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Cited by 45Iron Metabolism and DisordersFerroptosis and cancer prognosisTrace Elements in Health

Iron- and Hepcidin-Independent Downregulation of the Iron Exporter Ferroportin in Macrophages during Salmonella Infection

Open access
Willemetz, Alexandra, Beatty, Sean, Richer, Etienne, Rubio, Aude, Auriac, Anne, Milkereit, Ruth J., Thibaudeau, Olivier, Vaulont, Sophie +2 more·2017·Frontiers in Immunology
DOI 10.3389/fimmu.2017.00498PMC PMC5410627
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Abstract

Retention of iron in tissues macrophages via upregulation of hepcidin (HAMP) and downregulation of the iron exporter ferroportin (FPN) is thought to participate in the establishment of anemia of inflammation after infection. However, an upregulation of FPN has been proposed to limit macrophages iron access to intracellular pathogens. We therefore studied the iron homeostasis and in particular the regulation of FPN after infection with Salmonella enterica serovar Typhimurium in mice presenting tissue macrophages with high iron (AcB61), basal iron (A/J and wildtype mice), or low iron (Hamp knock out, Hamp-/-) levels. The presence of iron in AcB61 macrophages due to extravascular hemolysis and strong erythrophagocytosis activity favored the proliferation of Salmonella in spleen and liver with a concomitant decrease of FPN protein expression. Despite systemic iron overload, no or slight increase in Salmonella burden was observed in Hamp-/ mice compared to controls. Importantly, FPN expression at both mRNA and protein levels was strongly decreased during Salmonella infection in Hamp-/- mice. The repression of Fpn mRNA was also observed in Salmonella infected cultured macrophages. In addition, the downregulation of FPN was associated with decreased iron stores in both liver and spleen in infected mice. Our findings show, that during Salmonella infection FPN is repressed through an iron and hepcidin independent mechanism. Such regulation likely provide the cellular iron indispensable for the growth of Salmonella inside the macrophages.

AI snapshot

Written from the paper’s full text.

TL;DR

During Salmonella infection, ferroportin (FPN) is downregulated in macrophages through an iron- and hepcidin-independent mechanism, even in Hamp−/− mice and cultured macrophages. This repression is associated with increased Salmonella burden in iron-loaded AcB61 mice but not with increased burden in systemically iron-overloaded Hamp−/− mice, suggesting macrophage iron availability rather than systemic iron drives intracellular Salmonella growth.

Key findings

  • Salmonella infection strongly decreased FPN protein in liver and spleen of AcB61 and A/J mice, with loss at the macrophage cell surface; bacterial load was significantly higher in AcB61 liver and spleen than in A/J.
  • In Hamp−/− mice, despite systemic iron overload (liver iron ~2,000 µg/g, decreasing by more than 50% after infection) and high hematocrit (60% falling to 50%), Salmonella CFUs in liver and spleen did not significantly differ from WT; FPN mRNA and protein were still strongly decreased after infection.
  • Fpn mRNA was significantly downregulated in WT and Hamp−/− liver after Salmonella Typhimurium infection and in infected BMDM as early as 4 h, with no significant Hamp change at that time; Hamp was slightly but significantly upregulated at 6 h in BMDM.
  • FPN downregulation was associated with decreased iron stores in liver and spleen in infected mice, most clearly in Hamp−/− liver (>50% decrease); in AcB61, FPN protein loss occurred without major changes in Fpn mRNA, suggesting posttranscriptional regulation.

Methods

  • Mice (AcB61, A/J, WT, Hamp−/−; 8–12 weeks) were infected intravenously with Salmonella enterica serovar Typhimurium strain Keller (~1,000 CFUs for A/J and AcB61; ~5,000 CFUs for WT and Hamp−/−), with samples collected at D0, D3/D5, or D10.
  • Murine bone marrow-derived macrophages (BMDM) from CD1 mice were infected with S. Typhimurium strain SL1344 at MOI 5–10 for 1 h, then treated with gentamicin and harvested for RNA at time points.
  • Analyses included blood parameters, tissue iron by acid digestion and Perls staining, immunohistofluorescence, western blot, and RT-qPCR; statistics used two-way ANOVA with Sidak’s multiple comparisons and unpaired t-tests for CFUs.

Limitations

  • The authors state that the role of NOS2/NO in FPN regulation needs further investigation, that the discrepancy between studies on FPN regulation during intracellular infection is unclear, and that different anti-FPN antibodies not all carefully characterized may contribute to differences.
  • The authors state it remains to be determined whether this HAMP-independent FPN downregulation occurs in different macrophage populations and is a general host response to other intracellular pathogens.
  • Evident from the text: conclusions rely on mouse models and one BMDM system, some supporting observations are “not shown” (e.g., Erfe mRNA, unpublished Listeria data), and infection doses/timepoints differ across models, limiting direct comparability.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fimmu.2017.00498

Cites

50 of 50 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 52 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In parallel to decreased FPN protein, Fpn mRNA level is significantly downregulated in both WT and Hamp−/− liver after ST infection.

    “In parallel to the decrease of FPN protein expression, the level of Fpn mRNA was significantly downregulated in both WT and Hamp−/− liver after ST infection (Figure 4B).”
    Quote in the paperSubstantial claim
  • Findingown data

    Downregulation of FPN is associated with decreased iron stores in both the liver and spleen in infected mice.

    “In addition, the downregulation of FPN was associated with decreased iron stores in both the liver and spleen in infected mice.”
    Quote in the paperSubstantial claim
  • Findingown data

    After ST infection, in both WT and Hamp−/− mice, FPN expression is strongly decreased in hepatocytes and Kupffer cells compared to uninfected tissues.

    “After ST infection, in both WT and Hamp−/−, the expression of FPN was strongly decreased in hepatocytes and Kupffer cells when compared to uninfected tissues (Figure 4A).”
    Quote in the paperSubstantial claim
  • Findingown data

    Bacterial load is significantly higher in the liver of AcB61 mice than in A/J mice after Salmonella infection.

    “Importantly, the bacterial load was significantly higher in the liver of AcB61 mice when compared to A/J (Figure 1F).”
    Quote in the paperNot a substantial claim
  • Findingown data

    In WT spleen, Perls staining indicates a decrease in macrophage iron after Salmonella infection.

    “In WT spleen, Perls staining indicated a decrease in macrophage iron after Salmonella infection (Figure 3G; Figure S2 in Supplementary Material).”
    Quote in the paperSubstantial claim
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 28

  • May combine two claims.

    Findingown data

    FPN is mostly detected in Kupffer cells (F4/80+) in WT liver, whereas in Hamp−/− liver FPN is strongly expressed by both Kupffer cells (F4/80+) and hepatocytes (F4/80−).

    “FPN was mostly detected in Kupffer cells (F4/80+) in WT liver, whereas in Hamp−/− liver, FPN was strongly expressed by both Kupffer cells (F4/80+) and hepatocytes (F4/80−) (Figure 4A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In liver of uninfected Hamp−/− mice, iron accumulates in hepatocytes of centrilobular zones, whereas periportal areas are not stained.

    “In the liver of uninfected Hamp−/− mice, iron accumulation was observed in hepatocytes of CL zones, whereas PP areas were not stained (Figure 3I).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In liver at day 0, AcB61 mice show strong iron accumulation compared to A/J; liver iron levels do not change significantly in A/J or AcB61 after infection.

    “In the liver at D0, Perls staining of tissue sections (Figure 1D) and quantitative determination of iron (Figure 1E) indicated a strong iron accumulation in AcB61 liver when compared to A/J liver. We did not observe any significant changes in iron levels in the liver of both A/J and AcB61 after infection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    During Salmonella infection, HMOX1 protein expression increases in the liver of both A/J and AcB61 mice and in the spleen of AcB61 mice; HMOX1 expression is maintained in the spleen of infected A/J mice.

    “During Salmonella infection, protein expression of HMOX1 increased in the liver (Figures 2A,C) of both of A/J and AcB61 mice and in the spleen (Figures 2B,D) of AcB61 mice. HMOX1 expression was maintained in spleen of infected A/J (Figure 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Salmonella infection leads to profound downregulation of FPN in the liver and spleen of AcB61 mice and in the spleen of A/J mice.

    “On the other hand, infection leads to a profound downregulation of FPN in the liver (Figure 2A, lower panels) and in the spleen (Figure 2B, lower panels) of AcB61. FPN downregulation was also observed in the spleen of A/J mice (Figure 2B, upper panels).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In AcB61 spleen before infection, there is strong expansion of red pulp and evidence of extramedullary erythropoiesis with numerous trapped RBC; iron accumulates in enlarged splenic macrophages of red pulp before and after infection.

    “Histologic examination of the spleen of AcB61 mice before infection revealed a strong expansion of the red pulp (RP) and evidence of extramedullary erythropoiesis with numerous trapped RBC (Figure 1K, lower panel). Important accumulation of iron in the AcB61 spleen was clearly detected in enlarged splenic macrophage of the RP before and after infection (Figure 1K; Figure S1A in Supplementary”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    After 10 days postinfection, WT mice present signs of anemia with hematocrit levels below 40% and decreased plasma iron levels.

    “After 10 days postinfection, WT mice present signs of anemia with hematocrit levels below 40% (Figure 3A) and decreased plasma iron levels (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In naïve AcB61 liver, iron accumulates mostly in sinusoid zones and centrilobular areas, with most periportal zones unstained; at cellular level, iron strongly accumulates in Kupffer cells with milder staining in surrounding hepatocytes.

    “Histological examination of Perls staining indicated iron accumulation mostly in sinusoid zones and centrilobular (CL) area of the naïve AcB61 liver, whereas most periportal (PP) zones were not stained (Figure 1J). At the cellular level, iron strongly accumulated (deep blue) in Kupffer cells with some milder iron staining (light blue) in surrounding hepatocytes (Figure 1J, lower panels; Figure S1A”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    No major changes in Fpn mRNA expression are observed in AcB61 liver during Salmonella infection, contrasting with strong protein downregulation.

    “Contrasting with the strong protein downregulation, no major changes of Fpn mRNA expression were observed in AcB61 liver during Salmonella infection.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Il6 expression strongly increases during ST infection in BMDM.

    “As a control of BMDM infection, Il6 expression strongly increased during ST infection (Figure 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Hamp−/− mice present higher hematocrit (60%) and higher concentrations of iron and ferritin in blood compared to WT mice.

    “As previously described (14), Hamp−/− mice present a higher hematocrit (60%) and a higher concentration of iron and ferritin in the blood when compared to WT (Figures 3A–C).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Lesbordes-Brion, 2006, Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis, Blood source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The negative regulation of Fpn is rapid, occurring after 4 h of infection, with no significant changes of Hamp level at that time.

    “Such negative regulation of Fpn was rapid occurring after 4 h of infection with no significant changes of Hamp level at that time.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Iron is strongly detected in Hamp−/− liver (around 2,000 μg/g) and decreases significantly by more than 50% after infection.

    “On the other hand, iron was strongly detected in Hamp−/− liver (around 2,000 µg/g) and decreased significantly by more than 50% after infection (Figure 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During Salmonella infection in Hamp−/− mice, hematocrit significantly decreases to 50% but is accompanied by a significant increase (around two times more) of plasma iron.

    “During Salmonella infection in Hamp−/− mice, the hematocrit significantly decreased to 50% but was accompanied with a significant increase (around two times more) of plasma iron (Figures 3A,B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FPN protein is strongly expressed in AcB61 tissues and localized at the cell surface of hepatic and splenic AcB61 macrophages that contain numerous engulfed RBC.

    “FPN protein was also strongly expressed in AcB61 tissues (Figure S1E in Supplementary Material) and was localized at the cell surface of both hepatic (Figure S1F in Supplementary Material) and splenic (Figure S1G in Supplementary Material) AcB61 macrophages, presenting numerous engulfed RBC.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Compared to A/J, AcB61 mice present hypoferremia and hyperferritinemia; with Salmonella infection, plasma iron and ferritin levels increase significantly in AcB61.

    “Compared to A/J, AcB61 mice presented hypoferremia (Figure 1B) and hyperferritinemia (Figure 1C). With Salmonella infection, both plasma iron and ferritin levels increase significantly in AcB61.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Concomitant with the decrease of Fpn, Nos2 mRNA expression increases with ST infection in both WT and Hamp−/− liver.

    “However, concomitant with the decrease of Fpn, an increase in the mRNA expression of Nos2 gene was observed with ST infection in both WT and Hamp−/− liver (Figure 4B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Perls staining of A/J spleen suggests a slight decrease in iron after infection.

    “On the other hand, the Perls staining of A/J spleen suggested a slight decrease in iron after infection (Figure 1G).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In WT liver, iron is present in Kupffer cells and tends to decrease after Salmonella infection.

    “A careful microscopy analysis of Perls staining confirmed the presence of iron (deep blue staining) in WT Kupffer cells, which tend to decrease (light blue staining) after Salmonella infection (Figure 3I).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In AcB61 spleen, iron level is significantly higher than in A/J and tends to slightly increase with infection, with marked iron accumulation in enlarged splenic macrophages.

    “In the AcB61 spleen, the iron level was significantly higher than the one detected in A/J and tended to slightly increase with infection (Figure 1H) with a marked iron accumulation in enlarged splenic macrophages (Figure 1G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    AcB61 mice show constitutive anemia at day 0 with lower hematocrit (35% versus 50% in A/J) that worsens during Salmonella infection (~20% in AcB61).

    “Consistent with previous reports (22), AcB61 mice showed a constitutive anemia at D0 with a lower hematocrit (35% in AcB61 versus 50% for A/J) that worsens during Salmonella infection (~20% in AcB61; Figure 1A).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [22] Roy, 2007, Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice, J Exp Med source

      Not checked in the free run.

  • May combine two claims.

    Findingreasoning

    As a consequence of enhanced EP activity in AcB61 macrophages, strong heme recycling is observed with increased bilirubinemia and enhanced expression of both HMOX1 and FPN.

    “As a consequence of enhanced EP activity in AcB61 macrophages, strong heme recycling is observed with increased bilirubinemia and enhanced expression of both HMOX1 and FPN.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Either no changes (AcB61 mice) or a decrease (WT mice) of hepatic Hamp expression is associated with anemia after ST infection, and even in Hamp−/− mice, a decreased hematocrit is still observed.

    “Indeed, either no changes (AcB61 mice) or a decrease (WT mice) of hepatic Hamp expression is associated with anemia after ST infection, and even in Hamp−/− mice, a decreased hematocrit is still observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We did not observe a decrease in Fpn mRNA expression in infected AcB61 mice despite the strong loss of FPN protein expression at the cell surface of AcB61 macrophages.

    “Of note, we did not observe a decrease in Fpn mRNA expression in infected AcB61 mice despite the strong loss of FPN protein expression at the cell surface of AcB61 macrophages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We observed extramedullary erythropoiesis and increase of Erfe mRNA levels (not shown) in the spleen of AcB61.

    “Indeed, we observed extramedullary erythropoiesis and increase of Erfe mRNA levels (not shown) in the spleen of AcB61.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In WT mice, we also observed a concomitant decrease in both FPN expression and plasma iron with a decrease in Hamp expression.

    “In WT mice, we also observed a concomitant decrease in both FPN expression and plasma iron with a decrease in Hamp expression.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The exacerbated susceptibility of AcB61 mice to Salmonella infection is not the consequence of a blunted immune response or a defect in the expression of the iron-siderophore binding protein lipocalin 2.

    “Other observations (not shown) indicate that the exacerbated susceptibility of AcB61 mice to Salmonella infection is not the consequence of a blunted immune response or a defect in the expression of the iron-siderophore binding protein lipocalin 2 and therefore likely reflects the accessibility to intracellular iron by the bacteria.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In our ST-infected BMDM as well as in the liver of infected mice, the FPN gene repression occurred with a concomitant increase of the NOS2 expression.

    “However, in our ST-infected BMDM as well as in the liver of infected mice, the FPN gene repression occurred with a concomitant increase of the NOS2 expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Such a decrease in tissues iron was strongly observed in Hamp−/− liver but was not seen in AcB61 tissues.

    “Such a decrease in tissues iron was strongly observed in Hamp−/− liver but was not seen in AcB61 tissues, likely masked by the exacerbated EP activity and heme iron uptake by macrophages in these tissues.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Iron sequestration in macrophages is a hallmark of anemia of inflammation and an efficient mechanism to deplete serum iron to limit growth of extracellular pathogens.

    “Iron sequestration in macrophages is a described hallmark of the AI and is an efficient mechanism to quickly deplete iron in the serum to limit the growth of extracellular pathogens (5).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Ganz, 2009, Iron sequestration and anemia of inflammation, Semin Hematol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hypoferremia contributes to the establishment of anemia of inflammation.

    “However, for the host, hypoferremia also contributes to the establishment of the so-called anemia of inflammation (AI) (4), an anemia difficult to treat and that can add substantially to the morbidity of the underlying infection.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Nemeth, 2014, Anemia of inflammation, Hematol Oncol Clin North Am source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The decrease in liver iron content and its redistribution during infection suggest that Salmonella alter mechanisms of iron storage or export.

    “The decrease in liver iron content and its redistribution during infection suggest that Salmonella alter mechanisms of iron storage or export.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Hamp−/− mice develop a specific iron phenotype with high serum iron concentration, excess iron deposition in hepatocytes, and low iron levels in tissue macrophages.

    “Hamp−/− mice have been shown to develop a specific iron phenotype with high serum iron concentration, excess iron deposition in hepatocytes, and low iron levels in tissue macrophages (14).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Lesbordes-Brion, 2006, Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis, Blood source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Nutritional immunity relies on the sequestration of essential molecules such as iron, preventing the growth of pathogens.

    “An important host defense strategy against infection, known as “nutritional immunity,” relies on the sequestration of essential molecules, such as iron, preventing the growth of pathogens (2).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Hood, 2012, Nutritional immunity: transition metals at the pathogen-host interface, Nat Rev Microbiol source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    In response to infection, patients commonly develop hypoferremia, a host response to limit iron availability to invading pathogens.

    “In response to infection, patients commonly develop hypoferremia (i.e., a decrease of iron in the circulation), a host response to limit iron availability to invading pathogens (3).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Cassat, 2013, Iron in infection and immunity, Cell Host Microbe source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In AcB61 mice, the presence of iron in macrophages due to extravascular hemolysis and strong erythrophagocytosis favors the proliferation of Salmonella in the spleen and liver with a concomitant decrease of FPN protein expression.

    “The presence of iron in AcB61 macrophages due to extravascular hemolysis and strong erythrophagocytosis activity favored the proliferation of Salmonella in the spleen and liver with a concomitant decrease of FPN protein expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The important iron storage and iron fluxes in AcB61 macrophages likely represent an advantage for the growth of Salmonella and contribute to the high susceptibility of AcB61.

    “The important iron storage and iron fluxes in AcB61 macrophages likely represent an advantage for the growth of Salmonella and contribute to the high susceptibility of AcB61.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Iron regulatory proteins (IRPs) have been shown to play a role during Salmonella infection.

    “Recently, iron regulatory proteins (IRPs) have been shown to play a role during Salmonella infection (39).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Nairz, 2015, Iron regulatory proteins mediate host resistance to Salmonella infection, Cell Host Microbe source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    FPN is quickly downregulated through endocytosis and degradation upon interaction with HAMP.

    “FPN is quickly downregulated through endocytosis and degradation upon interaction with HAMP (6, 8).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Delaby, 2005, Presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin, Blood source

      Not checked in the free run.

    • cites [8] Nemeth, 2004, Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization, Science source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Both heme and iron positively regulate macrophage FPN at both transcriptional and posttranscriptional levels.

    “Heme is known to be a potent inducer of HMOX1 transcription (25), and both heme and iron positively regulate macrophage FPN at both transcriptional and posttranscriptional levels (23, 26).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [23] Delaby, 2008, Sequential regulation of ferroportin expression after erythrophagocytosis in murine macrophages: early mRNA induction by haem, followed by iron-dependent protein expression, Biochem J source

      Not checked in the free run.

    • cites [26] Marro, 2010, Heme controls ferroportin1 (FPN1) transcription involving Bach1, Nrf2 and a MARE/ARE sequence motif at position -7007 of the FPN1 promoter, Haematologica source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In AcB61 mice challenged with Listeria monocytogenes, different strains of Mycobacterium bovis, and Legionella pneumophila, the Pklr mutation did not contribute to the clinical phenotype.

    “For all these models of infection, the Pklr mutation in AcB61 did not contribute to the clinical phenotype, and no further studies focusing on iron metabolism were performed.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [22] Roy, 2007, Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice, J Exp Med source

      Not checked in the free run.

    • cites [27] Di Pietrantonio, 2010, Strain-specific differences in the genetic control of two closely related mycobacteria, PLoS Pathog source

      Not checked in the free run.

    • cites [13] Fortin, 2001, Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits, Genomics source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Iron is an indispensable metal for the spread of virtually all human pathogens.

    “Iron is an indispensable metal for the spread of virtually all human pathogens (3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Cassat, 2013, Iron in infection and immunity, Cell Host Microbe source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    HAMP is produced mainly by hepatocytes in case of inflammation and also by infected macrophages.

    “HAMP is produced mainly by hepatocytes in case of inflammation and also by infected macrophages (9).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Peyssonnaux, 2006, TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens, Blood source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Reduction of spleen Fpn mRNA level by TLR4 agonist was shown to be HAMP independent.

    “Similarly, reduction of spleen Fpn mRNA level by TLR4 agonist was shown to be HAMP independent (38).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Deschemin, 2013, Role of hepcidin in the setting of hypoferremia during acute inflammation, PLoS One source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    AcB61 mice harbor a mutation in the gene Pklr leading to PK deficiency and resulting in chronic hemolytic anemia and tissue iron overload.

    “AcB61 mice harbor a mutation in the gene Pklr leading to PK deficiency and resulting in chronic hemolytic anemia and tissue iron overload (20–22).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [20] Min-Oo, 2003, Pyruvate kinase deficiency in mice protects against malaria, Nat Genet source

      Not checked in the free run.

    • cites [21] Min-Oo, 2004, Phenotypic expression of pyruvate kinase deficiency and protection against malaria in a mouse model, Genes Immun source

      Not checked in the free run.

    • cites [22] Roy, 2007, Pyruvate kinase deficiency confers susceptibility to Salmonella typhimurium infection in mice, J Exp Med source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    HAMP and FPN play key roles in decreasing systemic iron level by promoting macrophage iron sequestration during infection.

    “Two molecules, namely hepcidin (HAMP) and ferroportin (FPN), have been identified to play key roles in decreasing systemic iron level by promoting macrophage iron sequestration during infection (5).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Ganz, 2009, Iron sequestration and anemia of inflammation, Semin Hematol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Salmonella infection has been shown to initiate extramedullary erythropoiesis and splenomegaly with increases in RBC precursors and EPO production.

    “In addition, Salmonella infection has been shown to initiate extramedullary erythropoiesis and splenomegaly with increases in RBC precursors and EPO production (36).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Jackson, 2010, Innate immune activation during Salmonella infection initiates extramedullary erythropoiesis and splenomegaly, J Immunol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Iron deficiency, bone marrow suppression, and hemolysis are described to participate in the establishment of anemia in infectious diseases.

    “Iron deficiency, bone marrow suppression, and hemolysis are described to participate in the establishment of anemia in infectious diseases (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Nemeth, 2014, Anemia of inflammation, Hematol Oncol Clin North Am source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a model of the AI using a heat-killed Gram-negative bacteria, Brucella abortus, Hamp was shown to be upregulated in an early phase associated with erythropoietic suppression but was downregulated in a second later phase in parallel with an increase in EPO and erythropoiesis.

    “Interestingly, in a model of the AI using a heat-killed Gram-negative bacteria, Brucella abortus, Hamp was shown to be upregulated in an early phase associated with erythropoietic suppression but was downregulated in a second later phase in parallel with an increase in EPO and erythropoiesis (34, 35).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [34] Gardenghi, 2014, Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus, Blood source

      Not checked in the free run.

    • cites [35] Kim, 2014, A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin, Blood source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    FPN is the only known mammalian iron exporter and is expressed at the cell surface of macrophages.

    “FPN is the only known mammalian iron exporter and is expressed at the cell surface of macrophages (6, 7).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Delaby, 2005, Presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin, Blood source

      Not checked in the free run.

    • cites [7] Canonne-Hergaux, 2006, Comparative studies of duodenal and macrophage ferroportin proteins, Am J Physiol Gastrointest Liver Physiol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Differences observed between studies in the regulation of Hamp during Salmonella infection may rely on differences in the degree of compensatory erythropoiesis at the infection time.

    “These differences observed between studies in the regulation of Hamp during Salmonella infection may rely on differences in the degree of compensatory erythropoiesis at the infection time (negative regulation of Hamp via ERFE).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Heme is known to be a potent inducer of HMOX1 transcription.

    “Heme is known to be a potent inducer of HMOX1 transcription (25), and both heme and iron positively regulate macrophage FPN at both transcriptional and posttranscriptional levels (23, 26).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Sun, 2004, Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In vivo, the expression of Hamp in the hepatocyte is governed by stimulatory (iron overload and inflammation) and inhibitory factors (erythropoietic ERFE and hypoxia), the net effect of these factors defining the Hamp level in the organism.

    “In vivo, the expression of Hamp in the hepatocyte is governed by stimulatory (iron overload and inflammation) and inhibitory factors (erythropoietic ERFE and hypoxia), the net effect of these factors defining the Hamp level in the organism (31).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Rishi, 2015, Hepcidin: regulation of the master iron regulator, Biosci Rep source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Both iron overload and LPS/inflammation have been shown to induce apoptosis in hepatocytes.

    “Indeed both iron overload and LPS/inflammation have been shown to induce apoptosis in hepatocytes (47, 48).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [47] Zhao, 1997, Hepatocyte apoptosis in hepatic iron overload diseases, Histol Histopathol

      Not checked in the free run.

    • cites [48] Kudo, 2009, Lipopolysaccharide triggered TNF-alpha-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model, J Hepatol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Degradation of FPN resulted in increased macrophage bacterial growth in Salmonella-infected J774 macrophages.

    “This is consistent with in vitro studies showing that degradation of FPN resulted in increased macrophage bacterial growth in Salmonella-infected J774 macrophages (46).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Chlosta, 2006, The iron efflux protein ferroportin regulates the intracellular growth of Salmonella enterica, Infect Immun source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Downregulation of Fpn expression in macrophages was also reported with TLR2/6.

    “Moreover, downregulation of Fpn expression in macrophages was also reported with TLR2/6 (37, 45) expanding the FPN response to various pathogen-associated molecules.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Guida, 2015, A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia, Blood source

      Not checked in the free run.

    • cites [45] Ben-Othman, 2014, Leishmania-mediated inhibition of iron export promotes parasite replication in macrophages, PLoS Pathog source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Increases in FPN mRNA and protein expression in mouse macrophages cell lines RAW264.7 or thioglycollate-elicited peritoneal macrophages have been reported during Salmonella infection.

    “Increases in FPN mRNA and protein expression in mouse macrophages cell lines RAW264.7 or thioglycollate-elicited peritoneal macrophages have been reported during Salmonella infection (10).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Nairz, 2013, Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection, J Exp Med source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Premisecitation

    Other authors proposed that during infection with macrophage-tropic intracellular pathogens, macrophages respond by an upregulation of FPN to limit intracellular iron content.

    “In opposition to this cellular scenario, other authors proposed that during infection with macrophage-tropic intracellular pathogens, macrophages respond by an upregulation of FPN to limit intracellular iron content (10, 12).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] Nairz, 2013, Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection, J Exp Med source

      Not checked in the free run.

    • cites [12] Brown, 2015, Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by Salmonella enterica serovar Typhimurium infection in mice, Infect Immun source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The same authors have proposed that upregulation of FPN during Salmonella infection involves NO production by NOS2.

    “The same authors have proposed that upregulation of FPN during Salmonella infection involves NO production by NOS2 (10, 12).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] Nairz, 2013, Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection, J Exp Med source

      Not checked in the free run.

    • cites [12] Brown, 2015, Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by Salmonella enterica serovar Typhimurium infection in mice, Infect Immun source

      Not checked in the free run.

59 of 59

  • The paper relies on this without support.

    Iron is essential for both the host and the invading microbes and plays a critical role in host–pathogen interactions.

    “Iron is essential for both the host and the invading microbes and plays a critical role in host–pathogen interactions.”

  • The paper relies on this without support.

    Macrophages are a common niche for the replication of numerous intracellular pathogens including Salmonella.

    “macrophages are a common niche for the replication of numerous intracellular pathogens including Salmonella.”

  • The paper relies on this without support.

    Salmonella is a facultative intracellular bacterium.

    “despite the fact that Salmonella is a facultative intracellular bacterium”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Although there is evidence for this effect in cultured macrophages, mouse studies have not supported enhancement of intracellular infection by high hepcidin concentrations.

    “Although there is evidence for this effect in cultured macrophages [50], studies in mice have not supported the enhancement of intracellular infection by high concentrations of hepcidin [38,51].”

    From Iron and infection (2018) · cites this paper as [51]

  • During Salmonella infection, ferroportin expression is inhibited without hepcidin.

    “Although hepcidin-mediated down-regulation of ferroportin is probably the major mechanism leading to hypoferremia in response to infection (19), ferroportin expression is inhibited independently of hepcidin in the context of Salmonella infection (68).”

    From Innate Nutritional Immunity (2018) · cites this paper as [68]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 2D is identified as both liver and spleen

    Minor

    The Figure 2 caption describes panels C and D as images of liver, but the results describe panel D as showing splenic macrophages.

1 of 1

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Related

Authors
Willemetz, A., Beatty, S., Richer, E., Rubio, A., Auriac, A., Milkereit, R. J., Thibaudeau, O., Vaulont, S., Malo, D., & Canonne-Hergaux, F.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • McGill University (CA)
  • Université Toulouse III - Paul Sabatier (FR)
  • Hôpital Bichat-Claude-Bernard (FR)
  • Inserm (FR)
ORCID
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2017-05-01
Volume
8
Pages
498-498
ISSN
1664-3224 (ISSN-L)
DOI
10.3389/fimmu.2017.00498
PMC
PMC5410627
OpenAlex
W2611181829
Semantic Scholar
dee536df184f450c27e6b0f6af67da579908b7fd
Cited by
45
References
50
Citations by year
2026: 4 · 2025: 4 · 2024: 2 · 2023: 6 · 2022: 5 · 2021: 5 · 2020: 5 · 2019: 4 · 2018: 8 · 2017: 2
FWCI
4.00
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Immunology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Canadian Institutes of Health Research (MOP-15461)
  • Agence Nationale de la Recherche (ANR-10-MIDI-004)
Topics
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Ferroptosis and cancer prognosis
  • Health Sciences › Nursing › Nutrition and Dietetics › Trace Elements in Health
Keywords
ferroportin, hepcidin, iron homeostasis, macrophages, Salmonella typhimurium, iron export, anemia of chronic disease, iron overload, erythrophagocytosis, iron stores, intracellular bacterial growth

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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