Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection.

Nitric oxide (NO) generated by inducible NO synthase 2 (NOS2) affects cellular iron homeostasis, but the underlying molecular mechanisms and implications for NOS2-dependent pathogen control are incompletely understood. In this study, we found that NO up-regulated the expression of ferroportin-1 (Fpn1), the major cellular iron exporter, in mouse and human cells. Nos2(-/-) macrophages displayed increased iron content due to reduced Fpn1 expression and allowed for an enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium. Nos2 gene disruption or inhibition of NOS2 activity led to an accumulation of iron in the spleen and splenic macrophages. Lack of NO formation resulted in impaired nuclear factor erythroid 2-related factor-2 (Nrf2) expression, resulting in reduced Fpn1 transcription and diminished cellular iron egress. After infection of Nos2(-/-) macrophages or mice with S. typhimurium, the increased iron accumulation was paralleled by a reduced cytokine (TNF, IL-12, and IFN-γ) expression and impaired pathogen control, all of which were restored upon administration of the iron chelator deferasirox or hyperexpression of Fpn1 or Nrf2. Thus, the accumulation of iron in Nos2(-/-) macrophages counteracts a proinflammatory host immune response, and the protective effect of NO appears to partially result from its ability to prevent iron overload in macrophages.

Recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb.

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.

The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium.

In being both, a modifier of cellular immune effector pathways and an essential nutrient for microbes, iron is a critical determinant in host-pathogen interaction. Here, we investigated the metabolic changes of macrophage iron homeostasis and immune function following the infection of RAW264.7 murine macrophages with Salmonella typhimurium. We observed an enhanced expression of the principal iron export protein, ferroportin 1, and a subsequent increase of iron efflux in Salmonella-infected phagocytes. In parallel, the expression of haem oxygenase 1 and of the siderophore-binding peptide lipocalin 2 was markedly enhanced following pathogen entry. Collectively, these modulations reduced both the cytoplasmatic labile iron and the ferritin storage iron pool within macrophages, thus restricting the acquisition of iron by intramacrophage Salmonella. Correspondingly, limitation of macrophage iron decreased microbial survival, whereas iron supplementation impaired immune response pathways in Salmonella-infected macrophages (nitric oxide formation and tumour necrosis factor-alpha production) and promoted intracellular bacterial proliferation. Our findings suggest that the enhancement of ferroportin 1-mediated iron efflux, the upregulation of the haem-degrading enzyme haem oxygenase 1 and the induction of lipocalin 2 following infection concordantly aim at withholding iron from intracellular S. typhimurium and to increase antimicrobial immune effector pathways thus limiting pathogen proliferation.

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1.

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.

Short term protective effects of iron in a murine model of ischemia/reperfusion.

The role of iron in the pathogenesis of cardio-vascular disorders is still controversial. We studied the effects of iron perturbations on myocardial injury upon temporary ischemia/reperfusion. C57BL/6J male mice were injected with iron dextran for 2 weeks while controls received saline. Mice were then subjected to 30 min of myocardial ischemia and subsequent reperfusion for 6-24 h. Tissue damage was quantified histologically and by troponin T determination. The expressions of tumor necrosis factor-alpha (TNF-alpha), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were investigated in non-ischemic and ischemic regions of both groups. After myocardial ischemia and reperfusion, troponin T levels, as a marker of myocardial damage, were significantly reduced in iron-treated mice as compared to control mice (P < 0.05). Under the same conditions the infarction area and damage score were significantly lower in iron-treated animals. In parallel, TNF-alpha and SOD expressions were increased in infarcted regions of iron-treated mice as compared to controls, whereas myocardial iNOS expression was significantly lower in iron-treated mice. Although, iron challenge increased radical formation and TNF-alpha expression in vivo, this did not result in myocardial damage which may be linked to the parallel induction of SOD. Importantly, iron treatment inhibited iNOS expression. Since, an increased nitric oxide (NO) formation has been linked to cardiac damage after acute myocardial infarction, iron may exert short time cardio-protective effects after induction of ischemia/reperfusion via decreasing iNOS formation.

The kinase inhibitor imatinib mesylate inhibits TNF-{alpha} production in vitro and prevents TNF-dependent acute hepatic inflammation.

Imatinib exerts potent antileukemic effects in vitro and in vivo. Despite its well known antitumor activity, the potential of imatinib for the treatment of inflammatory diseases remains elusive so far. Our current report provides strong evidence that imatinib has potent antiinflammatory effects. It potently inhibits LPS- and Con A-induced TNF-alpha production by human myeloid cells in vitro (peripheral blood mononuclear cells, CD14-selected monocytes, and monocyte-derived macrophages). Of note, the production of the antiinflammatory cytokine IL-10 was not significantly regulated by imatinib. In line with this observation, phosphorylation of IkappaB and subsequent DNA binding of NF-kappaB, which is critically involved in TNF-alpha, but not IL-10 expression, was reduced by imatinib. Using several murine models of acute hepatitis, we could corroborate our in vitro findings, as imatinib prevented macrophage- and TNF-alpha-dependent inflammatory damage of the liver induced by injection of either Con A or d-galactosamine/LPS by inhibition of hepatic TNF-alpha production. Of note, d-galactosamine/TNF-induced hepatitis was not affected, showing that imatinib does not directly inhibit TNF-alpha-induced hepatocellular cell death. These findings suggest a potent antiinflammatory role of imatinib by modulation of TNF-alpha production in monocytes/macrophages. This observation might be of therapeutic value for the treatment of TNF-mediated diseases.

Pathways for the regulation of body iron homeostasis in response to experimental iron overload.

BACKGROUND/AIMS: Secondary iron overload is a frequent clinical condition found in association with multiple blood transfusions. METHODS: To gain insight into adaptive changes in the expression of iron genes in duodenum, liver and spleen upon experimental iron overload we studied C57BL/6 mice receiving repetitive daily injections of iron-dextran for up to 5 days. RESULTS: Iron initially accumulated in spleen macrophages but with subsequent increase in macrophage ferroportin and ferritin expression its content in the spleen decreased while a progressive storage of iron occurred within hepatocytes which was paralleled by a significant increase in hepcidin and hemojuvelin expression. Under these conditions, iron was still absorbed from the duodenal lumen as divalent metal transporter-1 expressions were high, however, most of the absorbed iron was incorporated into duodenal ferritin, while ferroportin expression drastically decreased and iron transfer to the circulation was reduced. CONCLUSIONS: Experimental iron overload results in iron accumulation in macrophages and later in hepatocytes. In parallel, the transfer of iron from the gut to the circulation is diminished which may be referred to interference of hepcidin with ferroportin mediated iron export, thus preventing body iron accumulation.

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis.

Although the recent identification of several genes has extended our knowledge on the maintenance of body iron homeostasis, their tissue specific expression patterns and the underlying regulatory networks are poorly understood. We studied C57black/Sv129 mice and HFE knockout (HFE -/-) variants thereof as a model for hemochromatosis, and investigated the expression of iron metabolism genes in the duodenum, liver, and kidney as a function of dietary iron challenge. In HFE +/+ mice dietary iron supplementation increased hepatic expression of hepcidin which was paralleled by decreased iron regulatory protein (IRP) activity, and reduced expression of divalent metal transporter-1 (DMT-1) and duodenal cytochrome b (Dcytb) in the enterocyte. In HFE -/- mice hepcidin formation was diminished upon iron challenge which was associated with decreased hepatic transferrin receptor (TfR)-2 levels. Accordingly, HFE -/- mice presented with high duodenal Dcytb and DMT-1 levels, and increased IRP and TfR expression, suggesting iron deficiency in the enterocyte and increased iron absorption. In parallel, HFE -/- resulted in reduced renal expression of Dcytb and DMT-1. Our data suggest that the feed back regulation of duodenal iron absorption by hepcidin is impaired in HFE -/- mice, a model for genetic hemochromatosis. This change may be linked to inappropriate iron sensing by the liver based on decreased TfR-2 expression, resulting in reduced circulating hepcidin levels and an inappropriate up-regulation of Dcytb and DMT-1 driven iron absorption. In addition, iron excretion/reabsorption by the kidneys may be altered, which may aggravate progressive iron overload.

Duodenal HFE expression and hepcidin levels determine body iron homeostasis: modulation by genetic diversity and dietary iron availability.

HFE affects the interaction of transferrin bound iron with transferrin receptors (TfR) thereby modulating iron uptake. To study genetically determined differences in HFE expression we examined individual HFE levels in C57BL/Sv129 mice and assessed their relationship to the regulation of iron homeostasis in the duodenum and the liver, and their regulation by diet. We found an up to 14-fold variation in inter-individual expression of HFE mRNA in the duodenum. Mice with high duodenal HFE mRNA expression presented with significantly higher levels of TfR and DMT-1 mRNAs and an increased IRP-1 binding affinity as compared to mice with low HFE levels. Duodenal HFE expression was positively associated with serum iron and liver HFE levels. Dietary iron supplementation decreased HFE in the duodenum but not in the liver. This was paralleled by reduced amounts of DMT-1 and FP-1 in the duodenum while the expression of DMT-1, FP-1, and hepcidin in the liver were increased with dietary iron overload. Duodenal and liver HFE levels are regulated by divergent penetration of as yet unelucidated modifier genes and to a much lesser extent by dietary iron. These measures control duodenal iron transport and liver iron homeostasis by modulating HFE expression either directly or via stimulation of iron sensitive regulatory molecules, such as hepcidin, which then exert their effects on body iron homeostasis.

Cytokine-mediated regulation of iron transport in human monocytic cells.

Under chronic inflammatory conditions cytokines induce a diversion of iron traffic, leading to hypoferremia and retention of the metal within the reticuloendothelial system. However, the regulatory pathways underlying these disturbances of iron homeostasis are poorly understood. We investigated transferrin receptor (TfR)-dependent and -independent iron transport mechanisms in cytokine-stimulated human monocytic cell lines THP-1 and U937. Combined treatment of cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) reduced TfR mRNA levels, surface expression, and iron uptake, and these effects were reversed by interleukin-10 (IL-10), thus stimulating TfR-mediated iron acquisition. IFN-gamma and LPS dose-dependently increased the cellular expression of divalent metal transporter-1, a transmembrane transporter of ferrous iron, and stimulated the uptake of nontransferrin bound iron (NTBI) into cells. At the same time, IFN-gamma and LPS down-regulated the expression of ferroportin mRNA, a putative iron exporter, and decreased iron release from monocytes. Preincubation with IL-10 partly counteracted these effects. Our results demonstrate that the proinflammatory stimuli IFN-gamma and LPS increase the uptake of NTBI via stimulation of divalent metal transporter-1 expression and cause retention of the metal within monocytes by down-regulating ferroportin synthesis. Opposite, the anti-inflammatory cytokine IL-10 stimulates TfR-mediated iron uptake into activated monocytes. The regulation of iron transport by cytokines is a key mechanism in the pathogenesis of anemia of chronic disease and a promising target for therapeutic intervention.