Figure 1.
Distribution of Mtb infection within monocytic cell populations in the lung.
Whole lung leukocytes were harvested from groups of mice with TB (4 and 10 weeks p.i.) and compared to uninfected controls. Monocytic cells were sorted into AM, RM and mDC as described in Materials and Methods. Ziehl-Neelsen staining was performed on cytospin preparations of sorted populations. (A) The proportion of AM, RM and mDC within the total lung monocytic cell population of uninfected mice and mice with pulmonary TB. (B) The proportion of AM, RM and mDC containing any AFB in mice with pulmonary TB. (C) The proportion of GFP-labeled lung leukocytes, GFP+ AM, RM and mDC, in uninfected mice and mice with TB. Lung leukocytes within the airspace were transduced by tracheal instillation of WT mice with CMV-GFP-W. After 8 weeks, one group of GFP-transduced mice was challenged by aerosol with 300 CFU of Mtb Erdman delivered to the lung. The category Other comprised cells that could not be categorized as AM, RM or mDC based on light scatter characteristics and CD11b/CD11c staining. By light microscopy, cells in the Other category included a small number of neutrophils that may have acquired GFP by efferocytosis, as well of cells with monocytic appearance that had very high intracellular Mtb burden and features of cell death. Monocytic cell subsets were classified by surface immunostaining as alveolar macrophages (AM; CD11b− CD11c+/hi), recruited monocyte/macrophages (RM; CD11b+/lo CD11clo/−) and myeloid dendritic cells (mDC; CD11b+/hi CD11c+/hi).
Figure 2.
Kinetics of intracellular Mtb growth in vivo.
BAL cells and whole lung leukocytes were harvested at 1, 2, 3 and 8 weeks after aerosol infection of mice with 300 CFU Mtb Erdman delivered to the lung. Cells from both sources were counted and cytospin preparations were made for Ziehl-Neelsen staining. (A) The total number of Mtb-infected (AFB+) cells was derived by multiplying the % AFB+ cells by the total number of cells in each sample. Results for individual mice are presented as log10 total AFB+ cells, with the line representing the mean. (B) The proportion of Mtb-infected phagocytes within the total sample was counted at each time point and expressed as % AFB+ cells. (C) Neutrophils infected with Mtb were identified by their typical nuclear morphology on Ziehl-Neelsen stained cytospin slides of BAL cells and whole lung leukocytes. The relative proportion of AFB in neutrophils versus monocytic phagocytes (AM, RM and mDC) is expressed as mean % AFB+ leukocytes ± SD from one representative experiment. All in vivo experiments were repeated twice.
Figure 3.
Enumeration of intracellular Mtb in lung phagocytes.
BAL cells and whole lung leukocytes were isolated from C57BL/6 mice 2 weeks after aerosol challenge with Mtb Erdman. Ziehl-Neelsen stained cytospin preparations were used to visualize and count intracellular AFB by light microscopy. Representative photomicrographs show examples of infected cells along with AFB counts as indicated (magnification, 400×).
Figure 4.
Distribution of intracellular bacillary load in lung phagocytes changes over time after aerogenic Mtb infection.
The number of AFB per cell was counted in BAL cells and lung leukocytes harvested at the indicated times after aerosol challenge with Mtb Erdman. Across all time points, Mtb burden per cell was interrogated in a total 5.7×106 individual phagocytes, with counts grouped into bins of 1–5, 6–10, 11–15, 16–20, or ≥21 AFB. Results are expressed as mean log10 AFB+ monocytic cells (A) or neutrophils (B) ± SD within each bin, counted in BAL cells or in whole lung leukocytes as indicated. All bins were compared at each time point and between time points as described in Materials and Methods. Statistically significant differences (p<0.05) are not indicated on the figure for the sake of clarity. Among statistically significant differences, monocytic cells with 1–10 AFB outnumbered cells with >10 AFB at all time points p.i., the proportion of monocytic cells containing >15 AFB was lower at week 8 than earlier time points, and the distribution of AFB loads was significantly different in neutrophils compared to monocytic cells.
Figure 5.
Kinetics of Mtb growth in IFN-γ-deficient mice.
BAL cells were harvested from WT and GKO mice 7, 11, 14, 18 and 21 days after aerosol challenge with 100 CFU Mtb Erdman delivered to the lung. Total cells from both sources were counted and cytospin preparations were made for Ziehl-Neelsen staining. (A) The total number of Mtb-infected (AFB+) cells was derived by multiplying the % AFB+ cells by the total number of cells in each sample. Results for individual mice are presented as log10 total AFB+ cells, with the line representing the mean. (B) The proportion of Mtb-infected phagocytes within the total sample was counted at each time point and expressed as % AFB+ cells with the bar indicating the mean. Data were analyzed as detailed in Materials and Methods. The number of AFB+ BAL cells from GKO mice was significantly different (p<0.05) from WT on days 14, 18 and 21 p.i. The % AFB+ BAL cells from GKO mice were significantly different from WT on day 21 p.i. Data are representative from one experiment with five mice per group. In vivo experiments were repeated twice.
Figure 6.
Lung leukocyte populations and distribution of intracellular Mtb in WT and GKO mice.
(A) The relative proportion of neutrophils and monocytic cells in BAL from WT and GKO mice following aerogenic infection with Mtb Erdman was determined by light microscopy. Results are expressed as mean % cells of either type ± SD at the indicated time points. All groups were compared within and between all time points. GKO mice had a significantly higher proportion of neutrophils to monocytic cells compared to WT at 14, 18 and 21 days p.i. (B) The distribution of Mtb infection between neutrophils and monocytic cells was determined by counting AFB+ cells of both types on Ziehl-Neelsen stained cytospins. Results are expressed as mean % AFB+ ± SD. There was a statistically significant difference in the number of AFB+ neutrophils between WT and GKO mice on day 18 p.i. There was also a significant increase in the number of AFB+ neutrophils at day 21 p.i. for GKO mice compared to earlier time points.
Figure 7.
Distribution of AFB loads in lung monocytic cells and neutrophils from WT and GKO mice with TB.
GKO and WT mice were challenged by aerosol with 100 CFU of Mtb Erdman delivered to the lung. BAL was performed 7, 11, 14, 18 and 21 days p.i. A total of 4.8×106 Ziehl-Neelsen stained cells were counted. Numbers of AFB per cell were grouped into 5 bins as indicated and counted separately in monocytic cells (A) and neutrophils (B) from GKO and WT mice. Results are presented as mean log10 AFB+ cells in each bin ± SD. GKO mice had a greater number of AFB+ monocytic cells in all bins at 7 days p.i. and a significantly higher proportion of cells with ≥11 AFB compared to WT mice at that time point. The proportion of cells within each bin was similar between WT and GKO mice on days 11–18 p.i. but by day 21 p.i. the proportion of cells with ≥11 AFB fell significantly in WT compared to GKO mice. On day 21 p.i. the number of AFB+ neutrophils from GKO mice was significantly higher than earlier time points. In contrast, the number of AFB+ neutrophils from WT mice was significantly lower on day 21 than day 18 p.i.
Figure 8.
Morphology of macrophage cell death in pulmonary TB.
(A) BMDM challenged with Mtb Erdman in vitro (MOI 25, 3 h) and BAL cells from WT mice challenged 3 weeks previously with a low aerosol dose of Mtb Erdman were stained with carbolfuchsin to visualize intracellular bacilli and stained with DAPI to visualize nuclear morphology (magnification, ×400). Heavily infected cells in both cases exhibited nuclear condensation without fragmentation (yellow arrows). (B) Representative scanning electron micrographs of BMDM infected with Mtb in vitro and BAL macrophages isolated from mice after 4 weeks of TB disease (magnification, ×5,000). Damaged cells in both cases exhibit disintegration of outer cell membranes (red arrows) with escape of intracellular bacilli. BAL cells were isolated from GKO mice 4 weeks after aerosol Mtb infection. Cytospin preparations were processed for Ziehl-Neelsen staining, immunostaining or scanning EM.