Article Figures & Data
Figures
- FIGURE 1.
Characterization of microbiologic features of i.t. instillation of low and high inocula of pneumococci. A, Bacteria in lung homogenates 24 h after i.t. instillation of 104 CFU type 1 or 2 (low Spn 1, low Spn 2) or 107 CFU type 1 or 2 (high Spn 1, high Spn 2) pneumococci, low Spn 1 vs high Spn 1, p < 0.05 (∗); low Spn 2 vs high Spn 2, p < 0.01 (∗∗), Kruskal-Wallis with Dunn’s post test. Bacteria were undetectable in the lungs of 69% of mice infected with low Spn 1 compared with 25% infected with high Spn 1 (p = 0.026) and 57% of mice infected with low Spn 2 compared with 0% infected with high Spn 2 (p = 0.007, Fisher’s exact test). B, Bacteria in blood 24 h after infection, low Spn 1 vs high Spn 1, p < 0.05 (∗); low Spn 2 vs high Spn 2, p < 0.01 (∗∗), Kruskal-Wallis with Dunn’s post test. Ninety-two percent of the mice were abacteremic following infection with low Spn 1 compared with 20% following high Spn 1 (p = 0.008) and 57% of the mice following infection with low Spn 2 compared with 8% following high Spn 2 (p = 0.031, Fisher’s exact test). C, The percentage of mice with detectable bacteria in the lung 2–192 h after infection with low Spn 1 (n = 4–16) for each time point. D, Cumulative survival after instillation of low Spn 1 or mock infection (PBS; n = 8 in each group); low Spn 1 vs PBS (p = 0.134, log rank analysis).
- FIGURE 2.
Characterization of inflammatory features in the lung of low- and high-dose pneumococcal infection. A, Absolute numbers of PMN (#PMN) in BAL 24 h after i.t. instillation of 104 CFU type 1 or 2 (low Spn 1, low Spn 2) or 107 CFU type 1 or 2 (high Spn 1, high Spn 2) pneumococci or mock infection (PBS, n = 9), mean + SEM. Low Spn 1 (n = 7) vs high Spn 1 (n = 10), p < 0.05 (∗); low Spn 2 (n = 7) vs high Spn 2 (n = 9), p < 0.05 (∗), Kruskal-Wallis with Dunn’s post test. B, Representative appearances of lung sections stained with H&E from mice 24 h after i.t. instillation of PBS (top left), low Spn 1 (top right), or high Spn 2 (bottom left), all ×10 objective. An enlargement of the marked area of inflammation from high Spn 2 is shown bottom right (×40 objective) to demonstrate PMN recruitment. C, #PMN in BAL 2–192 h after instillation of low Spn 1 (n = 3–9 for each time point, mean + SEM).
- FIGURE 3.
AM depletion modifies the course of low-dose pneumococcal infection in the lung. A, Absolute number of AM (# AM) in BAL 72 h after intranasal instillation of PBS (PBSAM+), liposome-encapsulated clodronate (AM−), or liposome-encapsulated PBS (LipAM+), mean + SEM. #AM was reduced by 68% in AM− compared with PBSAM+. AM− (n = 19) vs PBSAM+ (n = 19), p < 0.001 (∗∗∗); AM− vs LipAM+ (n = 19), p < 0.001 (∗∗∗), Kruskal-Wallis with Dunn’s post test. B, Bacteria in lung homogenates 24 h after i.t. instillation of 104 CFU type 1 pneumococci (Spn) in PBSAM+, AM−, or LipAM+. Bacteria were undetectable in the lung of 81% of the PBSAM+ group, 9% of the AM− group, and 42% of the LipAM+ group. AM− vs PBSAM+, p < 0.001 (∗∗∗); AM− vs LipAM+, p < 0.05 (∗), Kruskal-Wallis with Dunn’s post test. C, Absolute numbers of PMN (# PMN) in BAL 24 h after i.t. instillation of low-dose Spn 1 (Spn) or PBS in PBSAM+, AM−, or LipAM+ (mean + SEM). AM−/Spn vs AM−/PBS, p < 0.01 (∗∗); AM−/Spn vs PBSAM+/Spn, p < 0.01 (∗∗); AM−/Spn vs LipAM+/Spn, p < 0.05 (∗), Kruskal-Wallis with Dunn’s post test. D, Cumulative survival after infection with low-dose Spn 1 in PBSAM+, AM−, or LipAM+ (n = 12 in each group). PBSAM+ and LipAM+ had identical 100% survival (AM+), which was not significantly different from survival in the AM− group (p = 0.135, log rank analysis).
- FIGURE 4.
Infection with pneumococci is associated with increased numbers of apoptotic cells in the lung. A, Representative lung sections (×40 objective) stained by TUNEL and propidium iodide from mice 24 h after mock infection (PBS, left panel) or infection with 104 CFU type 1 pneumococci (low Spn, right panel); apoptotic TUNEL+ cells appear green. B, Quantification of the number of TUNEL+ cells in lung sections from low Spn 1 or PBS 24 h after infection. Mean + SEM TUNEL+ cells per lung field (×100 objective), low Spn 1 vs PBS, p < 0.05 (∗), Mann-Whitney U test; results are representative of three experiments. C, Cytospins obtained from the BAL 24 h after infection with low Spn 1 demonstrate apoptotic cells (left panel) and apoptotic bodies in macrophages (right panel). D, Total number of apoptotic events (apoptotic cells and bodies) in cytospins of BAL from mice 24 h after instillation of low-dose type 1 (low Spn 1, n = 9), high-dose type 1 (high Spn 1, n = 6) pneumococci or mock infection (PBS, n = 8), mean + SEM. PBS vs low Spn 1, p < 0.05 (∗); PBS vs high Spn 1, p < 0.001 (∗∗∗); low Spn 1 vs high Spn 1, p < 0.05 (∗), Kruskal-Wallis with Dunn’s post test.
- FIGURE 5.
Increased numbers of apoptotic macrophages are found in the lung after infection with pneumococci. A, Representative appearance of lung sections stained with H&E from mice 24 h after i.t. instillation of 104 CFU type 1 pneumococci (low Spn 1) showing an apoptotic macrophage (arrow) and normal macrophages (arrowheads) viewed by differential interference contrast microscopy (×40 objective). B, Cytospin of BAL 24 h after low Spn 1 infection stained with F4/80 FITC and Ho33342. Macrophages show positive F4/80 staining (green cells), one of which demonstrates a fragmented nucleus by Ho33342 staining (arrow). C, Representative dot plots depicting percentage of macrophage apoptosis in BAL 24 h after i.t. instillation of PBS or infection with low-dose Spn. FL2 represents Annexin VPE and FL-4 TOP-RO-3 staining. Apoptotic cells were Annexin VPE+/TO-PRO-3− and the percentage of apoptotic cells are recorded in the upper left quadrant. Cells treated with EDTA during staining are shown in the right-hand panel as a negative control for annexin V binding. D, Percentage of macrophage apoptosis (Annexin VPE+/TO-PRO-3−, flow cytometry) in BAL 24 h after i.t. instillation of low Spn 1 (n = 11), 107 CFU type 1 pneumococci (high Spn 1, n = 10), or mock infection (PBS, n = 11). Mean + SEM, PBS vs low Spn 1, p < 0.05 (∗); PBS vs high Spn 1, p < 0.01 (∗∗), Kruskal-Wallis with Dunn’s post test.
- FIGURE 6.
Inhibition of macrophage apoptosis is associated with increased bacteremia after low-dose pneumococcal infection. A, Percentage of apoptotic AM in BAL (AnnexinVPE+/TO-PRO-3−, flow cytometry) 24 h after infection with 104 CFU type 1 pneumococci (Spn) or mock infection (PBS) and treatment with zVADfmkfmk (zVADfmk) or vehicle control (2% DMSO, n = 7–9/group). Mean + SEM, Spn/DMSO vs Spn/zVADfmk, p < 0.05, Mann-Whitney U test (∗). B, Bacterial killing at 6 h by BMDM infected in vitro with type 1 pneumococci in the absence or presence of zVADfmk or zFAfmk (zFAfmk), zVADfmk vs zFAfmk, p < 0.05, Friedman test with Dunn’s post test comparison (∗). C, Bacteria in lung homogenates 24 h after i.t. instillation of Spn and i.p. 2% DMSO or zVADfmk. D, Bacteria in blood 24 h after infection and i.p. 2% DMSO or zVADfmk. One hundred percent of the Spn/DMSO group were abacteremic compared with 54% of the Spn/zVADfmk group, p = 0.046, Fisher’s exact test (∗). E, Cumulative survival after infection with Spn and i.p. DMSO or zVADfmk (n = 12 mice/group). There was no significant difference in survival, two deaths in the DMSO group compared with four in the zVADfmk group (p = 0.13, log rank analysis).
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