Figure 2.
Figure 2. Differential susceptibility to necrosis in macrophages leads to M. tuberculosis growth. (A) Quantification of the proportion of cells that had a PI-positive (PI+) nucleus in resting (blue dots) or activated (red dots) GM-CSF macrophages. A PI-positive nucleus indicates that the cell membrane has become permeable to the extracellular medium and therefore is likely to be dead. Each data point represents one independent experiment, and the bars show means ± SEM. (B) Same as in A, but with M-CSF macrophages. (C) Representative confocal images of macrophages infected with GFP-Mtb (green) and labeled with PI (red). Macrophages were infected at an MOI of 1 for 2 h. 7 d after infection, macrophages were stained with 25 µg/ml PI in RPMI for 15 min and fixed. PI-positive cells are outlined. (D) Same as in C, but with M-CSF macrophages. Bars, 10 µm. (E) Quantification of GFP-Mtb replication in PI-negative or PI-positive cells in resting (black squares) or activated (red squares) GM-CSF macrophages. Images were analyzed, and the GFP signal per cell was plotted. Data represent the means ± SEM of one representative experiment. (F) Same as in E, but with M-CSF macrophages. (G) Representative snapshots at the indicated time points (Video 2) of resting GM-CSF macrophages infected with GFP-Mtb (green) in the presence of PI. Macrophages were infected at an MOI of 1 for 2 h, and after 24-h infection, the media were replaced with RPMI medium containing 0.4 µg/ml PI. Cells were imaged for at least 7 d. Bar, 20 µm. (H) Comparison of GFP-Mtb growth rates as measured by live-cell imaging. Three conditions were tested: M. tuberculosis in RPMI medium alone (green lines), M. tuberculosis within GM-CSF–differentiated human monocyte-derived macrophages (HMDMs) before the moment the cell becomes PI positive (blue lines), and M. tuberculosis after human monocyte-derived macrophages become PI positive (red lines). Each line shows the growth rate of M. tuberculosis represented by the fold change in GFP signal (RAWIntDen) of the GFP-Mtb signal over time, normalized to the first frame in the video. (I) Quantification of intracellular replication of GFP-Mtb before and after membrane damage in resting or activated GM-CSF and M-CSF macrophages at the single-cell level. Red dashed lines indicate when the cell’s nucleus becomes PI positive. GFP-Mtb signal was monitored in three representative cells from each condition using ImageJ software. (J) Same as in I, but with M-CSF macrophages.

Differential susceptibility to necrosis in macrophages leads to M. tuberculosis growth. (A) Quantification of the proportion of cells that had a PI-positive (PI+) nucleus in resting (blue dots) or activated (red dots) GM-CSF macrophages. A PI-positive nucleus indicates that the cell membrane has become permeable to the extracellular medium and therefore is likely to be dead. Each data point represents one independent experiment, and the bars show means ± SEM. (B) Same as in A, but with M-CSF macrophages. (C) Representative confocal images of macrophages infected with GFP-Mtb (green) and labeled with PI (red). Macrophages were infected at an MOI of 1 for 2 h. 7 d after infection, macrophages were stained with 25 µg/ml PI in RPMI for 15 min and fixed. PI-positive cells are outlined. (D) Same as in C, but with M-CSF macrophages. Bars, 10 µm. (E) Quantification of GFP-Mtb replication in PI-negative or PI-positive cells in resting (black squares) or activated (red squares) GM-CSF macrophages. Images were analyzed, and the GFP signal per cell was plotted. Data represent the means ± SEM of one representative experiment. (F) Same as in E, but with M-CSF macrophages. (G) Representative snapshots at the indicated time points (Video 2) of resting GM-CSF macrophages infected with GFP-Mtb (green) in the presence of PI. Macrophages were infected at an MOI of 1 for 2 h, and after 24-h infection, the media were replaced with RPMI medium containing 0.4 µg/ml PI. Cells were imaged for at least 7 d. Bar, 20 µm. (H) Comparison of GFP-Mtb growth rates as measured by live-cell imaging. Three conditions were tested: M. tuberculosis in RPMI medium alone (green lines), M. tuberculosis within GM-CSF–differentiated human monocyte-derived macrophages (HMDMs) before the moment the cell becomes PI positive (blue lines), and M. tuberculosis after human monocyte-derived macrophages become PI positive (red lines). Each line shows the growth rate of M. tuberculosis represented by the fold change in GFP signal (RAWIntDen) of the GFP-Mtb signal over time, normalized to the first frame in the video. (I) Quantification of intracellular replication of GFP-Mtb before and after membrane damage in resting or activated GM-CSF and M-CSF macrophages at the single-cell level. Red dashed lines indicate when the cell’s nucleus becomes PI positive. GFP-Mtb signal was monitored in three representative cells from each condition using ImageJ software. (J) Same as in I, but with M-CSF macrophages.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal