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Low plasma iron (hypoferremia) induced by hepcidin is a conserved inflammatory response that protects against infections but inhibits erythropoiesis. How hypoferremia influences leukocytogenesis is unclear. Using proteomic data, we predicted that neutrophil production would be profoundly more iron-demanding than generation of other white blood cell types. Accordingly in mice, hepcidin-mediated hypoferremia substantially reduced numbers of granulocytes but not monocytes, lymphocytes, or dendritic cells. Neutrophil rebound after anti-Gr-1–induced neutropenia was blunted during hypoferremia but was rescued by supplemental iron. Similarly, hypoferremia markedly inhibited pharmacologically stimulated granulopoiesis mediated by granulocyte colony-stimulating factor and inflammation-induced accumulation of neutrophils in the spleen and peritoneal cavity. Furthermore, hypoferremia specifically altered neutrophil effector functions, suppressing antibacterial mechanisms but enhancing mitochondrial reactive oxygen species–dependent NETosis associated with chronic inflammation. Notably, antagonizing endogenous hepcidin during acute inflammation enhanced production of neutrophils. We propose plasma iron modulates the profile of innate immunity by controlling monocyte-to-neutrophil ratio and neutrophil activity in a therapeutically targetable system.

Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. Yet, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory proteins (IRP)-1 and −2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.

SummarySystem-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo eRIC (enhanced RNA interactome capture) to characterize the poly(A)RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

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.

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of pro-senescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound anti-viral and anti-proliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, pro-senescent and pro-apoptotic agents in both normoxic and hypoxic environments. This article is protected by copyright. All rights reserved. published

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

Living organisms face the dual challenge of acquiring enough iron to perform biological functions while preventing toxic iron accretion. A study now shows that sensing of iron-catalysed free radicals by a druggable gene-regulatory pathway helps the body avoid iron poisoning.

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.

200 billion red blood cells (RBCs) are produced every day, requiring more than 2 × 3 1015 iron atoms every second to maintain adequate erythropoiesis. These numbers translate into 20 mL of blood being produced each day, containing 6 g of hemoglobin and 20 mg of iron. These impressive numbers illustrate why the making and breaking of RBCs is at the heart of iron physiology, providing an ideal context to discuss recent progress in understanding the systemic and cellular mechanisms that underlie the regulation of iron homeostasis and its disorders.

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.

SummaryMacrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

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.

Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1*) using Cre/Lox technology. Systemic activation of the IRP1* transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1* activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson’s disease or Friedreich’s ataxia. Electronic supplementary material The online version of this article (doi:10.1007/s00109-013-1008-2) contains supplementary material, which is available to authorized users.

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.

Aims Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

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.

SummaryMitochondria supply cells with ATP, heme, and iron sulfur clusters (ISC), and mitochondrial energy metabolism involves both heme- and ISC-dependent enzymes. Here, we show that mitochondrial iron supply and function require iron regulatory proteins (IRP), cytosolic RNA-binding proteins that control mRNA translation and stability. Mice lacking both IRP1 and IRP2 in their hepatocytes suffer from mitochondrial iron deficiency and dysfunction associated with alterations of the ISC and heme biosynthetic pathways, leading to liver failure and death. These results uncover a major role of the IRPs in cell biology: to ensure adequate iron supply to the mitochondrion for proper function of this critical organelle.

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.

SummaryHereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.

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.

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 proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5′- or 3′-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3′-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE- and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IRE-containing mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.

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)

Central aspects of cellular iron metabolism are controlled by IRP1 and IRP2, which are ubiquitously expressed in mouse organs and cells. Total and constitutive deficiency of both IRPs causes embryonic lethality in the mouse. To bypass the early lethality and to study organ-specific and/or temporal functions of IRP1 and/or IRP2 we generated Irp1 and Irp2 conditional alleles. We used mouse lines where a betaGeo gene trap construct was inserted into the second intron of the Irp1 and the Irp2 gene, generating hypomorphic alleles by interrupting the corresponding open reading frame near the amino-termini. The gene trap cassettes are flanked by Frt sites and were co-inserted with LoxP sites flanking exon 3. Flp-mediated removal of the gene trap construct generates floxed alleles with wildtype functions. For both Irp genes, Cre-assisted deletion of exon 3 generates complete null alleles that, in the case of IRP2, are associated with altered body iron distribution and compromised hematopoiesis. If not removed, the gene trap construct causes partially penetrant embryonic lethality unrelated to IRP deficiency when inserted within the Irp1 but not the Irp2 locus. We discuss the implications for functional genomics in the mouse.

The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.

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).

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

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).

Fibroblast growth factor-2 (FGF-2) is a powerful mitogen and angiogenic factor whose expression is strongly regulated at the translational level. The constitutive upregulation of FGF-2 isoforms in transformed cells prompted us to investigate the post-transcriptional effects of a tumour suppressor, p53, on FGF-2 expression. We show here in human primary skin fibroblasts that the cell density-dependent variation of FGF-2 mRNA translatability was inversely correlated with endogenous p53 expression. Transient cell transfection revealed an inhibitory effect of wild-type p53 on the expression of chimeric FGF--CAT proteins. RNAse mapping experiments ruled out any effect of p53 on FGF--CAT mRNA accumulation, suggesting a translational inhibition. This inhibition was mediated by the FGF-2 mRNA leader, but not by vascular endothelial growth factor or platelet derived growth factor mRNA leaders. Neither p53-like protein p73, nor p21/waf had any inhibitory activity. Furthermore a set of hot spot mutants of p53 bearing mutations in the DNA binding domain had no post-transcriptional inhibitory effect. In contrast a p53 mutant of the transactivating domain was still able to block FGF--CAT expression, indicating that the post-transcriptional activity of p53 described here was independent of the trans-activation of target genes. Such data reveal a novel mechanism by which p53 efficiently blocks the expression of a major proliferating, anti-apoptotic and angiogenic gene.

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.

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.