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International audience; The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a β(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike β-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Divalent metal transporter 1 (DMT1) is essential for dietary iron assimilation and erythroid iron acquisition. The 3’ untranslated region of the murine DMT1 mRNA contains an iron responsive element (IRE) that is conserved in humans but whose functional role remains unclear. We generated and analyzed mice with targeted disruption of the DMT1 3’IRE. These animals display hypoferremia during the suckling period, associated with a reduction of DMT1 mRNA and protein in the intestine. In contrast, adult mice exhibit hyperferremia, accompanied by enlargement of hepatic and splenic iron stores. Intriguingly, disruption of the DMT1 3’IRE in adult animals augments intestinal DMT1 expression, in part due to increased mRNA translation. Hence, during postnatal growth, the DMT1 3’IRE promotes intestinal DMT1 expression and secures iron sufficiency; in adulthood, it suppresses DMT1 and prevents systemic iron loading. This work demonstrates that the 3’IRE of DMT1 plays a role in the control of DMT1 expression and systemic iron homeostasis, and reveals an age-dependent switch in its activity.Key pointsTargeted mutagenesis of the 3’IRE of DMT1 in mice reveals its importance for maintenance of systemic iron homeostasis.The 3’IRE stimulates intestinal DMT1 expression and prevents hypoferremia during early life, but exerts opposite effects in adulthood

HIV‐1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV‐1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac‐infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV‐1 latency. We found that PML NBs are hyper‐SUMOylated and that PML protein is degraded via the ubiquitin–proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV‐1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment., Activation of cellular oxidative stress responses and iron import upon HIV‐1 infection triggers latency establishment via remodeling of PML nuclear bodies.

SummaryMetabolic alterations, such as oxidative stress, are hallmarks of HIV-1 infection. However, their influence on the development of viral latency, and thus on HIV-1 persistence during antiretroviral therapy (ART), have just begun to be explored. We analyzed omics profiles of in-vitro and in-vivo models of infection by HIV-1 and its simian homolog SIVmac. We found that cells survive retroviral replication by upregulating antioxidant pathways and intertwined iron import pathways. These changes are associated with remodeling of the redox sensitive promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML is depleted in productively infected cells and restored by ART. Moreover, we identified intracellular iron as a key link between oxidative stress and PML depletion, thus supporting iron metabolism modulators as pharmacological tools to impair latency establishment.

Summary Background How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. Methods We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. Findings We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. Conclusions Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. Funding Medical Research Council, UK, Graphical Abstract, Highlights Low serum iron caused by hepcidin impairs primary and memory immune responses Activated T-cells demand iron and iron scarcity inhibits mitochondrial metabolism Patients with mutant TMPRSS6 have high hepcidin and lower IgG against pathogens High hepcidin during viral infection inhibits T- and B-cells and inflames disease, Context and Significance Iron deficiency is very common in humans and animals. Frost et al demonstrate that low concentrations of iron in serum, caused by the hormone hepcidin, inhibit the body’s response to vaccines and infections; conversely, increasing iron can boost immunity., Iron deficiency is very common in humans and animals. Frost et al. demonstrate that low concentrations of iron in serum, caused by the hormone hepcidin, inhibit the body’s response to vaccines and infections; conversely, increasing iron can boost immunity.

25 mg of iron are required every day to sustain hematopoiesis in the bone marrow. Most iron is consumed by erythroid cells that take up transferrin-bound iron to satisfy their demand for hemoglobin biogenesis. Unexpectedly, erythroblasts express high levels of the iron exporter Ferroportin (Fpn). Fpn surface expression and activity is controlled by Hepcidin, a small liver peptide hormone, produced in response to elevated systemic and tissue iron availability. Resistance of ferroportin to hepcidin binding is caused by a gain of function mutation in the FpnC326S residue, which is the reason for iron overload in patients with Hereditary Hemochromatosis type 4. Some years ago we generated the corresponding mouse model hallmarked by this constitutively active iron exporter. We now applied bone marrow transplantation to investigate the role of the hepcidin/ferroportin regulatory system in cell types of the bone marrow (BM-FpnC326S). CD45.1 host mice were irradiated twice with 500 cGy 4h apart and injected with 2 million of BM cells obtained from C57BL/6N mice (control) or from C57BL/6N congenic constitutive FpnC326S animals. Mice were analyzed three months after transplantation, whereby only animals with an engraftment higher than 95% were included for further analyses. Analysis of the erythroid hematological parameters revealed mild macrocytosis in the presence of unaltered red blood cell count (RBC), hematocrit (HCT), hemoglobin (Hb) and mean corpuscular hemoglobin (MCH) values. Serum iron content, transferrin saturation and serum hepcidin levels remained unchanged, despite a strong decrease in splenic iron levels within the red pulp, where reticuloendothelial macrophages are located. The latter phenotype is consistent with high ferroportin surface expression in macrophages derived from FpnC326S monocytes that causes increased iron export and cellular iron deficiency. Cytokine production (IL1β, TNFα and IL6) in the spleen was unchanged suggesting that iron deficiency in splenic macrophages did not cause inflammation. In contrast to the spleen iron content was not changed in other organs analyzed, including the liver, suggesting that de novo monocyte infiltration is not a major feature in these organs. Interestingly, histological analysis of the femurs revealed a marked decrease in bone marrow iron content; we were unable to detect a single iron-stained cell in the bone marrow of BM-FpnC326S mice, contrasting results from control mice. To investigate whether iron deficiency in bone marrow cells alters numbers of immune cells, we performed a detailed characterization by FACS. Unexpectedly, iron deficiency in the bone marrow did not cause changes in total bone marrow cellularity, total numbers of Ter119+ erythroid cells and the percentage of different erythroblast subpopulations, the number of hematopoietic stem cells (HSC) and of common lymphoid (CLP), myeloid (CMP) and megakaryocyte/erythroid (MEP) progenitors. Taken together, our data clearly demonstrate that at steady state, the hepcidin/ferroportin regulatory circuitry in the bone marrow is dispensable and that iron deficiency in bone marrow cells is not altering the normal hematopoietic process. Future studies will have to extend the analysis to stress conditions. Disclosures Muckenthaler: Novartis: Research Funding; Silence Therapeutics: Consultancy.

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

SummaryMitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron regulatory protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool. Surprisingly, our data indicate that IRP1 activation sustains mitochondrial iron supply and function rather than driving detrimental iron overload. Mitochondrial iron accumulation is shown to depend on mitochondrial dysfunction and heme-dependent upregulation of the mitochondrial iron importer mitoferrin-2. Our results uncover an unexpected protective role of IRP1 in pathological conditions associated with altered Fe-S metabolism.

Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis-regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 (Pfn2) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 39 untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice., This work was supported by grant SAF2015-70412-R from the Spanish Secretary of Research, Development, and Innovation (Ministerio de Economía, Industria y Competitividad [MINECO]) and grant DJCLS R14/04 from Deutsche José Carreras Leukämie Stiftung, grant 2014 SGR225 (Grups de Recerca Emergent, from Generalitat de Catalunya) from Generalitat de Catalunya, and financial support from Fundació Internacional Josep Carreras and from Obra Social “la Caixa” Spain (M. Sanchez). All work on the Pfn2 KO mouse model was supported by the Deutsche Forschungsgemeinschaft grant SFB1089 and SPP1464 (W.W.). S.L. was supported by the EMBO Short Term Fellowship ASTF 301-2013 for her work on the Pfn2−/− mice at the Institute of Genetics, University of Bonn, Bonn, Germany.

SummaryThe regulatory axis between the iron hormone hepcidin and its receptor, the iron exporter ferroportin (FPN), is central to iron homeostasis. Mutations preventing hepcidin-mediated degradation of FPN cause systemic iron overload. We have introduced a point mutation (C326S) into the murine Fpn locus, resembling human hereditary hemochromatosis type 4, including elevated plasma iron and ferritin levels, high transferrin saturation, hepatic iron overload, and iron depletion of duodenal enterocytes and reticuloendothelial macrophages. Unlike other mouse models of iron overload, homozygous C326S mice die between 7 and 14 months of age. Pancreatic acinar cells display marked iron accumulation, oxidative damage and degeneration, associated with failure of the exocrine pancreas and severe body weight loss. Rescue experiments reveal iron overload and exocrine pancreatic failure as leading causes of death. This work uncovers the critical importance of the hepcidin-ferroportin regulatory axis for life and unveils the sensitivity of the exocrine pancreas to iron overload.

SummaryMammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated “mucosal block.” We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin “mucosal block” and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.

Red blood cell production is a finely tuned process that requires coordinated oxygen- and iron-dependent regulation of cell differentiation and iron metabolism. Here, we show that translational regulation of hypoxia-inducible factor 2α (HIF-2α) synthesis by iron regulatory protein 1 (IRP1) is critical for controlling erythrocyte number. IRP1-null (Irp1(-/-)) mice display a marked transient polycythemia. HIF-2α messenger RNA (mRNA) is derepressed in kidneys of Irp1(-/-) mice but not in kidneys of Irp2(-/-) mice, leading to increased renal erythropoietin (Epo) mRNA and inappropriately elevated serum Epo levels. Expression of the iron transport genes DCytb, Dmt1, and ferroportin, as well as other HIF-2α targets, is enhanced in Irp1(-/-) duodenum. Analysis of mRNA translation state in the liver revealed IRP1-dependent dysregulation of HIF-2α mRNA translation, whereas IRP2 deficiency derepressed translation of all other known 5' iron response element (IRE)-containing mRNAs expressed in the liver. These results uncover separable physiological roles of each IRP and identify IRP1 as a therapeutic target for manipulating HIF-2α action in hematologic, oncologic, and other disorders.

The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway.

Disruptions in iron homeostasis from both iron deficiency and overload account for some of the most common human diseases. Iron metabolism is balanced by two regulatory systems, one that functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and another that predominantly controls cellular iron metabolism through iron-regulatory proteins that bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct systems function and how they “tango” together in a coordinated manner. We also highlight some of the current questions in mammalian iron metabolism and discuss therapeutic opportunities arising from a better understanding of the underlying biological principles.

The maintenance of iron homeostasis is critical as both iron deficiency and iron excess are deleterious. In mammals, iron homeostasis is regulated systemically by the iron-hormone hepcidin, an acute-phase protein secreted by the liver which inhibits iron absorption and recycling. Cellularly, the interaction of iron regulatory proteins (IRP) 1 and 2 with iron-responsive elements controls the expression of target mRNAs encoding proteins of iron acquisition, storage, utilization, and export. These processes critically affect iron levels, which in turn impact on numerous aspects of inflammation. To explore the role of IRP1 and IRP2 in inflammation, IRP-deficient mice, i.e., mice with total and constitutive deficiency of either IRP, were subjected to acute aseptic local inflammation. Turpentine oil injection increases the expression of acute phase proteins in the liver and interleukin 6 levels in the serum of control mice. Both IRP-deficient mouse models mount the same responses, indicating that the treatment was efficient in all animals and that the acute phase response does not require expression of both IRPs. As expected, turpentine oil treatment enhances hepcidin mRNA expression in the liver of wild-type mice, associated with decreased serum iron levels. Importantly, Irp1 −/− and Irp2 −/− animals, respectively, display quantitatively similar hepcidin mRNA induction and the appropriate reduction of the serum iron values. Our data indicate that the response of Irp1 −/− and Irp2 −/− mice to acute local inflammation is largely preserved.

Because ferroportin (Fpn) is the only known mammalian cellular iron exporter, understanding its localization and regulation within the retina would shed light on the direction of retinal iron flux. The hormone hepcidin may regulate retinal Fpn, as it triggers Fpn degradation in the gut. Immunofluorescence was used to label Fpn in retinas of mice with 4 different genotypes (wild type; Fpn C326S, a hepcidin-resistant Fpn; hepcidin knockout; and ceruloplasmin/hephaestin double knockout). No significant difference in Fpn levels was observed in these retinas. Fpn localized to the abluminal side of the outer plexiform vascular endothelial cells, Müller glia cells, and the basolateral side of the retinal pigment epithelium. Adeno-associated virus (AAV)-hepcidin was injected into the eyes of hepcidin knockout mice, while AAV-lacZ was injected into the contralateral eyes as a control. AAV-hepcidin injected eyes had increased ferritin immunolabeling in retinal vascular endothelial cells. Fpn C326S mice had systemic iron overload compared to wild type and had the fastest retinal iron accumulation of any hereditary model studied to date. The results suggest that physiologic hepcidin levels are insufficient to alter Fpn levels within the retinal pigment epithelium and Müller cells, but may limit iron transport into the retina from vascular endothelial cells.—Theurl, M., Song, D., Clark, E., Sterling, J., Grieco, S., Altamura, S., Galy, B., Hentze, M., Muckenthaler, M. U., Dunaief, J. L. Mice with hepcidin-resistant ferroportin accumulate iron in the retina.

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1. (Blood. 2005;106: 2580-2589)

Iron is an essential nutrient but, when present in excess, may turn into a biohazard by catalyzing the generation of aggressive radicals. Bacteria and eukaryotes have developed elegant homeostatic mechanisms to satisfy their metabolic needs for iron and to prevent the toxicity of iron overload. In mammals, the expression of several proteins of iron metabolism is coordinately regulated at the posttranscriptional level by a mechanism which involves binding of iron-regulatory proteins IRP1 and IRP2 to mRNA “iron-responsive elements” (IREs) (reviewed in references 3, 13, and 29). In iron-starved cells, IRE-IRP interactions stabilize transferrin receptor (TfR) mRNA and inhibit translation of ferritin (H and L) mRNAs, thereby promoting cellular iron uptake and preventing iron sequestration. By contrast, IRE-binding activity decreases in iron-replete cells, resulting in opposite homeostatic responses. Under these conditions, the IRE-binding activity of IRP1 is inactivated upon assembly of a cubane iron-sulfur cluster (9), while IRP2 undergoes degradation by the proteasome (8, 16). The regulatory function of IRP1 and IRP2 in mammalian iron metabolism has been examined by gene-targeting experiments. IRP1−/− mice have been reported to lack any obvious phenotype (30), while IRP2−/− mice display aberrant iron homeostasis and accumulate iron in the intestinal mucosa and the central nervous system (23). Moreover, these mice develop a progressive neurodegenerative disorder, suggesting a prominent role of IRP2 in controlling neuronal iron metabolism. IRP1 and IRP2 share extensive sequence homology and belong to the family of iron-sulfur cluster isomerases (6). By analogy to mitochondrial aconitase, a well-characterized enzyme of the citric acid cycle and a member of this family, both IRP1 and IRP2 are projected to contain three compact domains, linked to a fourth domain by a flexible hinge region. However, IRP2 contains an insertion of 73 amino acids (aa), which is encoded by a unique exon, within its domain 1. This cysteine- and proline-rich sequence has been implicated in the mechanism for iron sensing by IRP2 and its iron-mediated degradation. In particular, removal of the 73 aa from IRP2 has been reported to yield a stable molecule (16). Conversely, insertion of this sequence into an equivalent position within domain 1 apparently rendered IRP1 sensitive to iron-dependent degradation (16). Further mapping of the 73-aa domain showed that mutation of the cysteines at positions 168, 174, and 178 to serines abolished the iron-dependent instability of the IRP1-IRP2 hybrid molecule (16). These data paved the way for the “cysteine oxidation model” to explain the iron-dependent degradation of IRP2 (15). According to this model, iron mediates the site-specific oxidation of C168, C174, and C178, which tags the protein for ubiquitination and degradation by the proteasome (15). In support of this model, recent experiments showed that iron can directly bind to the 73-aa domain in vitro and that this interaction yields oxidized cysteine species (19). It should also be noted that heme has earlier (7) and more recently (38) been proposed to serve as a signal for IRP2 degradation. To better understand the function of IRP2 as an iron sensor in vivo, we generated stably transfected cell lines expressing epitope-tagged wild-type or mutated IRP2. We provide evidence that neither the three cysteines C168, C174, and C178 nor the entire 73 aa domain is critical for the iron-dependent degradation of IRP2. Instead, we demonstrate that the machinery for IRP2 degradation can be saturated when the protein is expressed at high levels, which may help to reconcile previous data. We show that, unexpectedly, antioxidants can also drive IRP2 toward degradation, and we reveal that the iron-mediated degradation of IRP2 may involve the activity of 2-oxoglutarate-dependent oxygenase(s).

Paper presented at the European Iron Club, which was held in Verona (Italy) on 12-14th September 2014., [Objetive] The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis., [Materials and Methods] Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes., [Results] Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction., [Conclusions] Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 modulates iron homeostasis in cell lines and mice., Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Pathologies associated with increased circulating iron levels include primary iron overload disorders, such as hereditary hemochromatosis, as well as secondary iron overload disorders, such as thalassemia, sickle cell anemia and myelodysplastic syndromes. In the latter elevated circulating iron levels due to enhanced intestinal iron absorption in support of ineffective erythropoiesis are further increased by the need of chronic blood transfusions. Organ toxicity and increased patient mortality under these conditions are mainly attributed to iron overload and the occurrence of non-transferrin-bound iron (NTBI). So far, epidemiological data and studies in animal models have provided conflicting evidence regarding a role of excess iron in atherogenesis and thrombosis. Here we investigate the role of high circulating iron levels in the development of cardiovascular disease. A mouse model of type IV hereditary hemochromatosis, in which the hepcidin/ferroportin regulatory circuitry is disrupted due to a point mutation in the iron exporter ferroportin (Altamura et al., Cell Metabolism 2014), was interbred with ApoE-null mice, to study its susceptibility to atherosclerosis. As expected, this novel mouse model shows close to 100% transferrin saturation as well as NTBI. The mice were studied at 3, 6 and 12 months of age. We observed a mildly decreased lesion size and number at 3 months of age (6.15±1.25 vs 1.84±0.86 % aortic lesion area, P=0.02), while the same parameters were strongly increased in 6 to 12 month-old mice (6 months: 1.44±0.23 vs 5±0.53 % aortic lesion area, P=0.0001; 12 months: 10.24±1.21 vs 20.44±2.69 % aortic lesion area, P=0.0065). The atherosclerotic phenotype positively correlates with higher levels of circulating iron (12 months: 122.2±6.57 vs 337±19.22 mg iron/dl serum, P Our data suggest that in young mice (3 months) a less severe atherosclerotic phenotype may be explained by iron depletion of macrophages. With increasing age, high circulating iron levels strongly enhance the severity of the atherosclerotic phenotype, indicating that systemic iron overload is a risk factor for atherosclerosis progression and predisposes to cardiovascular disease. Our findings have potential implications for those pathological conditions with elevated systemic iron levels, ranging from patients with hemochromatosis to anemic patients dependent on chronic blood transfusions, as well as for individuals subjected to intravenous iron administration (e.g. patients undergoing hemodialysis). Disclosures No relevant conflicts of interest to declare.

International audience; In mammals, iron regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of genes involved in iron metabolism, including transferrin receptor 1, the ferritin (Ft) H and L subunits, and ferroportin by binding mRNA motifs called iron responsive elements (IREs). IRP1 is a bifunctional protein that mostly exists in a non-IRE-binding, [4Fe-4S] cluster aconitase form, whereas IRP2, which does not assemble an Fe-S cluster, spontaneously binds IREs. Although both IRPs fulfill a trans-regulatory function, only mice lacking IRP2 misregulate iron metabolism. NO stimulates the IRE-binding activity of IRP1 by targeting its Fe-S cluster. IRP2 has also been reported to sense NO, but the intrinsic function of IRP1 and IRP2 in NO-mediated regulation of cellular iron metabolism is controversial. In this study, we exposed bone marrow macrophages from Irp1(-/-) and Irp2(-/-) mice to NO and showed that the generated apo-IRP1 was entirely responsible for the posttranscriptional regulation of transferrin receptor 1, H-Ft, L-Ft, and ferroportin. The powerful action of NO on IRP1 also remedies the defects of iron storage found in IRP2-null bone marrow macrophages by efficiently reducing Ft overexpression. We also found that NO-dependent IRP1 activation, resulting in increased iron uptake and reduced iron sequestration and export, maintains enough intracellular iron to fuel the Fe-S cluster biosynthetic pathway for efficient restoration of the citric acid cycle aconitase in mitochondria. Thus, IRP1 is the dominant sensor and transducer of NO for posttranscriptional regulation of iron metabolism and participates in Fe-S cluster repair after exposure to NO.

The regulation and maintenance of systemic iron homeostasis is critical to human health. Iron overload and deficiency diseases belong to the most common nutrition-related pathologies across the globe. It is now well appreciated that the hormonal hepcidin/ferroportin system plays an important regulatory role for systemic iron metabolism. We review recent data that uncover the importance of the cellular iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network in systemic iron homeostasis. We also discuss how the IRE/IRP regulatory system communicates with the hepcidin/ferroportin system to connect the control networks for systemic and cellular iron balance.

RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To identify novel target mRNAs of RBPs, we incubate total RNA with recombinant RBP and immunoselect the messenger-ribonucleoproteins using a specific anti-RBP antibody. The mRNA composition of the supernatant and/or immunoprecipitated fraction is analyzed using dual-color microarrays in comparison with control reaction. From start to finish, the protocol takes approximately 6 d.

Mutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the “iron hormone” hepcidin which then controls intestinal iron absorption.

Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of a conserved, functional iron-responsive element (IRE) in the 5' untranslated region of the messenger RNA encoding endothelial PAS domain protein-1, EPAS1 (also called hypoxia-inducible factor-2alpha, HIF2alpha). Via this IRE, iron regulatory protein binding controls EPAS1 mRNA translation in response to cellular iron availability. Our results uncover a regulatory link that permits feedback control between iron availability and the expression of a key transcription factor promoting iron utilization. They also show that an IRE that is structurally distinct from, for example, the ferritin mRNA IRE and that has been missed by in silico approaches, can mediate mechanistically similar responses.

Iron aggravates the cardiotoxicity of doxorubicin (DOX), a widely used anticancer anthracycline. The amount of iron in the cell is regulated by the iron regulatory proteins (IRPs)-1 and -2 that control the posttranscriptional expression of key iron metabolism genes. In vitro and cell culture studies revealed the ability of DOX to modulate the activity of both IRPs. However, conflicting data were obtained from different cell types and experimental conditions. To investigate the connection between acute DOX cardiotoxicity and the IRPs in a mammalian organism, we analyzed IRP activity and the expression of IRP target genes in the heart of mice subjected to DOX treatment. DOX exposure elicits a differential modulation of the two IRPs with reduced IRP2 activity and unchanged IRP1 activity. IRP2 downmodulation is associated with the upregulation of the ferritin L and H genes and decreased expression of the transferrin receptor 1 (TfR1). To directly test the role of IRP1 in DOX cardiotoxicity, the DOX response was analyzed in mice lacking IRP1. DOX-mediated IRP2 downmodulation and regulation of ferritin and TfR1 expression is identical in Irp1 (-/-) mice compared to wild type, as is the degree of oxidative damage of the heart assessed by thioredoxin and thiobarbituric acid reactive substance levels and by brain natriuretic peptide mRNA expression. These data demonstrate that the alterations of cardiac iron homeostasis related to acute anthracycline cardiotoxicity occur independently of IRP1. The observed IRP2 downmodulation could serve as a means to counteract DOX cardiotoxicity by reducing the "free" cellular iron pool.

Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009). To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease. Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl’s Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted. Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms. Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61. Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443. Disclosures: No relevant conflicts of interest to declare.

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia. For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice. This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.

Erythroid cells are the major consumers of iron in the human body. Differentiating erythroid cells shuttle the metal with very high efficiency towards mitochondria for the formation of heme. To satisfy their high iron needs, developing red blood cells (RBC) have to sustain high expression of transferrin receptor 1 (TfR) despite increasing cellular iron concentration. Moreover, synthesis of ferritin must not be activated by incoming iron, since this would represent a counterproductive storage during the phase of high iron demand. Recently we have demonstrated that during terminal differentiation primary erythroid cells satisfy their exceptionally high requirements for iron by switching to a mode where the post-transcriptional, iron-dependent regulatory system, formed by iron responsive proteins (IRP1 and IRP2) and iron responsive elements (IREs), seems to sense a low-iron state. This occurs despite a massive net increase of iron import into the cell (Schranzhofer et al., Blood107:4159, 2006). To examine the hypothesis that erythroid cells have low non-heme iron levels in their cytosol, we experimentally increased the cytosolic iron pool by either inhibiting heme biosynthesis or overloading cells with iron. Both block of heme synthesis by either succinylacetone or isonicotinic acid hydrazide (INH) or administration of ferric ammonium citrate, resulted in a clear increase in ferritin levels. This increase was directly proportional to the increase in the cellular concentration of non-heme-iron. Moreover, the effect of INH, the inhibitor of 5-aminolevulinic acid (ALA) synthase, could be reversed by the addition of ALA. Strikingly, increases in ferritin expression upon perturbation of cellular iron homeostasis strongly correlated with the loss of IRE-binding activity of IRP2 but not IRP1, as determined by mobility shift assays. This suggests that IRP2 is the major regulator of ferritin expression in erythroid cells. To further elaborate on this observation, we cultured primary erythroblasts derived from IRP1−/− and IRP2−/− mice (kindly provided by Drs. M. Hentze and B. Galy). In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization. Moreover, only IRP2−/− cells showed a significant increase in ferritin expression, whereas developing RBC lacking IRP1 had levels of ferritin protein equal to wild type cells. We conclude that in erythroid cells efficient shuttling of incoming iron towards mitochondria and its prompt use for heme formation is important to keep the cytosol in an iron-deprived state and consequently ferritin protein levels low. This translational repression seems to be mainly achieved by IRP2. Together with the observation that surface expression of TfR was reduced in IRP2−/− erythroblasts during self renewal but not during terminal differentiation, our results suggest that not only down-regulation of TfR, but also up-regulation of ferritin may be a major factor for the anemic phenotype observed in IRP2−/− mice.

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder hallmarked by intestinal iron hyperabsorption, hyperferremia and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the ubiquitously expressed HFE/Hfe gene. To understand the molecular mechanism(s) underlying HH and to identify the cell type in which Hfe acts to prevent HH we generated mice with tissue-specific Hfe ablations. We demonstrate that hepatocyte-specific Hfe deficiency fully recapitulates the phenotype observed in Hfe−/− mice with severe liver iron accumulation, reduced splenic iron content, increased Tf saturation, hyperferremia and reduced hepcidin expression. These findings unambiguously and directly show that Hfe acts in hepatocytes to prevent hemochromatosis.

Hereditary haemochromatosis (HH) is a highly prevalent iron-overload disease that is mainly associated with recessive mutations in the gene encoding HFE, a MHC class I-like protein. Interestingly, non-HFE haemochromatosis has also been reported and two other loci have been recently identified. One maps to chromosome 1q and is responsible for a juvenile form of the disease. The other, mapped to 7q22, contains the gene encoding transferrin receptor 2; a C→G transversion in this gene introduces a nonsense mutation. In addition, the genes encoding ferroportin and ferritin H are now to be added to the list. Ferroportin is a basolateral iron transporter involved in the movement of iron across the intestinal enterocytes to the bloodstream. Linkage analysis in a large Dutch family with inherited iron overload led to the identification of a single A→C transversion at position 734 (Ref. 1xA mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Njajou, O.T. et al. Nat. Genet. 2001; 28: 213–214CrossRef | PubMed | Scopus (356)See all References1). This mutation substitutes an uncharged Asn residue at position 144, located within one of the predicted transmembrane domains of the protein, with a charged His residue. The expected reduction in the hydrophobicity of this domain could affect the folding of the molecule. Ferritin H is an iron-storage protein whose expression is essentially controlled at the translational level via the association of a protein trans-regulator with a cis-regulatory RNA element in the mRNA leader. A single mutation in this RNA structure has been found in a Japanese family 2xA mutation, in the iron-responsive element of H Ferritin mRNA, causing autosomal dominant iron overload. Kato, J. et al. Am. J. Hum. Genet. 2001; 69: 191–197Abstract | Full Text | Full Text PDF | PubMed | Scopus (141)See all References2. Strikingly, this mutation seems to increase the affinity of the RNA motif for the protein trans-regulator, resulting in reduced translation of the mutant mRNA.One interesting feature of these two families is that HH segregates as a dominant trait, raising questions about the mechanisms that underlie iron overload in these patients. Njajou and co-workers hypothesize that the ferroportin mutation could be an activating one, resulting in excessive transport of iron out of the enterocytes into the circulation. This would emphasize the importance of this basolateral transport for body iron homeostasis. Further work is needed to validate this interesting supposition. As regards ferritin H, this subunit bears the ferroxidase activity that is required for incorporation of iron into the ferritin H–L complex. It has consequently been proposed that reduction of ferritin H translation would impair the capacity of the cell to sequester iron. How this mutation can be a dominant trait requires clarification.Independent groups have shown that HFE knockout mice develop iron overload that resembles human haemochromatosis. Similarly, animal models with the ferroportin or ferritin H mutations will undoubtedly help our study of the causative role of these genetic alterations in non-HFE HH. These mutations might also be useful for the molecular diagnosis of non-HFE HH. However, additional family studies are required to establish their prevalence.

Summary Iron regulatory proteins (IRPs) orchestrate the posttranscriptional regulation of critical iron metabolism proteins at the cellular level. Redundancy between IRP1 and IRP2 associated with embryonic lethality of doubly IRP-deficient mice has precluded the study of IRP function in vivo. Here we use Cre/Lox technology to generate viable organisms lacking IRP expression in a single tissue, the intestine. Mice lacking intestinal IRP expression develop intestinal malabsorption and dehydration postnatally and die within 4 weeks of birth. We demonstrate that IRPs control the expression of divalent metal transporter 1 (DMT1) mRNA and protein, a limiting intestinal iron importer. IRPs are also shown to be critically important to secure physiological levels of the basolateral iron exporter ferroportin. IRPs are thus essential for intestinal function and organismal survival and coordinate the synthesis of key iron metabolism proteins in the duodenum.