Expression of Fpn limits the growth of L pneumophila. (A) Bone marrow macrophages isolated from A/J mice were iron loaded as described in Figure 1, infected with L pneumophila for 2 hours, and incubated with or without hepcidin (hep25) or hep20 for 18 hours. Following hepcidin treatment, cells were fixed and processed for immunofluorescence using mouse anti–L pneumophila followed by Alexa 488–conjugated goat anti–mouse IgG. Bar represents 10 μm. (B) Quantification of intracellular L pneumophila expressed as bacteria foci/cell calculated from panel A where 10 fields of cells were examined per sample with approximately 10 to 15 cells present per field. The error bars represent the standard deviation of the mean from 3 separate experiments. (C) Cells treated as in panel A were lysed to release intracellular bacteria and lysates plated on CYET at either 10 or 25 μL. Bacterial colonies (CFUs) grown at 37°C were quantified 3 days after plating. The error bars represent the standard deviation of the mean from 3 separate experiments.