Figure 1.
Higher numbers of CD11b+ cells in the lungs of NOS2-/- KO mice.
Flow cytometry analysis was performed on single cell suspensions obtained from lungs of WT C57/BL6 or NOS2-/- mice. In these mice increased numbers of CD11b+ cells but not CD11b+CD11c+ cells could be observed as the infection progressed (A, B, C). In contrast, differences for CD11c+ and CD4+ cells only became evident at later time points (B, D). No significant differences were observed for CD8+ cells (E). Results are expressed as the mean values of log mean cell number (± SEM, n=5) in the lung. Bacterial burden (F) was enumerated at different time points after a low dose aerosol infection with M. tuberculosis. As observed, bacterial burden in both murine strains is similar at early time points after infection and diverges after 30 days of infection. Results are expressed as the mean Log 10 CFUs (±SEM, n=5) in the lungs. Student t-test, * p<0.05, ** p<0.01, *** p<0.001.
Figure 2.
Two populations of CD11b+ cells based on FSC analysis.
As suggested by FSC analysis, the increased number of CD11b+ cells present in NOS2 -/- mice could be separated in two populations based on cell size (A, B). Both populations of CD11b+ FSChigh cells and CD11b+FSClow cells are significantly increased in NOS2 -/- mice compared to C57/BL/6 mice (A, B). Contour plot is representative of day 45 after infection. FSC analysis clearly identified two CD11b+ populations in the lungs of NOS2 -/- mice (C). Results are expressed as the mean values fluorescence intensity (± SEM, n=5) in the Lung, Student t-test, *** p<0.001. Necrotic granulomas are only present in the lungs of NOS2 -/- mice (D). At different time points after infection, lungs were harvested and stained with H&E after fixation and paraffin embedding. For both murine strains, lesions are not present at day 15 after infection. Starting day 30, necrotic granulomas become evident in the lungs of NOS2 -/- mice but not WT C57BL/6. As observed at day 45 and 60, necrotic granulomas coalesce leading to severe lung consolidation at day 60 when most NOS2 -/- are moribund.
Figure 3.
Gr1int CD11b+ FSClow cells predominate in the lungs of NOS2-/- mice.
Further analysis of CD11b+FSClow and CD11b+FSChigh cells was performed by staining with anti-Gr1 antibody. As determined by mean fluorescence intensity, CD11b+FSChigh had significantly higher Gr1 expression than CD11b+FSClow (A). Starting at early time points NOS2-/- mice but not WT C57BL/6 mice have a significant number of cells giving an intermediate staining pattern for Gr-1 (B). At later time points, the influx of CD11b+FSChigh cells into the lungs of NOS2 -/- mice is also enhanced (C). Results are expressed as the Log mean cell number (± SEM, n=5) in the lung. Gr1high CD11b+ FSChigh cells express markers compatible with a monocytic lineage. Both populations of Gr1 expressing cells were further analyzed by flow cytometry using the monocytic markers Ly6C, CD14 and F4/80 (D). As determined by the mean fluorescence intensity for these markers, a more precise phenotype for these two cellular populations would be Gr1high CD11b+ FSChigh Ly6Chigh CD14+ F4/80+ and Gr1int CD11b+ FSClow Ly6Clow CD14low F4/80low, compatible with a monocytic and granulocytic lineage, respectively. Results are expressed as the log mean cell number (± SEM, n=5) in the Lung. ***Student t test, * p<0.05, ** p<0.01, *** p<0.001.
Figure 4.
Peripheral localization and distinct morphology of Gr1+ cells present in necrotic lungs.
Anti-Gr1 immunohistochemistry was performed to determine the location of Gr1+ cells surrounding necrotic lesions in the NOS2 -/- mice. As observed in low magnification (4x), anti-Gr1 labeling was predominantly localized in the periphery or rim of these structures (red, arrows, A). At higher magnification (100x), two distinct cellular morphologies could be observed for cells staining with anti-Gr1. In the left panel, neutrophils with multi-lobed nucleus could readily be seen. However, particularly in close apposition to the fibrotic capsule, cells were seen that had abundant and vacuolated cytoplasms as well as a unilobed nucleus, consistent with the morphology of MDSCs. Arginase activity was biochemically detected in the lungs of NOS2 -/- mice but minimally WT controls (B). As disease progressed in NOS2 -/-, more arginase activity could be detected compared to wild-type mice (B).Results are expressed as the mean values of arginase activity (± SEM, n=5) in the Lung. **Student t-test, p<0.01 and ***Student t-test, p<0.001. Anti-arginase-1 immunohistochemistry was performed to determine its expression by inflammatory cells in the lungs of NOS2-/- mice. Similar to the anti-Gr1 staining, at low magnification (4x), robust arginase-1 expression was detected in cells located in the rim of necrotic granulomas (C, left panel). At higher magnification (100x), arginase-1 expression was limited to highly vacuolated cells containing abundant cytoplasm (C, right panel). Panel D shows co-localized staining of both Gr1+ and arginase-1 in NOS2 -/- mouse lung tissues after 45 days of infection. At higher magnification (100x), co-localized staining of Gr1+ and arginase-1 demonstrates that Gr1+ cells (D lower right panel, arrow, red) and arginase-1 staining (D, arrow, brown) are clearly co-localized on the same cell.
Figure 5.
Significant numbers of Gr1int CD11b+ cells were also found in RAG2-/- and C3HeB/FeJ mice.
The influx of Gr1intCD11b+ cells was evaluated in immunocompromised RAG-/- (A), and immunocompetent C3HeB/FeJ mice (C) and NOS-/- (F). Again, large numbers of Gr1intCD11b+ cells were observed in these three mouse strains and significant differences were observed in the Gr1+ MFI (B, D, G). Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). In contrast, C3H/HeOuJ did not have major arginase activity. Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). Results are expressed as the mean values of mean fluorescence intensity (MFI) or arginase activity (± SEM, n=5) in the Lung. **Student t-test, p<0.001.
Figure 6.
Gr1int and Gr1hi cells sorted from M. tuberculosis infected NOS-/- mice expressed arginase I and IL-17.
Specific populations of Gr1int or Gr1hi cells were sorted from infected NOS-/- mice in an attempt to further characterize Gr1int and Gr1hi cell function during tuberculosis using flow cytometry. Panel A, demonstrates pre-sorted Gr1+CD11b+ cells which were then further characterized. Gr1hi and Gr1int sorted cells (B, C) both demonstrated high expression of arginase I and IL-17. Panel D shows the isotype controls for arginase-1 and IL-17A.
Figure 7.
Gr1int and Gr1hi cells sorted from M. tuberculosis infected NOS-/- mice activate Th17 cells and induce proliferation of CD4+ T cells.
Specific populations of Gr1int or Gr1hi cells were sorted from infected NOS-/- mice and placed into in vitro cultures with increasing numbers of either stimulated naïve or infected splenocytes. After 2 days of incubation T cells were evaluated for activation and memory profiles (A-D). Panel A -D shows lower ratios of Gr1high (A and C) or Gr1int (B and D) induce increased the percentage of activation of CD4+CD69+IL-17+ and memory CD4+CD44+IL-17+, when co-incubated with naïve (A and B) or M. tuberculosis infected splenocytes (C and D), respectively. Panel E and F shows Gr1 high and Gr1int induced of proliferating CD4 T cells. However, as the splenocyte to Gr1high or Gr1int ratio increased the percentage of CD4+ proliferation was reduced. Results are expressed as the mean values of mean percentage of CD4+CD69+IL-17+, CD4+CD44+IL-17+ and proliferation. ANOVA and the Tukey post-test. *p<0.05, **p<0.01, ***p<0.001.