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PreprintCited by 37Iron Metabolism and DisordersImmune responses and vaccinationsImmune Response and Inflammation

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Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

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Abreu, Rodrigo, Essler, Lauren, Giri, Pramod, Quinn, Fred·2020·PLoS ONE
DOI 10.1101/2020.10.07.329474PMC PMC7723272

Preprint — published as 10.1371/journal.pone.0240949 · Paper page

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Abstract

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis , these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-γ) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-γ decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-γ regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica , and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-γ significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-γ-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-γ significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

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Fig 1 Interferon-gamma regulates iron-related genes to favor iron export. A) transcriptional expression levels of SLC40A1, and (B) HAMP genes in THP-1 macrophages treated overnight with 200U/ml IFN-γ measured by qRT-PCR and compared to untreated controls. C) Ferroportin protein expression assessed in THP-1 macrophages treated as in A. D) Hepcidin secretion levels in THP-1 macrophages treated as in B (magnification = 63X). E) Transcriptional expression levels of the FTH gene in THP-1 macrophages treated as in A and B. **p<0.01, ***p<0.001. All data were from three independent experiments.
Fig 1 Interferon-gamma regulates iron-related genes to favor iron export. A) transcriptional expression levels of SLC40A1, and (B) HAMP genes in THP-1 macrophages treated overnight with 200U/ml IFN-γ measured by qRT-PCR and compared to untreated controls. C) Ferroportin protein expression assessed in THP-1 macrophages treated as in A. D) Hepcidin secretion levels in THP-1 macrophages treated as in B (magnification = 63X). E) Transcriptional expression levels of the FTH gene in THP-1 macrophages treated as in A and B. **p<0.01, ***p<0.001. All data were from three independent experiments.CC BY · PubMed Central
Fig 2 Intracellular pathogens modulate iron-related proteins to favor intracellular iron sequestration in macrophages. A) Hepcidin secretion from THP-1 macrophages after infection with M. tuberculosis (24 hours), M. bovis BCG (24 hours), L monocytogenes (eight hours) or S. enterica (16 hours) bacilli. B) Ferroportin levels in THP- 1 macrophages eight hours post-infection with L. monocytogenes (magnification = 40X). C) Ferroportin expression in hepcidin silenced THP-1 macrophages eight hours post infection with L. monocytogenes. Hepcidin gene silencing in THP-1 cells was achieved by lentiviral based shRNA transduction and Scramble short hairpin RNAs (ShScram) were used as a negative control (magnification = 40X). ***p<0.001. All data were from three independent experiments.
Fig 2 Intracellular pathogens modulate iron-related proteins to favor intracellular iron sequestration in macrophages. A) Hepcidin secretion from THP-1 macrophages after infection with M. tuberculosis (24 hours), M. bovis BCG (24 hours), L monocytogenes (eight hours) or S. enterica (16 hours) bacilli. B) Ferroportin levels in THP- 1 macrophages eight hours post-infection with L. monocytogenes (magnification = 40X). C) Ferroportin expression in hepcidin silenced THP-1 macrophages eight hours post infection with L. monocytogenes. Hepcidin gene silencing in THP-1 cells was achieved by lentiviral based shRNA transduction and Scramble short hairpin RNAs (ShScram) were used as a negative control (magnification = 40X). ***p<0.001. All data were from three independent experiments.CC BY · PubMed Central
Fig 3 Interferon-gamma treatment decreases pathogen-associated intracellular iron sequestration. A) Intracellular iron levels assessed by Prussian Blue staining in untreated and IFN-γ activated THP-1 macrophages, and (B-D) infected with three siderophilic bacteria (magnification = 100X). Percentage total area of Prussian Blue (PB) pixels in macrophages after infection with (E) L. monocytogenes, (F) M. bovis BCG, or (G) S. enterica with or without IFN-γ treatment. *p<0.05, **p<0.01, ***p<0.0001.
Fig 3 Interferon-gamma treatment decreases pathogen-associated intracellular iron sequestration. A) Intracellular iron levels assessed by Prussian Blue staining in untreated and IFN-γ activated THP-1 macrophages, and (B-D) infected with three siderophilic bacteria (magnification = 100X). Percentage total area of Prussian Blue (PB) pixels in macrophages after infection with (E) L. monocytogenes, (F) M. bovis BCG, or (G) S. enterica with or without IFN-γ treatment. *p<0.05, **p<0.01, ***p<0.0001.CC BY · PubMed Central
Fig 4 Interferon-gamma prevents pathogen associated iron modulation in macrophages. A) Ferroportin expression in IFN-γ-activated (200U/ml) macrophages eight hours post infection with L. monocytogenes, (B) 48 hours post infection with M. bovis BCG, or (C) 16 hours post infection with S. enterica (magnification = 40X). D) Hepcidin secretion in the culture supernatants of IFN-γ-activated macrophages eight hours post infection with L. monocytogenes, (E) 24 hours post infection with M. bovis BCG, or (F) 16 hours post infection with S. enterica. **p<0.01, ***p<0.001. All data were from three independent experiments.
Fig 4 Interferon-gamma prevents pathogen associated iron modulation in macrophages. A) Ferroportin expression in IFN-γ-activated (200U/ml) macrophages eight hours post infection with L. monocytogenes, (B) 48 hours post infection with M. bovis BCG, or (C) 16 hours post infection with S. enterica (magnification = 40X). D) Hepcidin secretion in the culture supernatants of IFN-γ-activated macrophages eight hours post infection with L. monocytogenes, (E) 24 hours post infection with M. bovis BCG, or (F) 16 hours post infection with S. enterica. **p<0.01, ***p<0.001. All data were from three independent experiments.CC BY · PubMed Central
Fig 5 Hepcidin inhibition limits intracellular M. tuberculosis and S. enterica bacterial replication in macrophages. Salmonella enterica (A), M. tuberculosis (B) and L. monocytogenes (C) intracellular burden in ShHAMP THP-1 macrophages and respective ShScram controls. Salmonella enterica (D), M. tuberculosis (E) and L. monocytogenes (F) intracellular burden in IFN-γ-activated macrophages (200U/ml) and respective untreated controls. *p<0.05, ***p<0.001. All data were from three independent experiments.
Fig 5 Hepcidin inhibition limits intracellular M. tuberculosis and S. enterica bacterial replication in macrophages. Salmonella enterica (A), M. tuberculosis (B) and L. monocytogenes (C) intracellular burden in ShHAMP THP-1 macrophages and respective ShScram controls. Salmonella enterica (D), M. tuberculosis (E) and L. monocytogenes (F) intracellular burden in IFN-γ-activated macrophages (200U/ml) and respective untreated controls. *p<0.05, ***p<0.001. All data were from three independent experiments.CC BY · PubMed Central

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Citations

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Metadata

Authors
Abreu, R., Essler, L., Giri, P., & Quinn, F.
Affiliations
University of Georgia (US)
ORCID
Venue
PLoS ONE
Publisher
openRxiv
Published
2020-10-07
Volume
15
Issue
12
Pages
e0240949-e0240949
ISSN
1932-6203 (ISSN-L)
DOI
10.1101/2020.10.07.329474
PMC
PMC7723272
OpenAlex
W3112899019
Cited by
37
References
2
Citations by year
2026: 3 · 2025: 3 · 2024: 9 · 2023: 7 · 2022: 7 · 2021: 8
FWCI
3.47
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Amgen
  • University of Georgia
Topics
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
interferon-gamma, iron export, macrophages, intracellular bacterial pathogens, hepcidin, ferroportin, ferritin, iron sequestration, Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis, bacterial replication, host-pathogen interaction
MeSH terms
THP-1 Cells, Host Microbial Interactions, Ferroportin, Biological Transport, Active, Ferritins, Humans, Immunity, Innate, Interferon-gamma, Iron, Listeria monocytogenes, Macrophages, Mycobacterium tuberculosis, Recombinant Proteins, Salmonella enterica, Cation Transport Proteins, Hepcidins

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