Mature neutrophil phagocytosis of S. pneumoniae in the splenic RP. (a) Representative spinning-disk confocal image of neutrophils (red) and RP macrophages (purple) containing S. pneumoniae (green) at 30 min after infection. Data are representative of n = 6 from three independent experiments. Bar, 50 µm. White arrows, bacteria containing neutrophils; blue arrows, bacterial containing RP macrophages. (b) Proportion of splenic neutrophil populations that bound S. pneumoniae at 30 min after infection. There were four fields of view per animal. n = 6 pooled from three independent experiments. (c) Time-lapse spinning-disk confocal images of S. pneumoniae (green) cell adhesion (white arrow) to RP macrophage (purple) and subsequent phagocytosis by neutrophils (red). Data are representative of n = 4 from three independent experiments. Bar, 10 µm. (d) 3D reconstruction of intracellular S. pneumoniae (green) within neutrophil (red) at 30–60 min after infection. Transparency of neutrophils is increased from left to right. Bar, 23.3 µm. Data are representative of n = 3 from two independent experiments. White arrows indicate intracellular bacteria. (e) IVM and flow cytometric quantification of S. pneumoniae phagocytosis by neutrophils (Neu) and RP macrophages (RPM) from 30–60 min after i.v. infection in wild-type (black bars) and C3-deficient (blue bars) animals. n = 3–6 pooled from three and two independent experiments for IVM and flow cytometry, respectively. FOV, field of view; S. p., S. pneumoniae. (f) Survival curve for S. pneumoniae i.v. infection at 10⁴-CFU dose in wild-type (black line) or C3 KO (blue line) mice. n = 5 from one experiment. (g) S. pneumoniae bacterial counts in the spleen at 1 and 24 h after i.v. infection in isotype antibody (black circles)– and Ly6G (1A8) antibody (1A8 Ab; blue triangles)–treated animals. n = 4–6 pooled from two independent experiments. Red lines show the median. (h) Time-lapse spinning-disk confocal images of OxyBURST probe activation (green) after S. pneumoniae (red) phagocytosis by mature splenic neutrophil (blue). Data are representative of n = 6 from two independent experiments. Bar, 5 µm. White arrows indicate OxyBURST activation on the surface of intracellular bacteria. (i) Quantification of intracellular OxyBURST probe activation within neutrophils in wild-type (blue line) and Cybb^−/− (gray line) mice. n = 3–6 pooled from two independent experiments. AU, arbitrary units. (j) S. pneumoniae bacterial counts in the spleen at 24 h after i.v. infection in wild-type (black circles), Cybb^−/− (gray squares), and CatC^−/− (blue triangles) animals. n = 4–6 pooled from two independent experiments. Black lines show the median. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (b, e, and i), log-rank Mantel-Cox test (f), and Mann-Whitney test (g and j) statistical analyses were performed. Data are represented as mean ± SEM.