Fig 2: c-Abl inhibitors activate TFEB, enhance lysosome biogenesis, and improve control of intracellular Mtb in cultured primary macrophages.a Torin1 (mTOR inhibitor) and two c-Abl kinase inhibitors (imatinib and nilotinib) increase nuclear translocation of TFEB. BMDM were treated with DMSO or the indicated small molecules for 4 h. Then, cells were fixed and stained with an anti-TFEB antibody. Scale bar, 20 µm.b Quantification of nuclear TFEB MFI per cell from >70 cells for each condition in (a) using ImageJ.c Imatinib and nilotinib increase lysosome gene expression. qPCR results of lysosome genes for BMDM treated with DMSO or the indicated small molecules for 24 h.d BMDM were transfected with the designated siRNA (20 nM) for 48 h, then treated with nilotinib (5 µM) for 18 h. The mRNA levels of lysosome genes were detected by qPCR. siNT: non-targeting siRNA control.e Histograms and MFI of LAMP1 and ATP6V1B2 for uninfected BMDM treated with small molecules for 24 h.f Histograms and MFI of cresyl violet for uninfected BMDM treated with small molecules for 24 h or 72 h.g Imatinib and nilotinib induce cathepsin B enzymatic activity. Histograms and MFI of fluorogenic cathepsin B product for H37Rv-infected BMDM treated with inhibitors for 72 h.h MFI of fluorogenic cathepsin B product in siRNA transfected BMDM.i Imatinib and nilotinib enhance lysosome acidification. BMDM were infected with H37Rv-ZsGreen (MOI=2), then treated with DMSO, imatinib (10 µM), or nilotinib (5 µM). BMDM were stained with DAPI and lysotracker at 24 hpi. Scale bar, 10 µm.j Imatinib and nilotinib enhance Mtb phagolysosome maturation in cultured primary macrophages. Quantification of Mtb co-localizing with lysotracker for (i) from >20 images per condition using JACoP in ImageJ.k Imatinib and nilotinib enhance control of Mtb in primary macrophages. BMDM were infected with H37Rv at MOI=1, then treated with DMSO or indicated small molecules for 4 days. Cells were then lysed and plated for CFU assay.l siRNA-transfected BMDM were infected with Mtb H37Rv (MOI=1) and then treated with nilotinib (2 µM) for 4d. Viable intracellular bacteria were quantitated by CFU assay.Results are presented as mean ± SD, n=3–4 replicates, representative of 2–3 independent experiments. **p<0.01, ***p<0.001, ****p<0.0001 by multiple unpaired Student’s t test (c), or unpaired Student’s t test (b, d, e-i).

Fig 3: MNC1 are deficient in lysosomal cathepsin B and LAMP1 protein content.C57BL/6 mice were infected by aerosol with ~100 Mtb H37Rv-ZsGreen or H37Rv-mCherry. At 28 dpi, mouse lungs were harvested for flow cytometry analysis, live cell sorting, and immunofluorescence staining.a Immunofluorescence analysis of CTSB protein in AM and MNC1 sorted from H37Rv-mCherry-infected mice (28 dpi). Representative images were taken by confocal microscopy. Scale bars, 10 µm.b Quantification of CTSB protein immunostaining MFI per cell for (a), >100 cells per subset were analyzed using ImageJ.c Immunofluorescence analysis of LAMP1 in AM and MNC1 sorted from H37Rv-mCherry-infected mice (28 dpi). Representative images were taken by confocal microscopy. Scale bars, 10 µm.d Quantification of LAMP1 MFI per cell for (c), >100 cells per subset were analyzed using ImageJ.e Representative histograms and MFI of intracellular LAMP1 analyzed by flow cytometry for AM and MNC1 from mice infected with Mtb H37Rv-ZsGreen (28 dpi).f-g LAMP1, CTSB and SiglecF staining of lung sections prepared from mice infected with H37Rv-mCherry (28 dpi). Scale bars, 20 µm. Green arrow indicating SiglecF+ AM.****p<0.0001 by unpaired Student’s t test. Results are presented as mean ± SD, representative of 2–3 independent experiments.

Fig 4: c-Abl inhibitors activate TFEB, enhance lysosome biogenesis, and improve control of intracellular Mtb in cultured primary macrophages.(A) Torin1 (mTOR inhibitor) and two c-Abl kinase inhibitors (imatinib and nilotinib) increase nuclear translocation of TFEB. BMDM were treated with DMSO or the indicated small molecules for 4 h. Then, cells were fixed and stained with an anti-TFEB antibody. Scale bar, 20 µm.(B) Quantification of nuclear TFEB MFI per cell from >70 cells for each condition in (A) using ImageJ.(C) Imatinib and nilotinib increase lysosome gene expression. qPCR results of lysosome genes for BMDM treated with DMSO or the indicated small molecules for 24 h.(D) BMDM were transfected with the designated siRNA (20 nM) for 48 h, then treated with nilotinib (5 µM) for 18 h. The mRNA levels of lysosome genes were detected by qPCR. siNT: non-targeting siRNA control.(E) Histograms and MFI of LAMP1 and ATP6V1B2 for uninfected BMDM treated with small molecules for 24 h.(F) Histograms and MFI of cresyl violet for uninfected BMDM treated with small molecules for 24 h or 72 h.(G) Imatinib and nilotinib induce cathepsin B enzymatic activity. Histograms and MFI of fluorogenic cathepsin B product for H37Rv-infected BMDM treated with inhibitors for 72 h.(H) MFI of fluorogenic cathepsin B product in siRNA transfected BMDM.(I) Imatinib and nilotinib enhance lysosome acidification. BMDM were infected with H37Rv-ZsGreen (MOI=2), then treated with DMSO, imatinib (10 µM), or nilotinib (5 µM). BMDM were stained with DAPI and lysotracker at 24 hpi. Scale bar, 10 µm.(J) Imatinib and nilotinib enhance Mtb phagolysosome maturation in cultured primary macrophages. Quantification of Mtb co-localizing with lysotracker for (G) from >20 images per condition using JACoP in ImageJ.(K) Imatinib and nilotinib enhance control of Mtb in primary macrophages. BMDM were infected with H37Rv at MOI=1, then treated with DMSO or indicated small molecules for 4 days. Cells were then lysed and plated for CFU assay.(L) siRNA-transfected BMDM were infected with Mtb H37Rv (MOI=1) and then treated with nilotinib (2 µM) for 4d. Viable intracellular bacteria were quantitated by CFU assay.Results are presented as mean ± SD, n=3–4 replicates, representative of 2–3 independent experiments. **p<0.01, ***p<0.001, ****p<0.0001 by multiple unpaired Student’s t test (C), or unpaired Student’s t test (B, D, E-I).

Fig 5: MNC1 are deficient in lysosomal cathepsin B and LAMP1 protein content.C57BL/6 mice were infected by aerosol with ~100 Mtb H37Rv-ZsGreen or H37Rv-mCherry. At 28 dpi, mouse lungs were harvested for flow cytometry analysis, live cell sorting, and immunofluorescence staining.(A) Immunofluorescence analysis of CTSB protein in AM and MNC1 sorted from H37Rv-mCherry-infected mice (28 dpi). Representative images were taken by confocal microscopy. Scale bars, 10 µm.(B) Quantification of CTSB protein immunostaining MFI per cell for (A), >100 cells per subset were analyzed using ImageJ.(C) Immunofluorescence analysis of LAMP1 in AM and MNC1 sorted from H37Rv-mCherry-infected mice (28 dpi). Representative images were taken by confocal microscopy. Scale bars, 10 µm.(D) Quantification of LAMP1 MFI per cell for (C), >100 cells per subset were analyzed using ImageJ.(E) Representative histograms and MFI of intracellular LAMP1 analyzed by flow cytometry for AM and MNC1 from mice infected with Mtb H37Rv-ZsGreen (28 dpi).(F-G) LAMP1, CTSB and SiglecF staining of lung sections prepared from mice infected with H37Rv-mCherry (28 dpi). Scale bars, 20 µm. Green arrow indicating SiglecF+ AM.****p<0.0001 by unpaired Student’s t test. Results are presented as mean ± SD, representative of 2–3 independent experiments.