Figure 1.
Polycaspase activation in M. tuberculosis infected RAW macrophages over 72 hours.
RAW macrophages were infected with M. tuberculosis GC1237 wild-type and trafficking mutant strains Tn::moaC1 and Tn::Rv1503c. After 24, 48 and 72 hours, nuclei were stained with Hoechst stain (blue), and active caspases stained with fluorescent polycaspase inhibitor (FLICA; red), and the cells analysed by automated confocal microscopy. Caspase staining (A) (surface of caspase/number of nuclei) and number of nuclei (B) were measured using Image Mining Software developed at the Institut Pasteur, Korea. Bar graphs are Mean+SEM of triplicate samples. *p = <0.05, **p = <0.01, ***p = <0.001 by 2-way ANOVA, with the upper and lower p values for GC1237 vs Tn::moaC1 and Tn::Rv1503c respectively. (C) Representative microscopy at 72 hours is shown. Results are representative of 3 independent experiments with similar results.
Figure 2.
Characterisation of cell death fates during M. tuberculosis infection.
Parallel cultures of RAW267.4 macrophages were infected with M. tuberculosis (MOI 10) for 48 hours. (a) Cell yield recovered from the cultures was determined and expressed as a percentage of control, uninfected cells (n = 4). (b) Apoptosis and necrosis were determined by staining with Apo-BrdU-FITC (n = 3) and 7aaD (n = 4) respectively. Results are representative of two similar experiments. Bar graphs are Mean+SEM. *p = <0.05, **p = <0.01, ***p = <0.001 by unpaired 2-tailed T-test. Asterisks above white bars are vs GC1237 ApoBrdU; asterisks above grey filled bars are vs GC1237 7-AAD control.
Figure 3.
Live mycobacteria inhibit doxorubicin-induced apoptosis in a dose-dependent manner.
Macrophages were induced to undergo apoptosis with different doses of doxorubicin, and cell survival enumerated at 3,6 and 20 hours using a calcein-AM (a). Macrophages infected with mycobacterial strains at MOI 1–10 (b) or MOI 10 (c–d) for 2.5 hours were induced to undergo apoptosis with doxorubicin (2.5 µM). After 20 hours, live cells were enumerated using Calcein-AM (b–c). (d) ApoBrdU TUNEL staining was performed on uninfected (grey fill) and GC1237-infected (black line) macrophages after 20 hrs treatment with 2.5 µM doxorubicin, and cells analysed by flow cytometry (representative plots of quadruplicate samples are shown). Bar graphs are Mean+SEM, n = 10, and are representative of two independent experiments. *p = <0.05, **p = <0.01, ***p = <0.001 by unpaired 2-tailed T-test.
Figure 4.
Mycobacterial strains inhibit apoptotic cell death induced by the chemical agents doxorubicin and cyclohexamide.
Macrophages infected with mycobacterial strains for 2.5 hours (MOI 10) were induced to undergo apoptosis with doxorubicin (2.5 µM) (a,b) or cyclohexamide (9 µM) (a,c). After 20 hours, live cells were enumerated using Calcein-AM. (a) A representative comparison of cell death induced by doxorubicin and cyclohexamide in uninfected and H37Rv-infected macrophages. Survival is expressed as a percentage of control untreated cells. (b,c) A comparison of the ability of mycobacterial strains to inhibit doxorubicin- (b) and cyclohexamide- (c) induced apoptotic cell death. Survival of drug treated infected cells is expressed as a percentage of untreated, infected cells. (Bar graphs are Mean+SEM, n = 10, and are representative of two independent experiments. *p = <0.05, **p = <0.01, ***p = <0.001 by unpaired 2-tailed T-test.