p < 0.001 vs. DMSO). (C) A representative image from DMSO or rapamycin pretreated BMDM shown cell nucleus stained with DAPI and parasites in green. Inserts show an area from the image (arrowhead) at higher magnification, indicating infected macrophages.