Fig. 5.

ViMs are protective against bacterial infection in neutropenic hosts. (A) BrdU incorporation into lung macrophages in untreated mice or mice at 4 wk after vaccination ± CY (n = 5–7 per group). Mice were labeled i.p. with BrdU for 3 d before lung harvest. Representative flow data and BrdU⁺ fractions in macrophage (Mac) gates are shown for AMs (Left) and ViMs (Right). Data are representative of two independent experiments. Each symbol represents one mouse, and error bars represent median with interquartile ranges. ***P < 0.001 by the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test for AMs. **P < 0.01 by the Mann–Whitney test for ViMs. (B) Phagocytic activity of AMs and ViMs in vitro. Macrophages isolated from the lungs pooled from Vac+CY-treated mice (n = 17–25 animals) were incubated in vitro with CypHer5E-labeled P. aeruginosa strain IT4 (in vivo challenge strain, LPS O-antigens heterologous to the vaccine strain) for 60 min in the presence of either intact or heat-inactivated sera from PAO1ΔaroA-immunized mice. Representative flow data (Left) and the fluorescence-positive fractions in macrophage gates in the presence of intact sera (Right) are shown. Bars represent mean of data from two independent experiments (three replicates for each). Error bars represent SEM. **P < 0.01 by unpaired t test. (C) Effect of macrophage adoptive transfer on survival (no. alive/total as indicated). AMs or ViMs isolated from the lungs pooled from immunized plus CY-treated mice were intratracheally administered (1 × 10⁵ cells per mouse) to unimmunized CY-treated mice. Recipients were challenged 14 h after the transfer. Unimmunized mice or actively immunized mice (both treated with CY) that did not receive the macrophages were controls. Data combined from two independent experiments (challenge doses of 59–63 cfu) are shown. ^#P = 0.03023 by log-rank test comparing ViM transfer vs. no transfer.