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β-thalassemias result from mutations in β-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of β-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated β-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in β-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.

Testosterone stimulates iron-dependent erythropoiesis and suppresses hepcidin. To clarify the role of iron in mediating testosterone's effects on erythropoiesis, we induced iron deficiency in mice by feeding low iron diet. Iron-replete and iron-deficient mice were treated weekly with testosterone propionate or vehicle for 3 weeks. Testosterone treatment increased red cell count in iron-replete mice, but, surprisingly, testosterone reduced red cell count in iron-deficient mice. Splenic stress erythropoiesis was stimulated in iron-deficient mice relative to iron-replete mice, and further increased by testosterone treatment, as indicated by the increase in red pulp area, the number of nucleated erythroblasts, and expression levels of TfR1, GATA1, and other erythroid genes. Testosterone treatment of iron-deficient mice increased the ratio of early-to-late erythroblasts in the spleen and bone marrow, and serum LDH level, consistent with ineffective erythropoiesis. In iron-deficient mice, erythropoietin levels were higher but erythropoietin-regulated genes were generally downregulated relative to iron-replete mice, suggesting erythropoietin resistance. Conclusion: Testosterone treatment stimulates splenic stress erythropoiesis in iron-replete as well as iron-deficient mice. However, testosterone worsens anemia in iron-deficient mice because of ineffective erythropoiesis possibly due to erythropoietin resistance associated with iron deficiency. Iron plays an important role in mediating testosterone's effects on erythropoiesis.

Reduced ferrochelatase activity in erythropoietic protoporphyria (EPP) causes the accumulation of protoporphyrin IX (PPIX) leading to acute cutaneous photosensitivity and liver injury. Many EPP patients also have a mild hypochromic, microcytic anemia and iron deficiency. Iron deficiency can lead to decreased PPIX accumulation in another erythropoietic porphyria, congenital erythropoietic porphyria (CEP). Expression of the iron regulatory peptide hepcidin is negatively regulated by the serine protease TMPRSS6. Hepcidin induction by siRNA-mediated inhibition of TMPRSS6 expression reduces iron availability and induces iron deficiency. To interrogate the therapeutic potential of iron deficiency to modify EPP, we treated an ethylnitrosourea-induced mouse model of EPP, Fech m1Pas , with a GalNAc-conjugated Tmprss6 siRNA and PPIX levels, anemia and iron parameters were monitored. The GalNAc-RNAi therapeutic reduces Tmprss6 expression and induces mild iron deficiency in Fech m1Pas animals. However, decreases in erythrocyte PPIX levels and liver PPIX accumulation were not seen. These results indicate short-term induction of iron deficiency, at least in a murine model of EPP, does not lead to decreased PPIX production.

Ncoa4 mediates autophagic degradation of ferritin, the cytosolic iron storage complex, to maintain intracellular iron homeostasis. Recent evidence also supports a role for Ncoa4 in systemic iron homeostasis and erythropoiesis. However, the specific contribution and temporal importance of Ncoa4-mediated ferritinophagy in regulating systemic iron homeostasis and erythropoiesis is unclear. Here, we show that Ncoa4 has a critical role in basal systemic iron homeostasis and both cell autonomous and non-autonomous roles in murine erythropoiesis. Using an inducible murine model of Ncoa4 knockout, acute systemic disruption of Ncoa4 impaired systemic iron homeostasis leading to tissue ferritin and iron accumulation, a decrease in serum iron, and anemia. Mice acutely depleted of Ncoa4 engaged the Hif2a-erythropoietin system to compensate for anemia. Mice with targeted deletion of Ncoa4 specifically in the erythroid compartment developed a pronounced anemia in the immediate postnatal stage, a mild hypochromic microcytic anemia at adult stages, and were more sensitive to hemolysis with higher requirements for the Hif2a-erythropoietin axis and extramedullary erythropoiesis during recovery. These studies demonstrate the importance of Ncoa4-mediated ferritinophagy as a regulator of systemic iron homeostasis and define the relative cell autonomous and non-autonomous contributions of Ncoa4 in supporting erythropoiesis in vivo.

Diminished β-globin synthesis in β-thalassemia is associated with ineffective erythropoiesis, leading to secondary iron overload caused by inappropriately low levels of hepcidin and to splenomegaly in the symptomatic thalassemias. Splenectomy is often employed in patients with β-thalassemia to reduce hemolysis. Expression of the iron regulatory peptide hormone hepcidin is repressed by the serine protease TMPRSS6. Hepcidin induction by RNAi-mediated inhibition of TMPRSS6 expression reduces iron overload and mitigates anemia in murine models of β-thalassemia intermedia. To interrogate the efficacy of RNAi-mediated reduction of Tmprss6 in splenectomized β-thalassemia, splenectomized β-thalassemic Hbbth3/+ animals were treated with a GalNAc-conjugated siRNA targeting Tmprss6 (GalNAc-Tmprss6) and their hematological and iron parameters monitored. We demonstrate that treatment with GalNAc-Tmprss6 significantly diminishes Tmprss6 expression and appropriately elevates hepcidin expression in splenectomized Hbbth3/+ animals. Similar to unsplenectomized animals, treated animals have markedly improved anemia due to diminished ineffective erythropoiesis and reduced iron loading in both serum and tissue. These results suggest that RNAi-mediated reduction of Tmprss6 may have positive outcomes even in splenectomized β-thalassemia patients.

Introduction. Pyridoxal 5' Phosphate (PLP) is the cofactor form of vitamin B6 in ~60 human enzymes. The first enzyme of the heme biosynthesis pathway, delta-aminolevulinic acid synthase (ALAS), that catalyzes the condensation of glycine and succinyl-CoA to form 5-ALA the sole precursor of porphyrins and heme, is PLP dependent. The erythroid specific isoform of ALAS is ALAS2. Inherited ALAS2 mutations cause two rare diseases: X-linked Sideroblastic Anemia (XLSA), due to loss-of-function mutations located throughout the gene, and X-linked Protoporphyria (XLPP), due to gain-of-function mutations specifically located in the C-terminal domain. Male XLSA patients have a microcytic hypochromic anemia of variable severity characterized by abnormal erythroid mitochondrial iron deposits, in nucleated and enucleate cells (ring sideroblasts and siderocytes). XLPP patients develop acute photosensitivity, due to an abnormal erythroid accumulation of free protoporphyrin IX (PPIX), the substrate of ferrochelatase, the last enzyme in the pathway. In two-thirds of XLSA patients, the anemia is responsive to oral pyridoxine supplementation. Isoniazid, an antituberculosis agent, that can cause sideroblastic anemia by limiting PLP availability to ALAS2, may limit free PPIX accumulation in protoporphyric patients. While being well tolerated, isoniazid treatment of protoporphyric patients did not reduce erythroid free PPIX accumulation. Given these clinical findings, we sought to explore the effects of dietary supplementation and restriction of vitamin B6 in animal models of the diseases. Methodology. Using CRISPR-CAS9 editing technology, we generated C57BL/6N mouse models of p.R170H, p.R452H and p.R411H found in XLSA patients with B6-sensitive or -refractory disease, and p. Q548X, a XLPP allele. Fed our normal chow containing 8ppm of pyridoxine, XLSA mouse males develop phenotypes ranging from severe (p.R411H) to trivial (p.R170H) anemia; XLPP animals have the expected protoporphyric phenotype. At weaning, we fed XLSA, XLPP and control male littermates with diets with defined amounts of B6 (Envigo: 0ppm, 2ppm or 10ppm). We performed complete blood counts (CBC) and quantified erythroid free PPIX by flow cytometry after 2, 5 and 8 weeks on diet and evaluated steady state and stress erythropoiesis by flow cytometry at 8 weeks. Results. B6-depleted animals have a growth delay that is more severe in the XLSA animals. Similarly treated control and XLPP animals develop a mild microcytic anemia with siderocytes only after 8 weeks. XLPP depleted animals accumulate less free PPIX compared to normal diet, while a 2 ppm B6 diet did not affect free PPIX accumulation. On 0 ppm B6, all XLSA depleted animals developed a very severe anemia characterized by profound reticulocytopenia and massive splenomegaly. Blood smears revealed many fragmented red blood cells and siderocytes. Flow cytometry analyses reveal a blockage of erythropoiesis, at early stages of differentiation, in both the marrow and the spleen. Feeding with 2ppm B6, demonstrated variable responses in each of the three mutants, with the p.R411H being the most severe and the R170H being the least. Conclusion. All XLSA mutations are sensitive to B6 depletion. Thus, the tendency to develop B6 deficiency with age may account for later clinical presentations in patients with pyridoxine-sensitive mutations. The limited PPIX response and development of siderocytic anemia in B6 deficient XLPP animals may suggest why the B6 inhibitor isoniazid had limited clinical efficacy. Thus our novel XLSA and XLPP mice model the each disease accurately and have demonstrated their potential for evaluating experimental treatments. Disclosures Schmidt: Disc Medicine, Inc.: Research Funding. Fleming: Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.

Human-specific duplications at chromosome 16p11.2 mediate recurrent pathogenic 600 kbp BP4-BP5 copy number variations, one of the most common genetic causes of autism. These copy number polymorphic duplications are under positive selection and include 3–8 copies of BOLA2, a gene involved in the maturation of cytosolic iron-sulfur proteins. To investigate the potential advantage provided by the rapid expansion of BOLA2, we assessed hematological traits and anemia prevalence in 379,385 controls and individuals who have lost or gained copies of BOLA2: 89 chromosome 16p11.2 BP4-BP5 deletion and 56 reciprocal duplication carriers in the UK Biobank. We found that the 16p11.2 deletion is associated with anemia (18/89 carriers, 20%, P=4e-7, OR=5), particularly iron-deficiency anemia. We observed similar enrichments in two clinical 16p11.2 deletion cohorts, with 6/63 (10%) and 7/20 (35%) unrelated individuals with anemia, microcytosis, low serum iron, or low blood hemoglobin. Upon stratification by BOLA2 copy number, we found an association between low BOLA2 dosage and the above phenotypes (8/15 individuals with three copies, 53%, P=1e-4). In parallel, we analyzed hematological traits in mice carrying the 16p11.2 orthologous deletion or duplication, as well as Bola2+/- and Bola2-/- animals. The deletion and Bola2-deficient mice showed early evidence of iron deficiency, including a mild decrease in hemoglobin, lower plasma iron, microcytosis, and an increased red blood cell zinc protoporphyrin to heme ratio. Our results indicate that BOLA2 participates in iron homeostasis in vivo and its expansion has a potential adaptive role in protecting against iron deficiency.

Iron is a redox-active metal required as a cofactor in multiple metalloproteins essential for a host of life processes. The metal is highly toxic when present in excess and must be strictly regulated to prevent tissue and organ damage. Hepcidin, a molecule first characterized as an antimicrobial peptide, plays a critical role in the regulation of iron homeostasis. Multiple stimuli positively influence the expression of hepcidin, including iron, inflammation, and infection by pathogens. In this Minireview, I will discuss how inflammation regulates hepcidin transcription, allowing for sufficient concentrations of iron for organismal needs while sequestering the metal from infectious pathogens.

β-thalassemias result from diminished β-globin synthesis and are associated with ineffective erythropoiesis and secondary iron overload caused by inappropriately low levels of the iron regulatory hormone hepcidin. The serine protease TMPRSS6 attenuates hepcidin production in response to iron stores. Hepcidin induction reduces iron overload and mitigates anemia in murine models of β-thalassemia intermedia. To further interrogate the efficacy of an RNAi-therapeutic downregulating Tmprss6, β-thalassemic Hbbth3/+ animals on an iron replete, an iron deficient, or an iron replete diet also containing the iron chelator deferiprone were treated with Tmprss6 siRNA. We demonstrate that the total body iron burden is markedly improved in Hbbth3/+ animals treated with siRNA and chelated with oral deferiprone, representing a significant improvement compared to either compound alone. These data indicate that siRNA suppression of Tmprss6, in conjunction with oral iron chelation therapy, may prove superior for treatment of anemia and secondary iron loading seen in β-thalassemia intermedia. Am. J. Hematol. 90:310–313, 2015. © 2015 The Authors. American Journal of Hematology Published by Wiley Periodicals, Inc.

INTRODUCTION The reticulocyte–red blood cell transition is a canonical example of terminal differentiation. The mature red blood cell has one of the simplest cellular proteomes known, with hemoglobin remarkably concentrated to ~98% of soluble protein. During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of cell type–specific proteins such as hemoglobin. The mechanisms that drive rapid turnover of soluble and normally stable proteins in terminally differentiating cells remain largely unclear. RATIONALE The ubiquitin-proteasome system (UPS) was discovered in reticulocytes, where it is highly active. However, its function in this developmental context has not been established. UBE2O is an E2 (ubiquitin-conjugating) enzyme that is co-induced with globin and expressed at elevated levels late in the erythroid lineage. We identified an anemic mouse line with a null mutation in Ube2o , and used multiplexed quantitative proteomics to identify candidate substrates of UBE2O in an unbiased and global manner. We found that the protein compositions of mutant and wild-type reticulocytes differed markedly, suggesting that UBE2O-dependent ubiquitination might target its substrates for degradation to effect remodeling of the proteome. To test whether UBE2O was sufficient for proteome remodeling, we engineered a non-erythroid cell line to inducibly express UBE2O above its basal level. Upon induction, we observed the decline of hundreds of proteins from these cells, in many cases the same proteins as those eliminated from reticulocytes. Overexpression of an active-site mutant of UBE2O did not show these effects. Therefore, a major component of the specificity underlying differentiation-linked proteome remodeling appears to be carried by UBE2O itself. These results also indicate that UBE2O may function as a hybrid enzyme with both E2 and E3 (ubiquitin-ligating) activities. In support of this model, candidate substrates identified by proteomics were ubiquitinated by purified UBE2O without the assistance of additional specificity factors. RESULTS The most prominent phenotypes of the Ube2o mutant are an anemia characterized by small cells with low hemoglobin content (microcytic hypochromic anemia), and a defect in the elimination of ribosomes, the latter being a key aspect of reticulocyte maturation. When we added recombinant UBE2O protein to reticulocyte lysates from the null mutant, ubiquitin was conjugated primarily to ribosomal proteins. Moreover, immunoblot analysis and quantitative proteomics revealed elevated levels of multiple ribosomal proteins in mutant reticulocytes. Sucrose gradient analysis indicated the persistence not only of ribosomal proteins but of ribosomes themselves during ex vivo differentiation of mutant reticulocytes. Accordingly, ribosomes were eliminated upon induction of UBE2O in non-erythroid cells. The elimination of organelles from reticulocytes, as exemplified by that of mitochondria, was not affected in the Ube2o mutant, indicating the specificity of its effects on programmed protein turnover. Free ribosomal proteins were ubiquitinated by purified UBE2O, which suggests that these proteins are true substrates of the enzyme. However, UBE2O substrates are diverse in nature and not limited to ribosomal proteins. Individual domains of UBE2O bound substrates with distinct specificities. Thus, the broad specificity of UBE2O reflects the presence of multiple substrate recognition domains within the enzyme. Proteasome inhibitors blocked the degradation of UBE2O-dependent substrates in reticulocytes, although UBE2O does not form polyubiquitin chains. Rather, UBE2O adds single ubiquitin groups to substrates at multiple sites. Proteasome inhibitor treatment ex vivo led to depletion of the pools of many amino acids; this result implies that the flux of ubiquitinated substrates through the reticulocyte proteasome is sufficient to supply amino acids needed for late-stage translation of mRNA. In late erythropoiesis, several ubiquitin-conjugating enzymes and ligases are induced together with Ube2o while most components of the UPS disappear. We propose that the UPS is not simply amplified during erythroid maturation, but is instead broadly reconfigured to promote remodeling of the proteome. CONCLUSION A highly specialized UPS is expressed in the reticulocyte and is used to remodel the proteome of these cells on a global scale. UBE2O, a hybrid E2-E3 enzyme, functions as a major specificity factor in this process. In reticulocytes, and perhaps in other differentiated cells such as in the lens, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface.

In this article, the authors discuss new approaches to treating iron overload diseases using hepcidin mimetics or by modulating endogenous hepcidin expression. In particular, the authors discuss lipid nanoparticle encapsulated siRNA and antisense oligonucleotide-mediated inhibition of TMPRSS6, an upstream regulator of hepcidin, and treatment with transferrin or hepcidin mimetics, including the recently described minihepcidins. In each case, in animal models of β-thalassemia, not only do the interventions affect iron absorption but they also act as disease-modifying agents that ameliorate the ineffective erythropoiesis.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

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.

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow.

Many past and recent advances in the field of iron metabolism have relied upon the use of mouse models of disease. These models have arisen spontaneously in breeder colonies or have been engineered for global or conditional ablation or overexpression of select genes. Full phenotypic characterization of these models typically involves maintenance on iron-loaded or -deficient diets, treatment with oxidative or hemolytic agents, breeding to other mutant lines or other stresses. In this review, we focus on systemic iron biology and the contributions that mouse model-based studies have made to the field. We have divided the field into three broad areas of research: dietary iron absorption, regulation of hepcidin expression and cellular iron metabolism. For each area, we begin with an overview of the current understanding of key molecular and cellular determinants then discuss recent advances. Finally, we conclude with brief comments on prospects for future study. This article is part of a Special Issue entitled: Cell Biology of Metals.

Paul Schmidt was born in 1925 into the Greatest Generation. Events during military service decided him on the study of medicine. Early research training in red cell preservation that continued during his medical studies opened a 20-year career at the National Institutes of Health (NIH). Beginning in 1954 at the Blood Bank of the NIH Clinical Center, he had exposure to the pioneers who had translated transfusion's wartime beginnings into civilian applications. Work inside the unique NIH clinical research atmosphere together with many of his students provided a fertile field for the growth of what has become transfusion medicine. Topics described range from early studies on platelets and on hepatitis to the background in Washington health politics leading to the National Blood Policy. National and global organizational activity and a second career in community blood service added to his 65 years of experience. The story as transfusion history is presented as a template for future progress.

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2Y245X/Y245X mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, coimmunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner. Am. J. Hematol. 87:588–595, 2012. © 2012 Wiley Periodicals, Inc.

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1−/− animals. Pus1−/− mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1−/− mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1−/− mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1−/− mice.

The politics of health were never tested more than when AIDS surfaced at the beginning of the 1980s in the industrialized nations. In those countries, it became the most important medical crisis of the last half of the 20th century. Today, the significance of AIDS remains as not only an unrelenting disease but also as a disease that continues to affect social and political life throughout the entire world. The connection between blood transfusion and AIDS is now under control in the industrialized countries but only because of lessons that took too long to learn over the past 25 years. That process had different roots and effects depending on the various national blood programs and policies in different countries. That is illustrated by comparing events in France, Japan, Canada, and the United States that differed in donor and patient populations and on decisions made and secrets kept. Some of the problems persist to this day in parts of the world. Overall, the lessons learned from what happened with blood early in the AIDS epidemic apply to other aspects of human disease and could help in facing the new problems that are sure to appear in the future.

Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins. To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C>A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct. K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B). We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis. Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and β2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. Conclusion: These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice. (Hepatology 2016;63:126–137)

Although it has been shown that red cell viability can be maintained for at least two years by storage in glycerol at −79° C, there is a progressive loss of viability after six months of storage at −20° C. Because of the practical objections to storage at −79° C, studies have been directed at the efficacy of storage at −45° C and at possible causes for the progressive deterioration observed during storage at −20° C. The in vivo survival of cells stored up to 18 months at −45° C is as good as for fresh cells. In addition, the damaging effect of oxygenation of red cells prior to storage at −20° C is described, as well as the prevention of this damage by the addition of anti-oxidants (glutathione, ascorbic acid) to the red cell suspension. The in vivo survival of red cells stored for one year at −20° C in the presence of ascorbic acid and glycerol is poor.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Sixty years ago, the premier blood grouping laboratory was that of Robert Race in London. Agglutination tests and blood grouping had provided breakthroughs in immunology, genetics, and the solution of clinical problems. The significance of immunohematology was recognized by the clinical hematology community as a potent force in the expanding field of disorders of the blood and blood-forming organs. The instructions by Race to his London workers entitled Hints to Blood Groupers provide a picture of the immunohematology laboratory even before automation and differed slightly from the American techniques that derived from Landsteiner. Before agglutination is replaced in the near future by the emergence of molecular methods, the detailed method of a superb laboratory is recorded.

Insertion of copper into superoxide dismutase 1 (SOD1) in vivo requires the copper chaperone for SOD1 (CCS). CCS encompasses three protein domains: copper binding Domains I and III at the amino and carboxyl termini, and a central Domain II homologous to SOD1. Using a yeast interaction mating system, yeast CCS was seen to physically interact with SOD1, and this interaction required sequences at the predicted dimer interface of CCS Domain II. Interactions with SOD1 also required sequences of Domain III, but not Domain I. Mutations were introduced at the dimer interface of yeast SOD1, and the corresponding mutant failed to interact with CCS. When loaded with copper independent of CCS, this mutant SOD1 exhibited superoxide scavenging activity, but was normally inactive in vivo because CCS failed to recognize the enzyme. Activation of SOD1 by CCS was also examined using an in vivo assay for copper incorporation into SOD1. Yeast CCS was observed to insert copper into a pre-existing pool of apoSOD1 without the need for new SOD1 synthesis or for protein unfolding by the major SSA cytosolic heat shock proteins. Our data are consistent with a model in which prefolded dimers of apoSOD1 serve as substrate for the CCS copper chaperone.

Objective Congenital heart valve disease is one of the most common abnormalities in children. There are limited technological solutions available for treating children with congenital heart valve diseases. The aim of this study is to provide the details of the consensus reached in terms of pediatric definitions, design approach, in vitro testing, and clinical trials, which may be used as guidance for developing prosthetic heart valves for the pediatric indication. Methods In stark contrast to the various designs of adult-sized replacement valves available in the market, there are no Food and Drug Administration (FDA)–approved prosthetic heart valves available for use in the pediatric population. There is a pressing need for FDA-approved pediatric valve devices in the United States. The pediatric patient population has been typically excluded from replacement heart valve trials for several reasons. In January 2010, heart valve manufacturers and pediatric clinicians collaborated with academicians and FDA staff in a workshop to suggest ways to successfully evaluate pediatric prosthetic valves and conduct pediatric clinical trials to provide acceptable heart valve replacement options for this patient population. Results Recommendations, derived from ISO 5840:2005 and the 2010 FDA Draft Replacement Heart Valve Guidance, are provided for hydrodynamic, durability, and fatigue testing. Conclusions The article specifically addresses in vitro and premarket and postmarket approval clinical studies that should be considered by a heart valve manufacturer for obtaining regulatory approval of pediatric sizes of prosthetic heart valve designs that are already approved for adult clinical use.

Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. -thalassemia is a congenital anemia caused by partial or complete loss of -globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe(-/-)) and -thalassemia intermedia (Hbb(th3/+)) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe(-/-) and Hbb(th3/+) mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbb(th3/+) mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of -thalassemia intermedia.

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow. We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies. Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy.

Mutations in HFE cause the most common form of hereditary hemochromatosis (HH). We previously showed that liver-specific, transgenic overexpression of murine Hfe stimulates production of the iron regulatory hormone hepcidin. Here, we developed several additional transgenic mouse strains to further interrogate the structural basis of HFE function in the pathophysiology of HH. We hypothesized that the small, cytoplasmic domain of HFE might be necessary for HFE-mediated induction of hepcidin. We demonstrate that, like the full-length protein, overexpression of Hfe proteins lacking the cytoplasmic domain leads to hepcidin induction, iron deficiency and a hypochromic, microcytic anemia. However, high-level expression of a liver-specific Hfe transgene carrying the mouse equivalent of the common HFE C282Y human disease-causing mutation (murine C294Y) did not cause iron deficiency. Furthermore, hepcidin induction by transgenes encoding both WT Hfe and Hfe lacking its cytoplasmic domain is greatly attenuated in the absence of hemojuvelin (Hjv). Our observations indicate that the extracellular and transmembrane domains of Hfe are sufficient, and Hjv is essential, for Hfe-mediated induction of hepcidin expression.

Background & Aims Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)–SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently shown to be impaired in Hfe knockout ( Hfe −/− ) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe −/− mice. Methods The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice ( Hfe Tg ) compared with wild-type (WT) mice. Hfe −/− and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters. Results Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, whereas anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe −/− mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites. Conclusions HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.

The ancient therapy of bloodletting that was universal in the West traveled to Japan 500 years ago on the trading vessels that carried physicians and barber-surgeons to care for the body and Christian missionaries to care for the soul. Then bloodletting was replaced by blood transfusion in the 19th century, only to return less than 50 years ago as apheresis. An understanding of those transitions can be gained from the story of the introduction of Western medicine to Japan and the events that have led to the practice of apheresis there today.

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.

SummaryHemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe−/− mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.

The transferrin receptor 1 (TfR1) has been identified as the cellular receptor for four New World arenaviruses — the Junin, Machupo, Guanarito and Sabia viruses. This class of arenaviruses is important because they cause fatal haemorrhagic fevers. Treating cultured cells with an antibody against TfR1 blocks viral entry and replication. Antibodies that limit arenavirus replication without interfering with host iron metabolism may be effective in controlling outbreaks of New World haemorrhagic fever. At least five arenaviruses cause viral haemorrhagic fevers in humans. Lassa virus, an Old World arenavirus, uses the cellular receptor α-dystroglycan to infect cells1. Machupo, Guanarito, Junin and Sabia viruses are New World haemorrhagic fever viruses that do not use α-dystroglycan2. Here we show a specific, high-affinity association between transferrin receptor 1 (TfR1) and the entry glycoprotein (GP) of Machupo virus. Expression of human TfR1, but not human transferrin receptor 2, in hamster cell lines markedly enhanced the infection of viruses pseudotyped with the GP of Machupo, Guanarito and Junin viruses, but not with those of Lassa or lymphocytic choriomeningitis viruses. An anti-TfR1 antibody efficiently inhibited the replication of Machupo, Guanarito, Junin and Sabia viruses, but not that of Lassa virus. Iron depletion of culture medium enhanced, and iron supplementation decreased, the efficiency of infection by Junin and Machupo but not Lassa pseudoviruses. These data indicate that TfR1 is a cellular receptor for New World haemorrhagic fever arenaviruses.