Fig 1.
MIP is a potent inducer of autophagy.
RAW 264.7 macrophages were infected with MIP (MOI-1:10) for 4 hrs, whole cell lysates were prepared and western blot was performed. (A) Shown is the representative blot depicting the level of lipidated LC3-II in Control, Rapamycin treated and MIP infected macrophages. (B) Shown is the mean fold change ± range in LC3-II level in Rapamycin treated and MIP infected cells as compared to uninfected control. (C) RAW cells expressing GFP-LC3 were infected overnight (MOI-1:10) with MIP. Images were taken at 63 X magnification. Shown are the merged images of GFP-LC3 (green) and DAPI (blue). ***: P<0.0001.
Fig 2.
Total autophagic flux is maintained in MIP infected macrophages.
RAW 264.7 macrophages were infected with MIP or treated with Rapamycin (1 μM) / Bafilomycin A1 (100 nM) and lysates were prepared at 4, 8, 12, 16 and 24 hrs and western blot was performed. Bafilomycin A1 was taken as positive control for fusion inhibition. (A) Western blot depicting the level of LC3-II in MIP infected and Rapamycin / Bafilomycin A1 treated cells. (B) RAW GFP-LC3 cells were treated with Rapamycin / Bafilomycin A1 or infected with MIP for 4, 8, 12, 16 and 24 hrs and imaging was done by confocal microscopy. Shown are the representative merged images of GFP-LC3 (green) and DAPI (blue) portraying LC3 puncta formation in various groups. (C) RAW 264.7 cells loaded with DQ-BSA were infected with MIP for 4 hrs and imaged at 63 X magnification. Merged images of DQ-BSA Red (red) and DAPI (blue), depicting the proteolysis of DQ-BSA are shown here. (D) DQ-BSA-loaded cells were infected with MIP for 4 hrs, and the percentage of DQ-BSA positive cells was determined by flow cytometry. **: P<0.001.
Fig 3.
M.tb induces autophagosome formation but inhibits its fusion with lysosome and proteolysis.
RAW 264.7 macrophages were infected with MIP/ M.tb H37Rv or treated with Bafilomycin A1 (100 nM) and lysates were prepared at 4 and 24 hrs followed by western blot. (A) Representative blot depicting LC3-II levels in MIP/ M.tb H37Rv infected and Bafilomycin A1 treated cells. (B) RAW GFP-LC3 cells were treated with Bafilomycin A1 or infected with MIP / M.tb H37Rv for 4 and 24 hrs and imaged by confocal microscopy. Shown are the representative merged images of GFP-LC3 (green) and DAPI (blue). (C) RAW 264.7 cells loaded with DQ-BSA were infected with MIP / M.tb H37Rv for 4 hrs and imaged at 63 X magnification. Shown here are the merged images of DQ-BSA Red (red) and DAPI (blue), depicting the proteolysis.
Fig 4.
MIP promotes maturation of M.tb containing phagosomes via autophagy.
Immunofluorescence images of macrophages stained for Rab5 (marker for early phagosomes) and Rab7 (marker present on late phagosomes). (A) Uninfected RAW 264.7 cells (B) RAW 264.7 macrophages infected with MIP and (C) with M.tb H37Rv (D) Co-infected with M.tb and MIP (E) treated with 3-MA (5 mM) for 1 hr to inhibit autophagy followed by M.tb and MIP infection. The cells were fixed and permeabilized followed by staining with Rab5 to Rab7 at different time points. Rab5 to Rab7 conversion was observed in MIP infected macrophages (4B) while M.tb was seen to inhibit the conversion process (4C). When M.tb infected cells were re-infected with MIP, it was seen that MIP promotes maturation of M.tb containing phagosomes (4D). Upon autophagy inhibition with 3-MA, maturation of M.tb containing phagosomes was inhibited even in the presence of MIP (4E).
Fig 5.
MIP induced autophagy results in enhanced phago-lysosome fusion in M.tb infected macrophages.
RAW 264.7 cells were co-infected with GFP expressing M.tb and MIP followed by LysoTracker-Red staining to stain the lysosomes. Slides were prepared and visualised by confocal microscopy to examine the co-localisation of GFP expressing M.tb within the lysosomes. 50 fields of each group were examined and green spots (M.tb located outside the lysosomes) and yellow spots (formed by co-localisation of GFP expressing M.tb with LysoTracker-Red giving a yellow fluorescence) were counted. (A) Confocal images showing co-localisation of GFP expressing M.tb within the lysosomes in different groups. (B) Graphical representation of the percentage of GFP expressing M.tb present within the lysosomes. (C) Representative images depicting the co-localisation of GFP expressing M.tb within the lysosomes of RAW 264.7 cells with basal level of autophagy. (D) Representative images showing the co-localisation of GFP expressing M.tb within the lysosomes of RAW 264.7 cells in which LC3 was knocked down using RNA interference technique. (E) Graphical representation of the percentage of GFP expressing M.tb present within the lysosomes after autophagy abrogation. ***: P<0.0001.
Fig 6.
Autophagy induction by MIP enhances clearance of M.tb from infected macrophages.
(A) RAW 264.7 macrophages were infected with either GFP expressing M.tb or co-infected with GFP expressing M.tb and MIP for 4 hrs followed by lysis. 100 μl of lysate from each group was plated in triplicates on 7H11-agar plates containing Hygromycin. Shown is the mean M.tb CFU count of 4 independent experiments. (B) Macrophages were infected with either M.tb or co-infected M.tb and MIP for 4, 8, 12, 24 hrs and lysed followed by plating in triplicates on 7H11-agar plates containing Hygromycin. Shown is the mean M.tb CFU count of 3 independent experiments. (C) RAW macrophages were treated with 3-MA for 1 hr to inhibit autophagy followed by M.tb and MIP infection. Graph showing CFU count of M.tb upon autophagy inhibition. (D) Autophagy was abrogated using SiRNA against LC3 and the same assay was performed. It was observed that when autophagy was abrogated by loss of LC3 protein, survival of M.tb was increased. This concludes that MIP induced autophagy plays an important role in M.tb clearance. ***: P<0.0001, **: P<0.001.