Autophagy Is an Innate Mechanism Associated with Leprosy Polarization
Fig 2
IFN-γ rescues M. leprae-mediated inhibition of autophagosome formation in skin lesion MΦs of L-lep patients.
(A) Macrophages (MΦs) were isolated from skin lesions of tuberculoid (T-lep) and lepromatous (L-lep) patients and treated with IFN-γ (10 ng/mL) or rapamycin (200 ng/mL) for 18 h. Cells were fixed and stained with the anti-LC3 antibody (green) and DAPI (blue). Non-stimulated (N.S.) MΦs from T-lep lesions showed enhanced LC3 puncta formation as compared to L-lep MΦs. IFN-γ treatment increased the number of LC3 puncta in both T-lep and L-lep skin-derived MΦs. The increase was more prominent in T-lep MΦs. A similar increase of LC3-positive vesicles was observed after addition of rapamycin to the cultures of T-lep and L-lep MΦs. Immunofluorescence images were quantified and bars represent the mean values of the number of LC3 puncta per cell ± SEM (T-lep, n = 3; L-lep, n = 3). *P < 0.05, **P < 0.01, Kruskal-Wallis test; ##P < 0.01, Mann-Whitney test. Scale bar: 25 μm. (B) Monocyte-derived THP-1 MΦs were infected or stimulated with live or dead PKH26-labeled M. leprae (red), respectively, at MOIs of 2, 10 and 50 mycobacteria per cell for 18 h. Cells were fixed and immunofluorescence for LC3 (green) and DAPI (blue) was performed. Stimulation with dead M. leprae directly triggers autophagy. In contrast, infection with live mycobacteria does not induce LC3 puncta accumulation in THP-1 MΦs. Rapamycin (200 ng/mL) treatment was used as a positive control. The number of LC3 puncta/cell was calculated. Colocalization profiles between M. leprae and LC3 were quantified and expressed as percentage of cell area. Results represent the mean ± SEM of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test; ##P < 0.01, ###P < 0.001, Mann-Whitney test. Scale bar: 10 μm.