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Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity

Flannagan, Ronald S., Farrell, Tayler J., Trothen, Steven M., Dikeakos, Jimmy D., Heinrichs, David E.·2021·Blood Advances
DOI 10.1182/bloodadvances.2020002833
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Abstract

Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the plasmalemma, can be decorated with FPN and, if functional, will move iron from the cytosol into the phagosome lumen. This serves to feed iron to phagocytosed microbes and would be counterproductive to the many other known host mechanisms working to starve microbes of this essential metal. To understand how FPN is regulated during phagocytosis, we expressed FPN as a green fluorescent protein–fusion protein in macrophages and monitored its localization during uptake of various phagocytic targets, including Staphylococcus aureus, Salmonella enterica serovar Typhimurium, human erythrocytes, and immunoglobulin G opsonized latex beads. We find that FPN is rapidly removed, independently of Vps34 and PI(3)P, from early phagosomes and does not follow recycling pathways that regulate transferrin receptor recycling. Live-cell video microscopy showed that FPN movement on the phagosome is dynamic, with punctate and tubular structures forming before FPN is trafficked back to the plasmalemma. N-ethylmaleimide–sensitive factor, which disrupts soluble NSF attachment protein receptor (SNARE)–mediated membrane fusion and trafficking, prevented FPN removal from the phagosome. Our data support the hypothesis that removal of FPN from the limiting phagosomal membrane will, at the cellular level, ensure that iron cannot be pumped into phagosomes. We propose this as yet another mechanism of host nutritional immunity to subvert microbial growth.

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Figures

Figure 1 FPN-GFP localizes to the plasmalemma and is responsive to hepcidin. (A) RAW cells were cotransfected with PM-RFP (red) and pTF1 (green) and then fixed. Scale bars, 20 m m. (B) Graph depicts the average number of phagocytosed beads per cell in untransfected (ie, GFP-negative) RAW cells or RAW cells transfected with pTF1 encoding FPN-GFP. Data are the mean 6 standard deviation (SD) derived from 3 independent experiments. Statistical significance was determined using an unpaired Student t test. (C) The responsiveness of wild-type FPN fused to GFP and a C326S mutant of FPN fused to GFP to hepcidin treatment is shown. RAW macrophages expressing each GFP fusion protein were treated for 2 hours with vehicle control or recombinant hepcidin (400 nM). Prior to the addition of hepcidin or vehicle control, RAW macrophages were treated with 50 m g/mL cycloheximide for 2 hours. Prior to fixation, macrophages were labeled with TRITC-WGA to mark the macrophage plasmalemma. (D) Quantitation of the mean GFP fluorescence normalized to TRITC-WGA fluorescence at the plasmalemma is shown. The data are the mean 6 standard error of the mean (SEM) of 3 independent experiments with at least 36 cells analyzed for each condition. Statistical significance was determined by a paired Student t test, * P , .05. Images represent fixed samples of the indicated conditions and were acquired using widefield fluorescence microscopy. N.S., not significant.
Figure 1 FPN-GFP localizes to the plasmalemma and is responsive to hepcidin. (A) RAW cells were cotransfected with PM-RFP (red) and pTF1 (green) and then fixed. Scale bars, 20 m m. (B) Graph depicts the average number of phagocytosed beads per cell in untransfected (ie, GFP-negative) RAW cells or RAW cells transfected with pTF1 encoding FPN-GFP. Data are the mean 6 standard deviation (SD) derived from 3 independent experiments. Statistical significance was determined using an unpaired Student t test. (C) The responsiveness of wild-type FPN fused to GFP and a C326S mutant of FPN fused to GFP to hepcidin treatment is shown. RAW macrophages expressing each GFP fusion protein were treated for 2 hours with vehicle control or recombinant hepcidin (400 nM). Prior to the addition of hepcidin or vehicle control, RAW macrophages were treated with 50 m g/mL cycloheximide for 2 hours. Prior to fixation, macrophages were labeled with TRITC-WGA to mark the macrophage plasmalemma. (D) Quantitation of the mean GFP fluorescence normalized to TRITC-WGA fluorescence at the plasmalemma is shown. The data are the mean 6 standard error of the mean (SEM) of 3 independent experiments with at least 36 cells analyzed for each condition. Statistical significance was determined by a paired Student t test, * P , .05. Images represent fixed samples of the indicated conditions and were acquired using widefield fluorescence microscopy. N.S., not significant.CC-BY-NC-ND · From the PDF
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Image 4
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Figure 5 FPN is removed from the early phagosome and differs from TfR recycling. (A) The laser scanning confocal fluorescent micrographs depict RAW macrophages expressing FPN-GFP (top row) or TfR-GFP (bottom row) (both in green) and the PI(3)P biosensor 2 3 FYVE-RFP (red) that were allowed to phagocytose IgG-coated beads for 10 minutes. After fixation, the presence of phagosomal FPN-GFP, TfR-GFP, and 2 3 FYVE-RFP was analyzed. Arrows indicate a TfR and 2 3 FYVEpositive phagosome, whereas arrowheads indicate FPN-negative but 2 3 FYVE-positive phagosomes. Scale bars, 10 m m. (B) The graph depicts the fraction of 2 3 FYVE-positive phagosomes that are also positive for either FPN- or TfR-GFP. The data represent the mean 6 SD derived from $ 56 phagosomes from at least 3 independent experiments. Statistical significance was determined using an unpaired Student t test with a Welch ' s correction. (C) The confocal fluorescent micrographs also depict RAW macrophages expressing either FPN-GFP or TfR-GFP (in green) that were fixed and immunostained for endogenous EEA1 (in red) using rabbit anti-EEA1 antibody followed by anti-rabbit Cy3-conjugated secondary. The cells were allowed to phagocytose IgG opsonized beads for 10 minutes prior to fixation and staining. Scale bars, 10 m m. The white arrows point to phagosomes that are TfR positive and EEA1 positive, whereas the arrowheads point to representative phagosomes that are FPN negative yet are EEA1 positive. Scale bars, 10 m m. (D) The graph depicts the fraction of EEA1-positive phagosomes at 10 minutes post -bead exposure that are either TfR-GFP or FPN-GFP positive. The data are the mean 6 SD of 3 independent experiments, and statistical significance was determined by an unpaired Student t test with a Welch ' s correction. ** P , .01; *** P , .001.
Figure 5 FPN is removed from the early phagosome and differs from TfR recycling. (A) The laser scanning confocal fluorescent micrographs depict RAW macrophages expressing FPN-GFP (top row) or TfR-GFP (bottom row) (both in green) and the PI(3)P biosensor 2 3 FYVE-RFP (red) that were allowed to phagocytose IgG-coated beads for 10 minutes. After fixation, the presence of phagosomal FPN-GFP, TfR-GFP, and 2 3 FYVE-RFP was analyzed. Arrows indicate a TfR and 2 3 FYVEpositive phagosome, whereas arrowheads indicate FPN-negative but 2 3 FYVE-positive phagosomes. Scale bars, 10 m m. (B) The graph depicts the fraction of 2 3 FYVE-positive phagosomes that are also positive for either FPN- or TfR-GFP. The data represent the mean 6 SD derived from $ 56 phagosomes from at least 3 independent experiments. Statistical significance was determined using an unpaired Student t test with a Welch ' s correction. (C) The confocal fluorescent micrographs also depict RAW macrophages expressing either FPN-GFP or TfR-GFP (in green) that were fixed and immunostained for endogenous EEA1 (in red) using rabbit anti-EEA1 antibody followed by anti-rabbit Cy3-conjugated secondary. The cells were allowed to phagocytose IgG opsonized beads for 10 minutes prior to fixation and staining. Scale bars, 10 m m. The white arrows point to phagosomes that are TfR positive and EEA1 positive, whereas the arrowheads point to representative phagosomes that are FPN negative yet are EEA1 positive. Scale bars, 10 m m. (D) The graph depicts the fraction of EEA1-positive phagosomes at 10 minutes post -bead exposure that are either TfR-GFP or FPN-GFP positive. The data are the mean 6 SD of 3 independent experiments, and statistical significance was determined by an unpaired Student t test with a Welch ' s correction. ** P , .01; *** P , .001.CC-BY-NC-ND · From the PDF
Image 6
CC-BY-NC-ND · From the PDF
Figure 7 Treatment of macrophages with NEM inhibits removal of FPN from the limiting phagosomal membrane. (A) RAW macrophages expressing FPN-GFP
Figure 7 Treatment of macrophages with NEM inhibits removal of FPN from the limiting phagosomal membrane. (A) RAW macrophages expressing FPN-GFPCC-BY-NC-ND · From the PDF

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Metadata

Authors
Flannagan, R. S., Farrell, T. J., Trothen, S. M., Dikeakos, J. D., & Heinrichs, D. E.
ORCID
Venue
Blood Advances
Publisher
American Society of Hematology
Published
2021-01-21
Volume
5
Issue
2
Pages
459-474
ISSN
  • 2473-9529
  • 2473-9537
DOI
10.1182/bloodadvances.2020002833
Cited by
31
References
14
Language
en

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Flannagan, R. S., Farrell, T. J., Trothen, S. M., Dikeakos, J. D., & Heinrichs, D. E. (2021). Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity. Blood Advances. https://doi.org/10.1182/bloodadvances.2020002833