Splenic localization ofS. pneumoniae. (a) Representative two-photon microscopy images of MZ macrophage interaction with S. pneumoniae during acute i.v. infection. Bar, 25 µm. Green, S. pneumoniae; red, MZ macrophages (MZM). White arrows indicate stationary bacteria. (b) Quantification of MZ dwell time of S. pneumoniae. n = 3 from three independent experiments. (c) Increased RP localization of S. pneumoniae 60 min after i.v. infection in MZ macrophage–depleted (low-dose CLLs; red bar) animals. n = 3–4 pooled from two independent experiments. Ctrl lipo, control liposome. (d) Representative spinning-disk confocal images of RP macrophage (RPM) interaction with S. pneumoniae during acute i.v. infection. Bars, 100 µm. Green, S. pneumoniae; purple, RP macrophages. (Inset) White arrows indicate stationary bacteria. Data are representative of n = 5 from three independent experiments. (e) S. pneumoniae counts per field of view (FOV) in the RP from 0–20 min after i.v. infection in wild-type (black line), control liposome (blue line)–, low-dose CLL (red line)–, or high-dose CLL (gray line)–treated animals. There were four fields of view per animal. n = 5 for WT, n = 4 for control liposomes, n = 3 for high-dose CLL, and n = 5 for low-dose CLL pooled from four independent experiments. (f) Representative composite 10× stitched image of fresh spleen sections at 20 min after i.v. S. pneumoniae infection. Bar, 110 µm. Green, S. pneumoniae; red, MZ macrophages; purple, RP macrophages. (Inset) White arrows indicate bacteria. (g) Localization of S. pneumoniae in both the MZ (blue line) and RP (red line) regions at 20 and 60 min after i.v. infection. n = 3 pooled from two independent experiments. (h) S. pneumoniae cell localization 30 minutes after infection in the spleen. MZ macrophages, CD11b⁺CD209b⁺F4/80⁻; RP macrophages, CD11b^lowCD209b⁻F4/80⁺; Neutrophils, CD11b⁺Ly6G⁺Ly6C^int; monocytes, CD11b⁺Ly6G⁻Ly6C^hi. n = 4 pooled from two independent experiments. (i) Flow cytometry quantification of total number of S. pneumoniae–GFP⁺ neutrophils in the blood (black bar) and spleen (teal bar). n = 4 pooled from two independent experiments. (j) Flow cytometry quantification of total neutrophil number in the spleen 30 min after saline (black bar) or S. pneumoniae (blue bar) injection. n = 3 for saline and n = 4 for S. pneumoniae pooled from two independent experiments. *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (c, i, and j) and two-way ANOVA (e and g) statistical analyses were performed. Data are represented as mean ± SEM.