The role of CD71+ erythroid cells in the regulation of the immune response.

Complex regulation of the immune response is necessary to support effective defense of an organism against hostile invaders and to maintain tolerance to harmless microorganisms and autoantigens. Recent studies revealed previously unappreciated roles of CD71+ erythroid cells (CECs) in regulation of the immune response. CECs physiologically reside in the bone marrow where erythropoiesis takes place. Under stress conditions, CECs are enriched in some organs outside of the bone marrow as a result of extramedullary erythropoiesis. However, the role of CECs goes well beyond the production of erythrocytes. In neonates, increased numbers of CECs contribute to their vulnerability to infectious diseases. On the other side, neonatal CECs suppress activation of immune cells in response to abrupt colonization with commensal microorganisms after delivery. CECs are also enriched in the peripheral blood of pregnant women as well as in the placenta and are responsible for the regulation of feto-maternal tolerance. In patients with cancer, anemia leads to increased frequency of CECs in the peripheral blood contributing to diminished antiviral and antibacterial immunity, as well as to accelerated cancer progression. Moreover, recent studies revealed the role of CECs in HIV and SARS-CoV-2 infections. CECs use a full arsenal of mechanisms to regulate immune response. These cells suppress proinflammatory responses of myeloid cells and T-cell proliferation by the depletion of ʟ-arginine by arginase. Moreover, CECs produce reactive oxygen species to decrease T-cell proliferation. CECs also secrete cytokines, including transforming growth factor ß (TGF-ß), which promotes T-cell differentiation into regulatory T-cells. Here, we comprehensively describe the role of CECs in orchestrating immune response and indicate some therapeutic approaches that might be used to regulate their effector functions in the treatment of human conditions.

Transferrin receptor targeting by de novo sheet extension.

The de novo design of polar protein-protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood-brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.

FLCN regulates transferrin receptor 1 transport and iron homeostasis.

Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

LncRNA PVT1 regulates ferroptosis through miR-214-mediated TFR1 and p53.

AIM: The study aims to investigate the roles of LncRNA and miRNA in ferroptosis in brain ischemia/reperfusion (I/R) in vivo and in vitro. MATERIALS AND METHODS: qPCR assay was used to analyze lncRNA PVT1 and miR-214 expressions in acute ischemic stroke (AIS) patients. Then, we established brain I/R mice models and OGD/R PC12 cell models to analyze the mechanism of ferroptosis. I/R mice were treated by lncRNA PVT silencing or miR-214 overexpressing lentivirus via lateral ventricles. Infarct size was analyzed by TTC staining, accompanied by the detection of ferroptosis indicators through Perls'Prussian blue staining, iron kit, MDA kit, glutathione kit, GPx activities kit and Western blotting (WB). Dual luciferase reporter assay was used to assess whether miR-214 bound to PVT1, TP53 or TFR1. Co-IP analyzed the interplay of p53 with SLC7A11. KEY FINDINGS: We found that the levels of PVT1 were upregulated and miR-214 levels were downregulated in plasma of AIS patients. NIHSS score was positively correlated with PVT1 levels but was negatively with miR-214 levels. PVT1 silencing or miR-214 overexpression significantly reduced infarct size and suppressed ferroptosis in vivo. miR-214 overexpression markedly decreased PVT1 levels. Specifically, miR-214 could bind to 3'untranslated region (3'UTR) of PVT1, TP53 or TFR1. PVT1 overexpression or miR-214 silencing markedly abolished the effects of Ferrostatin-1 on ferroptosis indicators except for TFR1 expression. Besides, miR-214 silencing counteracted the effects of PVT1 knockdown on the ferroptosis-related proteins. CONCLUSION: PVT1 regulated ferroptosis through miR-214-mediated TFR1 and TP53 expression. There was a positive feedback loop of lncRNA PVT1/miR-214/p53 possibly.

Type I interferon underlies severe disease associated with Junín virus infection in mice.

Junín virus (JUNV) is one of five New World mammarenaviruses (NWMs) that causes fatal hemorrhagic disease in humans and is the etiological agent of Argentine hemorrhagic fever (AHF). The pathogenesis underlying AHF is poorly understood; however, a prolonged, elevated interferon-α (IFN-α) response is associated with a negative disease outcome. A feature of all NWMs that cause viral hemorrhagic fever is the use of human transferrin receptor 1 (hTfR1) for cellular entry. Here, we show that mice expressing hTfR1 develop a lethal disease course marked by an increase in serum IFN-α concentration when challenged with JUNV. Further, we provide evidence that the type I IFN response is central to the development of severe JUNV disease in hTfR1 mice. Our findings identify hTfR1-mediated entry and the type I IFN response as key factors in the pathogenesis of JUNV infection in mice.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Early-Life Iron Deficiency Alters Glucose Transporter-1 Expression in the Adult Rodent Hippocampus.

BACKGROUND: Early-life iron deficiency (ID) impairs hippocampal energy production. Whether there are changes in glucose transporter (GLUT) expression is not known. OBJECTIVE: The aim of this study was to investigate whether early-life ID and the treatment iron dose alter brain regional GLUT expression in adult rats and mice. METHODS: In Study 1, ID was induced in male and female Sprague Dawley rat pups by feeding dams a 3-mg/kg iron diet during gestation and the first postnatal week, followed by treatment using low-iron [3-10 mg/kg; formerly iron-deficient (FID)-10 group], standard-iron (40-mg/kg; FID-40 group), or high-iron (400-mg/kg; FID-400 group) diets until weaning. The control group received the 40 mg/kg iron diet. GLUT1, GLUT3, hypoxia-inducible factor (HIF)-1α, and prolyl-hydroxylase-2 (PHD2) mRNA and protein expression in the cerebral cortex, hippocampus, striatum, cerebellum, and hypothalamus were determined at adulthood. In Study 2, the role of hippocampal ID in GLUT expression was examined by comparing the Glut1, Glut3, Hif1α, and Phd2 mRNA expression in adult male and female wild-type (WT) and nonanemic hippocampal iron-deficient and iron-replete dominant negative transferrin receptor 1 (DNTfR1-/-) transgenic mice. RESULTS: In Study 1, Glut1, Glut3, and Hif1α mRNA, and GLUT1 55-kDa protein expression was upregulated 20-33% in the hippocampus of the FID-10 group but not the FID-40 group, relative to the control group. Hippocampal Glut1 mRNA (-39%) and GLUT1 protein (-30%) expression was suppressed in the FID-400 group, relative to the control group. Glut1 and Glut3 mRNA expression was not altered in the other brain regions in the 3 FID groups. In Study 2, hippocampal Glut1 (+14%) and Hif1α (+147%) expression was upregulated in the iron-deficient DNTfR1-/- mice, but not in the iron-replete DNTfR1-/- mice, relative to the WT mice (P < 0.05, all). CONCLUSIONS: Early-life ID is associated with altered hippocampal GLUT1 expression in adult rodents. The mouse study suggests that tissue ID is potentially responsible.

Association of SNPs in transferrin and transferrin receptor genes with blood iron levels in human.

Iron is bound to mobile transferrin (TF) and ferritin in blood. TF receptors (TFRC and TFR2) regulate intracellular iron by delivering iron from TF into the cytoplasm. In this study, we examined the effects of 10 single nucleotide polymorphisms (SNPs) in each of the genes for TF and TF receptors on blood iron concentrations in Japanese subjects. Blood iron levels were determined by microwave plasma-atomic emission spectrometry and the SNPs were analyzed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Blood iron levels in males were significantly higher than those in females. Therefore, the analysis was performed only in males. Blood iron concentrations did not correlate with age and postmortem intervals in males. Among the 10 SNPs in TF, TFRC, and TFR2 genes, significant associations were observed between TF genotypes (rs12769) and male iron concentrations. Individuals with genotype GG in rs12769 had significantly higher blood iron concentrations than those with GA. Previous studies have shown the association between high tissue iron concentrations and disease, liver iron levels are higher in infants dying from sudden infant death syndrome and decreased blood iron concentrations were observed in critically ill children. Therefore, rs12769 in TF might be related to diseases and mortality risk.

Lower Abundance and Impaired Function of CD71+ Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy.

BACKGROUND AND AIMS: CD71+ erythroid cells are enriched during pregnancy with immuno suppressive properties. We investigated the frequency and functionality of CD71+ erythroid cells in peripheral blood, cord blood, and placenta of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. We aimed to determine their role in IBD pathogenesis during pregnancy. METHODS: Peripheral blood was collected at preconception, the first, second and third trimesters, and postpartum. Cord blood and placental tissues were collected at the time of birth. Cells from different specimens were subjected to immune-phenotyping and functional assays. CD71+ erythroid cells were purified for quantitative polymerase chain reaction [qPCR] analysis. Using an allogeneic mouse model of pregnancy, the effects of CD71+ erythroid cells depletion on intestinal homeostasis and dysbiosis was studied. RESULTS: IBD patients had lower CD71+ erythroid cells during pregnancy compared with HCs. Placenta and cord blood CD71+ erythroid cells from IBD patients exhibited impaired functionality and expressed lower inhibitory molecules including VISTA, TGF-ß, and reactive oxygen species [ROS]. Lower CD71+ erythroid cells were correlated with reduced regulatory T cells and increased immune-activation in IBD patients. Depletion of CD71+ erythroid cells in an allogeneic pregnancy model resulted in upregulation of TLRs, IL-6, and CXCL-1, and enhanced production of TNF-α, in intestinal tissues. In contrast, TGF-ß gene expression was reduced. Excessive inflammatory response in the gut [e.g. TNF-α] affects intestinal integrity and CD71+ erythroid cells impact on the gut's bacterial composition. CONCLUSIONS: Reduced frequency and/or impaired functionality of CD71+ erythroid cells during pregnancy may predispose IBD patients to a more pro-inflammatory milieu in their gastrointestinal tract, characterised by lower Tregs, higher IL-6, and TNF-α, and dysbiosis.

Secretory immunoglobulin A induces human lung fibroblasts to produce inflammatory cytokines and undergo activation.

Immunoglobulin (Ig)A is the most abundant immunoglobulin in humans, and in the airway mucosa secretory IgA (sIgA) plays a pivotal role in first-line defense against invading pathogens and antigens. IgA has been reported to also have pathogenic effects, including possible worsening of the prognosis of idiopathic pulmonary fibrosis (IPF). However, the precise effects of IgA on lung fibroblasts remain unclear, and we aimed to elucidate how IgA activates human lung fibroblasts. We found that sIgA, but not monomeric IgA (mIgA), induced interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production by normal human lung fibroblasts (NHLFs) at both the protein and mRNA levels. sIgA also promoted proliferation of NHLFs and collagen gel contraction comparable to with transforming growth factor (TGF)-ß, which is involved in fibrogenesis in IPF. Also, Western blot analysis and real-time quantitative polymerase chain reaction (PCR) revealed that sIgA enhanced production of α-smooth muscle actin (α-SMA) and collagen type I (Col I) by NHLFs. Flow cytometry showed that NHLFs bound sIgA, and among the known IgA receptors, NHLFs significantly expressed CD71 (transferrin receptor). Transfection of siRNA targeting CD71 partially but significantly suppressed cytokine production by NHLFs co-cultured with sIgA. Our findings suggest that sIgA may promote human lung inflammation and fibrosis by enhancing production of inflammatory or fibrogenic cytokines as well as extracellular matrix, inducing fibroblast differentiation into myofibroblasts and promoting human lung fibroblast proliferation. sIgA's enhancement of cytokine production may be due partially to its binding to CD71 or the secretory component.

CD71+VISTA+ erythroid cells promote the development and function of regulatory T cells through TGF-ß.

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- ß, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-ß compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-ß in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-ß1, TGF-ß2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.

Iron homeostasis: An anthropocentric perspective.

The regulation of iron metabolism in biological systems centers on providing adequate iron for cellular function while limiting iron toxicity. Because mammals cannot excrete iron, mechanisms have evolved to control iron acquisition, storage, and distribution at both systemic and cellular levels. Hepcidin, the master regulator of iron homeostasis, controls iron flows into plasma through inhibition of the only known mammalian cellular iron exporter ferroportin. Hepcidin is feedback-regulated by iron status and strongly modulated by inflammation and erythropoietic demand. This review highlights recent advances that have changed our understanding of iron metabolism and its regulation.

Quel pourrait être le futur de la prise en charge de l'anémie dans l'insuffisance rénale chronique ?

Erythropoietin (EPO) plays an essential role in the regulation of erythropoiesis. Its production is under the control of the Hypoxia Inducible Factor (HIF) protein whose stability varies according to the oxygen level. During chronic renal failure, EPO deficiency is the main cause of anemia, but other factors such as iron deficiency and inflammatory syndrome are also involved. More recently, it is hypothesized that other factors such an excess of GDF-11 production may be also involved. Thus, beside Epo treatment HIF and GDF-11 are potentially new therapeutic targets in anemia of chronic kidney disease.

Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron-deficient anemia.

Iron metabolism and erythropoiesis are inherently interlinked physiological processes. Regulation of iron metabolism is mediated by the iron-regulatory hormone hepcidin. Hepcidin limits the amount of iron released into the blood by binding to and causing the internalization of the iron exporter, ferroportin. A number of molecules and physiological stimuli, including erythropoiesis, are known to regulate hepcidin. An increase in erythropoietic demand decreases hepcidin, resulting in increased bioavailable iron in the blood. Transferrin receptor 2 (TFR2) is involved in the systemic regulation of iron metabolism. Patients and mice with mutations in TFR2 develop hemochromatosis due to inappropriate hepcidin levels relative to body iron. Recent studies from our laboratory and others have suggested an additional role for TFR2 in response to iron-restricted erythropoiesis. These studies used mouse models with perturbed systemic iron metabolism: anemic mice lacking matriptase-2 and Tfr2, or bone marrow transplants from iron-loaded Tfr2 null mice. We developed a novel transgenic mouse model which lacks Tfr2 in the hematopoietic compartment, enabling the delineation of the role of Tfr2 in erythroid development without interfering with its role in systemic iron metabolism. We show that in the absence of hematopoietic Tfr2 immature polychromatic erythroblasts accumulate with a concordant reduction in the percentage of mature erythroid cells in the spleen and bone marrow of anemic mice. These results demonstrate that erythroid Tfr2 is essential for an appropriate erythropoietic response in iron-deficient anemia. These findings may be of relevance in clinical situations in which an immediate and efficient erythropoietic response is required. Am. J. Hematol. 91:812-818, 2016. © 2016 Wiley Periodicals, Inc.

Transferrin Receptor 1 in Chronic Hypoxia-Induced Pulmonary Vascular Remodeling.

BACKGROUND: Iron is associated with the pathophysiology of several cardiovascular diseases, including pulmonary hypertension (PH). In addition, disrupted pulmonary iron homeostasis has been reported in several chronic lung diseases. Transferrin receptor 1 (TfR1) plays a key role in cellular iron transport. However, the role of TfR1 in the pathophysiology of PH has not been well characterized. In this study, we investigate the role of TfR1 in the development of hypoxia-induced pulmonary vascular remodeling. METHODS: PH was induced by exposing wild-type (WT) mice and TfR1 hetero knockout mice to hypoxia for 4 weeks and evaluated via assessment of pulmonary vascular remodeling, right ventricular (RV) systolic pressure, and RV hypertrophy. In addition, we assessed the functional role of TfR1 in pulmonary artery smooth muscle cells in vitro. RESULTS: The morphology of pulmonary arteries did not differ between WT mice and TfR1 hetero knockout mice under normoxic conditions. In contrast, TfR1 hetero knockout mice exposed to 4 weeks hypoxia showed attenuated pulmonary vascular remodeling, RV systolic pressure, and RV hypertrophy compared with WT mice. In addition, the depletion of TfR1 by RNA interference attenuated human pulmonary artery smooth muscle cells proliferation induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. CONCLUSIONS: These results suggest that TfR1 plays an important role in the development of hypoxia-induced pulmonary vascular remodeling.

Noncanonical role of transferrin receptor 1 is essential for intestinal homeostasis.

Transferrin receptor 1 (Tfr1) facilitates cellular iron uptake through receptor-mediated endocytosis of iron-loaded transferrin. It is expressed in the intestinal epithelium but not involved in dietary iron absorption. To investigate its role, we inactivated the Tfr1 gene selectively in murine intestinal epithelial cells. The mutant mice had severe disruption of the epithelial barrier and early death. There was impaired proliferation of intestinal epithelial cell progenitors, aberrant lipid handling, increased mRNA expression of stem cell markers, and striking induction of many genes associated with epithelial-to-mesenchymal transition. Administration of parenteral iron did not improve the phenotype. Surprisingly, however, enforced expression of a mutant allele of Tfr1 that is unable to serve as a receptor for iron-loaded transferrin appeared to fully rescue most animals. Our results implicate Tfr1 in homeostatic maintenance of the intestinal epithelium, acting through a role that is independent of its iron-uptake function.

Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

A critical role for murine transferrin receptor 2 in erythropoiesis during iron restriction.

Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6; also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.

The second transferrin receptor regulates red blood cell production in mice.

Transferrin receptor 2 (TFR2) contributes to hepcidin regulation in the liver and associates with erythropoietin receptor in erythroid cells. Nevertheless, TFR2 mutations cause iron overload (hemochromatosis type 3) without overt erythroid abnormalities. To clarify TFR2 erythroid function, we generated a mouse lacking Tfr2 exclusively in the bone marrow (Tfr2(BMKO)). Tfr2(BMKO) mice have normal iron parameters, reduced hepcidin levels, higher hemoglobin and red blood cell counts, and lower mean corpuscular volume than normal control mice, a phenotype that becomes more evident in iron deficiency. In Tfr2(BMKO) mice, the proportion of nucleated erythroid cells in the bone marrow is higher and the apoptosis lower than in controls, irrespective of comparable erythropoietin levels. Induction of moderate iron deficiency increases erythroblasts number, reduces apoptosis, and enhances erythropoietin (Epo) levels in controls, but not in Tfr2(BMKO) mice. Epo-target genes such as Bcl-xL and Epor are highly expressed in the spleen and in isolated erythroblasts from Tfr2(BMKO) mice. Low hepcidin expression in Tfr2(BMKO) is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity.