Iron depletion limits intracellular bacterial growth in macrophages

Abstract

Many intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

Figures

Figure 1

Expression of Fpn limits the growth of Chlamydia. (A) HEK293Fpn-GFP cells were incubated with FAC for 24 hours and induced to express Fpn-GFP (green) using ponasterone A. Eighteen hours after induction, cells were incubated with C psittaci at an MOI of 100 bacteria/cell for 2 hours, extracellular bacteria removed by extensive washing, and cells placed in growth medium for specified times. Twenty-four hours after infection, cells were fixed and processed for immunofluorescence using a mouse anti–C psittaci antibody followed by an Alexa 594–conjugated goat anti–mouse IgG (red). Cells that were successfully induced showed Fpn-GFP (green) at the plasma membrane and had small inclusions. Cells that did not express Fpn-GFP had large inclusions. Arrowheads denote large inclusions and arrows denote small inclusions. Bar represents 10 μm. (B) Bone marrow macrophages isolated from C57/B6 mice were iron loaded and infected with C psittaci for 2 hours as in panel A. Following infection, cells were incubated with or without 1 μg/mL hep25 for 18 hours. Cells were methanol fixed and processed for immunofluorescence using rabbit anti-Fpn and mouse anti–C psittaci followed by an Alexa 594–conjugated goat anti–rabbit IgG (red) and an Alexa 488–conjugated goat anti–mouse IgG (green). Arrowheads denote C psittaci and arrows denote plasma membrane Fpn. Bar represents 10 μm. (C) Cells treated as in panel B were solubilized in lysis buffer and applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Chlamydial infection was assessed by Western blot using mouse anti–C psittaci followed by peroxidase-conjugated goat anti–mouse IgG. Fpn levels were assessed using rabbit anti-Fpn followed by peroxidase-conjugated goat anti–rabbit IgG. Tubulin levels were detected using mouse antitubulin followed by peroxidase-conjugated goat anti–mouse IgG as a loading control. (D) Large (greater than 1 μ) and small inclusions were quantified from cells infected with C psittaci or C trachomatis. More than 200 cells were analyzed for inclusion size for each sample. Error bars represent the standard deviation of the mean of 3 independent experiments. (E) Macrophages were incubated with C psittaci for 2 hours, extracellular bacteria removed, and cells incubated in growth media for an additional 18 hours. Cells were solubilized in lysis buffer and applied to SDS-PAGE, and Fpn levels assessed as in panel D.

Figure 2

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.

Figure 3

The Fpn disease mouse model ffe/+ macrophages show increased C psittaci infections. (A,B) Wild-type (C3H) and ffe/+ mouse bone marrow macrophages were incubated with iron for 48 hours, infected with C psittaci for 2 hours, incubated with or without hep25 for 18 to 24 hours, and processed for C psittaci immunofluorescence as in Figure 1. (C) Ferritin levels were determined from cells infected as in panel A and then incubated with hep25, hep20, hep25 and deferriprone, or hep25 and desferasirox. Fpn levels were detected by Western blot using a rabbit anti–mouse Fpn followed by a peroxidase conjugated goat anti–mouse IgG. Error bars represent SD. (D) Bone marrow macrophages from ffe/+ mice treated as in panel C were processed for immunofluorescence. Bar represents 10 μm. Experiments were performed a minimum of 3 times and the error bars represent the standard deviation of the mean.

Figure 4

Iron chelators deferriprone and desferasirox remove iron stored in macrophages limiting intracellular L pneumophila growth. Bone marrow macrophages isolated from A/J mice were iron loaded as in Figure 1, infected with L pneumophila for 2 hours, and incubated with hepcidin with or without iron chelators deferriprone or desferasirox for 18 hours. Cells were either (A) lysed and lysates plated on CYET for 3 days and CFUs determined or (B) processed for immunofluorescence and bacterial foci/cell determined as described in Figure 2. Experiments were repeated a minimum of 3 times. Error bars represent the standard deviation of the mean.

Figure 5

Oral iron chelators desferasirox and deferriprone are more effective than DFO in chelating intracellular iron and limiting intracellular C psittaci and L pneumophila growth. Macrophages from either (A) wild-type mice (C3H) or (B) flatiron mice were grown in FAC for 48 hours and then incubated in the presence of different concentrations of DFO (•), desferasirox (△), or deferriprone (▲) for 18 hours. Cells were lysed and ferritin levels determined as described in “Methods.” (C) Flatiron macrophages grown in FAC for 48 hours were infected with C psittaci for 2 hours, extracellular bacteria washed away, and cells incubated in the presence of different concentrations of DFO (•), desferasirox (△), or deferriprone (▲). Twenty-fours hours after infection cells were fixed and processed for immunofluorescence, and the percentage of cells with large inclusions was determined as in Figure 1. (D) A/J macrophages grown in FAC for 48 hours were infected with L pneumophila for 2 hours, extracellular bacteria washed away, and cells incubated in the presence of different concentrations of DFO (•), desferasirox (△), or deferriprone (▲). Cells were lysed and lysates plated on CYET for 3 days and CFUs determined. Experiments were repeated a minimum of 3 times. Error bars represent the standard deviation of the mean.

Figure 6

Iron chelators limit the growth of Chlamydia by reducing intracellular iron. Macrophages from flatiron mice were grown in the presence (+) or absence (−) of FAC for 18 hours. Cells were infected with C psittaci for 2 hours, extracellular bacteria removed, and cells incubated in the presence of DFO, desferasirox, deferriprone, or desferasirox that was preincubated with 10 to 100 μM iron as FAC (+/+). Twenty-four hours after infection, cells were processed for immunofluorescence and large and small inclusions quantified as in Figure 1 (A), lysed for ferritin analysis as in Figure 5 (B), or lysed for RT-PCR analysis of C psittaci genes omcB, lcrH-1, and rs16 (C) as described in “Other procedures.” Error bars represent the standard deviation of the mean.