Hepcidin Suppression Research Articles

PF507 THE HEPCIDIN‐ERYTHROFERRONE AXIS AND ITS RELATIONSHIP WITH ERYTHROPOIESIS IN DIFFERENT TYPES OF ANEMIA

Background:The regulation of iron stores depends on hepcidin, while the adaptation mechanisms of iron availability in case of anemia are mediated by an erythroid factor that could be erythroferrone (ERFE). ERFE is produced by erythroid precursors in the bone marrow and the spleen in response to erythropoietin (EPO) stimulation and bleeding in animal models. ERFE induces hepcidin suppression during increased erythropoietic activity and thereby increases iron availability for new erythrocytes synthesis. Therefore, a negative correlation between serum ERFE concentrations and Hb level and serum ferritin were observed in pediatric patients with iron deficiency anemia (IDA). The role of so‐called hepcidin‐ERFE axis have not been confirmed in cases of increased and sustained erythropoiesis, like congenital hemolytic or dyserythropoietic anemias.Aims:To evaluate hepcidin and ERFE levels in different erythroid disorders to investigate their possible implications on iron metabolism.Methods:Multicentric study, in which 5 groups of patients were included: 1) healthy controls, 2) IDA, 3) congenital hemolytic anemias with biochemical data of active hemolysis, 4) congenital anemias with ineffective erythropoiesis (non‐transfusion dependent thalassemia, dyserythropoietic anemia) and 5) hereditary spherocytosis (HS) previously treated with splenectomy. In all groups, determination of hemoglobin, ferritin, hepcidin and ERFE levels was performed. The quantitative measurement of hepcidin in serum was a solid phase enzyme‐linked immunosorbent assay based on the principle of competitive binding, with a detection range 0.153–81 ng/ml and sensitivity 0.153 ng/ml. The quantitative measurement of ERFE (FAM132B) in serum was based on a standard sandwich enzyme‐linked immunosorbent assay technology with a detection range 156–10,000 pg/ml and sensitivity 75 pg/ml.Results:A total of 94 patients were included, of which 33 were excluded (distributed in all groups) because they did not have a valid ERFE value (not detectable with the method used). The 61 remaining cases distributed as follows: Healthy controls 26 (42.6%), IDA 9 (14.8%), congenital hemolytic anemia 13 (21.3%), ineffective erythropoiesis 4 (6.5%), splenectomized HS patients 9 (14.8%). The median values of Hb, ferritin, hepcidin and ERFE of the different groups are summarized in table 1.Summary/Conclusion:In IDA patients, a statistically significant decrease in hepcidin is observed; otherwise, there is an increase in ERFE, although not statistically significant, that could be explained by the low number of samples analyzed. In hemolytic anemias, despite an increase in erythropoiesis, there is no increase in ERFE, which may be in relation to the absence of anemia (compensated hemolytic anemia) and the increase of hepcidine could be explained by the high ferritin levels. In the ineffective erythropoiesis group, despite the anemia, no statistically significant ERFE‐hepcidin levels were observed. In splenectomized HS patients, ERFE/hepcidine levels were similar to those of the control group.imageThe newly identified ERFE hormone may act as physiological hepcidin suppressor in cases with IDA, but we have to establish this relationship in other types of anemia, for which it is necessary to evaluate more patients. The high number of patients that we had to exclude due to ERFE results outside the assay sensitivity range implies the need of validation and standardization of the method.

S148 TFR2-HAPLOINSUFFICIENCY ENHANCES THE BENEFICIAL EFFECT OF TMPRSS6-ANTISENSE OLIGONUCLEOTIDE TREATMENT IN BETA-THALASSEMIA MICE

Background: Transferrin receptor 2 (TFR2) is a type II transmembrane protein mainly expressed in the liver and in the erythroid compartment. In the liver it acts as an activator of the iron hormone hepcidin and, when mutated, is responsible for type 3 hereditary hemochromatosis. In erythroid cells TFR2 binds the erythropoietin (EPO) receptor and serves as a brake of the EPO-EPOR signaling pathway. We have recently demonstrated that the complete loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity and improves anemia, ineffective erythropoiesis and iron-overload in a β-thalassemia intermedia murine model (Hbbth3/+), without reducing splenomegaly. On the contrary, approaches based on the induction of iron restriction were proven to be effective in ameliorating splenomegaly and anemia in thalassemic mice. Among these, targeting the hepcidin inhibitor TMPRSS6 is one of the most promising strategies. Aims: The main aim of the present work is to investigate whether the combination of Tfr2-haploinsufficiency in the BM and TMPRSS6 antisense oligonucleotide (ASOs) administration will reduce splenomegaly and provide a synergistic benefit on thalassemic erythropoiesis and anemia. Methods: We generated thalassemic mice with both or a single Tfr2 allele in the BM by transplanting BM cells from Hbbth3/+ or Tfr2+/-/Hbbth3/+ mutants into lethally irradiated wild-type mice. Mice were treated with TMPRSS6-ASO (or control ASO) twice a week for 6 weeks starting 9 weeks after BM transplantation. Complete blood count was determined at the beginning of the treatment and after 3 and 6 weeks. At sacrifice, blood and organs were collected for full phenotypic analysis. Results: Deletion of a single Tfr2 allele in Hbbth3/+ mice BM mildly increased Red Blood Cells (RBC) count and Hemoglobin (Hb) levels and improved erythropoiesis, with a concomitant decrease in EPO levels, but no reduction of spleen size. A similar phenotype was induced by TMPRSS6-ASO treatment of Hbbth3/+ animals, with additional partial correction of splenomegaly. The combination of BM Tfr2 haploinsufficiency and TMPRSS6-ASO had a striking effect on the thalassemic phenotype: indeed, an impressive increase in RBC count (up to 11.2 ± 1.2∗106/μl) and Hb (∼3 g/L) compared with Hbbth3/+ was attained, accompanied by the normalization of reticulocyte and EPO levels, improved erythropoiesis and spleen size reduction. Mean corpuscular volume and hemoglobin were strongly reduced. This indicates a disproportionate erythropoietic expansion relative to the available iron, whose circulating levels are decreased relative to untreated thalassemic controls. Summary/Conclusion: While Tfr2 haploinsufficiency does not impair systemic iron homeostasis, here we demonstrate that deletion of a single Tfr2 allele in the BM is sufficient to improve the hematological parameters of thalassemic mice. This finding paves the way toward a potential targeted therapy based on partial Tfr2 inhibition that might be beneficial for thalassemia (and potentially for other forms of anemia) without inducing defective hepcidin activation and iron overload. In addition, our results prove that targeting Tfr2 in combination with an anti-TMPRSS6 therapy may be a valuable therapeutic approach that strongly improves the thalassemic phenotype. However, Tmprss6 inhibition must be tightly controlled to avoid excessive iron restriction. Further experiments will allow setting up the optimal conditions.

S1628 TARGETING THE IMMUNOPHILIN FKBP12 BY DRUG REPURPOSING OR RNASE‐BASED APPROACHES FOR BMP‐SMAD PATHWAY AND HEPCIDIN UPREGULATION IN HEREDITARY HEMOCHROMATOSIS

Background:The main regulator of iron homeostasis, the liver hormone hepcidin, is inappropriately low in Hereditary Hemochromatosis (HH). HH is caused by mutations in HJV, TFR2 and HFE that negatively affect the liver BMP‐SMAD pathway, the main signaling pathway regulating hepcidin, thus causing iron overload. Current phlebotomy‐based therapy is symptomatic, does not correct hepcidin deficiency and is not well tolerated by all patients. We recently identified a new function for a cytosolic protein, the immunophilin FKBP12, and demonstrated that it negatively controls the BMP‐SMAD pathway by binding the type I receptor ALK2 (Colucci et al., 2017). Therefore, FKBP12 may be a novel therapeutic target for increasing hepcidin expression.Aims:Since sequestration of FKBP12 by Tacrolimus (TAC) upregulates hepcidin in wild type (WT) mice, the aim of this work is to investigate whether reducing FKBP12 may restore hepcidin expression in HH mouse models.Methods:Hepatocytes were isolated from WT, Tfr2 and Hjv KO mice and treated with TAC. Hjv KO mice were treated for 28 days with TAC delivered by subcutaneous miniosmotic pumps. In vivo RNAse‐based Fkbp12 downregulation was achieved using antisense oligonucleotides (ASOs). Hepcidin, Id1, Smad7, Erfe expression were evaluated by qRT‐PCR. Iron parameters, CBC and terminal erythroid differentiation were analyzed by standard methods.Results:FKBP12 sequestration by TAC upregulates hepcidin through BMP‐SMAD pathway activation in Hjv and Tfr2 KO primary hepatocytes, suggesting that FKBP12 targeting may be effective in HH. Due to the transient effect of TAC‐mediated hepcidin upregulation, we treated Hjv KO mice with chronic TAC administration by miniosmotic pumps. TAC upregulated hepcidin through BMP‐SMAD pathway activation and caused spleen iron retention, demonstrating that the FKBP12 inhibitory function on ALK2 is also preserved in an in vivo HH model. Since TAC is an immunosuppressive drug, we are exploring Fkbp12 downregulation by ASOs. ASO‐Fkbp12 treatment of WT mice decreased Fkbp12 expression in liver (78.4%), spleen (66.3%) and kidney (56.4%) but not in the BM. Fkbp12 inactivation mildly upregulated Hamp, Id1 and Smad7, consistent with BMP‐SMAD pathway activation. Although hepcidin expression was not significantly increased, we observed a reduction in transferrin saturation and serum iron and increased spleen iron retention. Hemoglobin and MCV were reduced in ASO‐Fkbp12‐treated mice, characterized also by a trend towards a reduction in RBC number and HCT. Spleen size and EPO levels remained unchanged. Analysis of terminal erythroid differentiation demonstrated an increased percentage of immature erythroid precursors with comparable Ter119+ cells in the spleen of ASO‐Fkbp12‐treated mice but not in the BM, and accordingly Erfe, the erythroid hepcidin inhibitor, is upregulated.Summary/Conclusion:Fkbp12 downregulation in the liver upregulates the BMP‐SMAD pathway, although hepcidin expression is minimally affected. Fkbp12 downregulation in the spleen impairs erythropoiesis increasing Erfe expression and decreasing serum iron concentration. Considering the presence of two inhibitory signals (high Erfe, low iron), hepcidin is inappropriately high in ASO‐Fkbp12 treated mice. We conclude that to increase hepcidin expression in HH, FKBP12 must be specifically downregulated in the liver. In addition, our results reveal a potential effect of FKBP12 on erythropoiesis, uncovering a novel role for the immunophilin in the regulation of erythropoiesis.

Hepcidin is not essential for mediating testosterone's effects on erythropoiesis.

We have shown that testosterone administration suppresses hepcidin, stimulates iron-dependent erythropoiesis, and increases hemoglobin and hematocrit. We investigated whether testosterone-mediated suppression of hepcidin plays an essential role in mediating testosterone's stimulatory effects on erythropoiesis. We utilized two mouse models to elucidate the role of hepcidin as a mediator of testosterone's effects on erythropoiesis: First, we used a whole-body hepcidin knockout (HepKO) mouse. Because testosterone's effects on hepcidin expression are mediated through androgen receptor, we also utilized a liver-specific androgen receptor knockout mouse (L-ArKO). Effects of 6weeks of testosterone (50mg/kg weekly) administration relative to vehicle on hemoglobin and hematocrit, red blood cell indices, and markers of iron stores and availability were compared between wild-type (WT) and the two genetically modified mouse models. HepKO mice had significantly higher baseline levels of hemoglobin, hematocrit, serum and liver iron, and ferritin than WT mice. Compared to vehicle group, testosterone administration was associated with significant increases in hematocrit, hemoglobin, red cell counts, reticulocyte count, reticulocyte hemoglobin, and serum iron levels in both HepKO and WT mice. Baseline hematocrit levels did not differ between WT and L-ArKO mice. Compared to vehicle, testosterone treatment was associated with significantly greater increase in hematocrit, hemoglobin, red cell count, reticulocyte count, reticulocyte hemoglobin, and serum iron in WT and L-ArKO mice. Although hepcidin suppression by testosterone increases iron availability and erythropoiesis, hepcidin suppression is not essential for mediating testosterone's effects on erythropoiesis in healthy mice.

Serum Erythroferrone Levels Associate with Mortality and Cardiovascular Events in Hemodialysis and in CKD Patients: A Two Cohorts Study.

Erythroferrone (ERFE) is a hepcidin inhibitor whose synthesis is stimulated by erythropoietin, which increases iron absorption and mobilization. We studied the association between serum ERFE and mortality and non-fatal cardiovascular (CV) events in a cohort of 1123 hemodialysis patients and in a cohort of 745 stage 1–5 chronic kidney disease (CKD) patients. Erythroferrone was measured by a validated enzyme-linked immunosorbent assay (ELISA). In the hemodialysis cohort, serum ERFE associated directly with erythropoiesis stimulating agents (ESA) dose (p < 0.001) and inversely with serum iron and ferritin (p < 0.001). Erythroferrone associated with the combined outcome in an analysis adjusting for traditional risk factors, factors peculiar to end-stage kidney disease, serum ferritin, inflammation, and nutritional status (HR, hazard ratio, (5 ng/mL increase: 1.04, 95% confidence interval, CI: 1.01–1.08, p = 0.005). Furthermore, treatment with ESA modified the relationship between ERFE and the combined end-point in adjusted analyses (p for the effect modification = 0.018). Similarly, in CKD patients there was a linear increase in the risk for the same outcome in adjusted analyses (HR (2 ng/mL increase): 1.04, 95% CI: 1.0–1.07, p = 0.015). Serum ERFE is associated with mortality and CV events in CKD and in HD patients, and treatment by ESA amplifies the risk for this combined end-point in HD patients.

New insights into the links between hypoxia and iron homeostasis.

Purpose of reviewThis review outlines recent discoveries on the crosstalk between oxygen metabolism and iron homeostasis, focusing on the role of HIF-2 (hypoxia inducible factor-2) in the regulation of iron metabolism under physiopathological conditions.Recent findingsThe importance of the hepcidin/ferroportin axis in the modulation of intestinal HIF-2 to regulate iron absorption has been recently highlighted. Latest advances also reveal a direct titration of the bone morphogenetic proteins by the erythroferrone contributing to liver hepcidin suppression to increase iron availability. Iron is recycled thanks to erythrophagocytosis of senescent erythrocytes by macrophages. Hemolysis is frequent in sickle cell anemia, leading to increased erythrophagocytosis responsible of the macrophage polarization shift. New findings assessed the effects of hemolysis on macrophage polarization in the tumor microenvironment.SummaryHypoxia signaling links erythropoiesis with iron homeostasis. The use of HIF stabilizing or inhibiting drugs are promising therapeutic approaches in iron-associated diseases.

Iron Regulator Hepcidin Impairs Macrophage-Dependent Cardiac Repair After Injury.

Defective systemic and local iron metabolism correlates with cardiac disorders. Hepcidin, a master iron sensor, actively tunes iron trafficking. We hypothesized that hepcidin could play a key role to locally regulate cardiac homeostasis after acute myocardial infarction. Cardiac repair was analyzed in mice harboring specific cardiomyocyte or myeloid cell deficiency of hepcidin and challenged with acute myocardial infarction. We found that the expression of hepcidin was elevated after acute myocardial infarction and the specific deletion of hepcidin in cardiomyocytes failed to improve cardiac repair and function. However, transplantation of bone marrow-derived cells from hepcidin-deficient mice ( Hamp-/-) or from mice with specific deletion of hepcidin in myeloid cells (LysMCRE/+/ Hampf/f) improved cardiac function. This effect was associated with a robust reduction in the infarct size and tissue fibrosis in addition to favoring cardiomyocyte renewal. Macrophages lacking hepcidin promoted cardiomyocyte proliferation in a prototypic model of apical resection-induced cardiac regeneration in neonatal mice. Interleukin (IL)-6 increased hepcidin levels in inflammatory macrophages. Hepcidin deficiency enhanced the number of CD45+/CD11b+/F4/80+/CD64+/MHCIILow/chemokine (C-C motif) receptor 2 (CCR2)+ inflammatory macrophages and fostered signal transducer and activator of transcription factor-3 (STAT3) phosphorylation, an instrumental step in the release of IL-4 and IL-13. The combined genetic suppression of hepcidin and IL-4/IL-13 in macrophages failed to improve cardiac function in both adult and neonatal injured hearts. Hepcidin refrains macrophage-induced cardiac repair and regeneration through modulation of IL-4/IL-13 pathways.

Rapid growth is a dominant predictor of hepcidin suppression and declining ferritin in Gambian infants.

Iron deficiency and iron deficiency anemia are highly prevalent in low-income countries, especially among young children. Hepcidin is the major regulator of systemic iron homeostasis. It controls dietary iron absorption, dictates whether absorbed iron is made available in circulation for erythropoiesis and other iron-demanding processes, and predicts response to oral iron supplementation. Understanding how hepcidin is itself regulated is therefore important, especially in young children. We investigated how changes in iron-related parameters, inflammation and infection status, seasonality, and growth influenced plasma hepcidin and ferritin concentrations during infancy using longitudinal data from two birth cohorts of infants in rural Gambia (n=114 and n=193). This setting is characterized by extreme seasonality, prevalent childhood anemia, undernutrition, and frequent infection. Plasma was collected from infants at birth and at regular intervals, up to 12 months of age. Hepcidin, ferritin and plasma iron concentrations declined markedly during infancy, with reciprocal increases in soluble transferrin receptor and transferrin concentrations, indicating declining iron stores and increasing tissue iron demand. In cross-sectional analyses at 5 and 12 months of age, we identified expected relationships of hepcidin with iron and inflammatory markers, but also observed significant negative associations between hepcidin and antecedent weight gain. Correspondingly, longitudinal fixed effects modeling demonstrated weight gain to be the most notable dynamic predictor of decreasing hepcidin and ferritin through infancy across both cohorts. Infants who grow rapidly in this setting are at particular risk of depletion of iron stores, but since hepcidin concentrations decrease with weight gain, they may also be the most responsive to oral iron interventions.

The Effect of Vitamin D Supplementation on Hepcidin, Iron Status, and Inflammation in Pregnant Women in the United Kingdom.

Iron and vitamin D deficiencies are common during pregnancy. Our aim was to identify whether antenatal vitamin D3 supplementation affects iron status (via hepcidin suppression) and/or inflammation. Using a subset of the UK multicenter Maternal Vitamin D Osteoporosis Study (MAVIDOS)—a double-blinded, randomized, placebo-controlled trial (ISRCTN82927713; EudraCT2007-001716-23)—we performed a secondary laboratory analysis. Women with blood samples from early and late pregnancy (vitamin D3 (1000 IU/day from ~14 weeks gestation n = 93; placebo n = 102) who gave birth in the springtime (March–May) were selected as we anticipated seeing the greatest treatment group difference in change in 25-hydroxyvitamin D (25OHD) concentration. Outcomes were hepcidin, ferritin, C-reactive protein, and α1-acid glycoprotein concentration in late pregnancy (25OHD concentration was measured previously). By late pregnancy, 25OHD concentration increased by 17 nmol/L in the vitamin D3 group and decreased by 11 nmol/L in the placebo group; hepcidin, ferritin, and inflammatory markers decreased but no treatment group differences were seen. In late pregnancy, positive relationships between 25OHD and hepcidin and 25OHD and ferritin in the placebo group were observed but not in the treatment group (group × 25OHD interaction, p < 0.02). Vitamin D3 supplementation had no effect on hepcidin, ferritin, or inflammatory status suggesting no adjunctive value of vitamin D3 in reducing rates of antenatal iron deficiency.

The Role of Erythropoiesis on Hepcidin Level in Polytransfused b-Thalassemia Major Patients

AbstractBackground: Hepcidin peptide hormone is a main con-troller of iron homeostasis, itbecomes elevated in case of iron overload, however despite iron overload in b-Thalassemia major (b-TM) patients a contradictory decrease in Hepcidin occurs. The incompatible Hepcidin level is the main responsible factor causing the iron overload status in iron-loading anemias such as (b-TM) which contributes to organ dysfunctionand to iron toxicity.Aim of Study: To evaluate the conflicting effect of increased erythropoiesis in contrast to the effect of iron overloading on Hepcidin serum level and its correlation with iron status in (b-TM) patients.Subjects and Methods: For all patients and controls Complete Blood Count (CBC), serum assaysof Hepcidin, iron, ferritin, transferrin, HCVAbs, HBs Ag, CRP and serum level were performed.Results: Hepcidin level was significantly lowered in (b-TM) patients compared to controls with high significant difference (p=0.00). There was no correlation between serum Hepcidin and ferritin level, neither was between Hepcidin and serum iron, transferrin, Hb level and reticulocyte count in the study group. Hepcidin/Ferritin ratio showed high significance difference (p<0.000) with mean value in the study group 0.056 vs. 4.75 in controls pointing to an incom-patible levels of Hepcidin to iron overload status. Hepcidin level wasnegative correlatedwith age (p<0.05).Conclusion: Hepcidin was markedly decreased in the study group with no significant correlation between serum Hepcidin and ferritin level as a marker of iron overload in thalassemia major. Hepcidin deficiency is the main contributing factor of iron overload in b thalassemia which results from a strong suppressive effect of the high erythropoietic activity on Hepcidin expression. Hepcidin-ferritin ratio was markedly depressed <1 in all b thalassemia major patients which indicating suppression of Hepcidin out of proportion with the degree of iron loading.

Hepcidin Suppression By Momelotinib Is Associated with Increased Iron Availability and Erythropoiesis in Transfusion-Dependent Myelofibrosis Patients

Hepcidin Suppression By Momelotinib Is Associated with Increased Iron Availability and Erythropoiesis in Transfusion-Dependent Myelofibrosis Patients

Correcting Non-Transfusion Dependent β-Thalassemia by Utilizing a Combined Therapy that Modulates EPO Activity by Limiting Erythroid Cellular Iron Intake

Β-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Administration of agents that increase erythropoiesis (such as erythropoietin-Epo) can potentially improve anemia by enhancing the production of red blood cells (RBC). However, this improvement will likely result in exacerbation of splenomegaly and worsening of iron overload due to further suppression of hepcidin, the hormone that controls dietary iron absorption. However, we postulated that, following Epo administration, some level of iron restriction will limit the splenomegaly and iron overload without preventing the improvement of anemia.To increase circulating Epo levels and increase erythropoiesis, we infused animals with primary dermal fibroblasts transduced with a helper dependent adenoviral vector carrying the Epo gene (TARGTEpo). Pharmacokinetic studies indicated that 1e6 cells were sufficient to significantly increase Epo synthesis (up to 47.6%in Hbbth3/+ and 90% in WT). This led to an increase in hemoglobin levels in both mouse models (average of 3-4 g/dL) through the end of the treatment (6 weeks). In Hbbth3/+ mice, Hb levels rose to 10.7±1.0 g/dL in the first week and remained elevated through the end of the 6-weeks of treatment (compared to 7.7±0.7 g/dL in controls). As expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin.To test our hypothesis, we then combined TARGTEpo with low iron diet or antisense oligonucleotides targeting Tmprss6 mRNA (Tmprss6-ASO; the last treatment only for Hbbth3/+ animals). It has already been shown that inhibition of Tmprss6 increases hepcidin expression leading to decreased iron absorption and limited erythroid iron intake, improving anemia, IE, splenomegaly and iron overload in Hbbth3/+ mice (Guo et al, JCI, 2013).In WT animals, after three weeks, the combination of TARGTEpo with low iron diet significantly reduced Hb levels (-40%), RBC number (-38%) and reticulocytes (-80%), when compared to animals overexpressing Epo and receiving a normal iron diet. In contrast, Hbbth3/+ animals on an iron-deficient diet or treated with Tmprss6-ASO in the presence of TARGTEpo showed decreased hemichrome formation and improved anemia through the end of treatment. In particular, Hb levels increased (on average 4 g/dL), reaching levels of 11-12g/dl, corresponding to +36% more than baseline levels in Hbbth3/+ (7.7g/dL) and +25% more compared to Hbbth3/+ mice treated with Tmprss6-ASO alone (9.0g/dL). Animals receiving TARGTEpo with Tmprss6-ASO showed a significant reduction in splenomegaly when compared to baseline levels (-35%) and to animals treated only with TARGTEpo (-52.4%). Preliminary data indicate that organ iron concentration improved or normalized compared to control Hbbth3/+ mice when iron restriction was applied.We then inquired how these two treatments combined improved erythropoiesis. We quantified the number of nucleated erythroid cells positive for the phosphorylated form of pAkt and pJak2, proteins activated by Epo but whose activity is potentially limited by iron restriction. For pAkt+ cells in the spleen, the number of proerythroblasts were increased in Hbbth3/+ treated with TARGTEpo (+35% compared to control Hbbth3/+ mice), while they were reduced in Hbbth3/+ treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO (-72% and -43% respectively, compared to control Hbbth3/+ mice). Other erythroid populations showed a comparable profile for both pAkt+ and pJak2+ nucleated erythroid cells. The total number of progenitor erythroid cells (i.e. BFUe) also followed a similar trend. Cell cycle analysis of splenic erythroid progenitor cells showed a decrease in the percentage of nucleated erythroid cells in mice treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO compared to control or TARGTEpo-treated Hbbth3/+ mice. Given that the number of RBC in circulation was highest in mice treated with TARGTEpo+Tmprss6-ASO, this data suggests that co-administration of these two drugs maximize the ability of each erythroid cell to develop into a functional RBC.In conclusion, we propose that in β-thalassemia, combination of erythropoietic stimulating agents with iron restriction could maximize correction of anemia while improving splenomegaly and iron overload. DisclosuresCasu:Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Neil:Aevi Genomic Medicine, Inc: Employment, Other: Officer and share holder. Guo:Ionis Pharmaceuticals, Inc: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Disc Medicine: Consultancy; Protagonist: Consultancy.

Transmembrane Protease, Serine 6 (TMPRSS6) Antisense Oligonucleotide (IONIS-TMPRSS6-LRX) Reduces Plasma Iron Levels of Healthy Volunteers in a Phase 1 Clinical Study

Iron overload is the major cause of morbidity and mortality in beta-thalassemia patients. The low levels of beta-globin and ineffective erythropoiesis in these patients result in the suppression of hepcidin. The inappropriately low levels of hepcidin trigger an increased absorption of dietary iron and increased iron release from storage, causing iron overload. Expression of hepcidin, which is predominately produced in the liver, is negatively regulated by the transmembrane protease, serine 6 (TMPRSS6). Mouse and human genetic data indicated that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. Previously we identified a highly specific and potent antisense oligonucleotide (ASO) targeting either the murine (Guo et al. J Clin Invest. 2013; 123(4):1531-41) or the human (Aghajan et al, Blood 2016; 128:1013) TMPRSS6 mRNA. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation, leading to dramatic reductions in serum iron and transferrin saturation in animal models This in turn ameliorated the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (th3/+ mice), which recapitulates beta-thalassemia intermedia in humans.Herein, we are reporting the initial clinical safety and pharmacodynamics of IONIS-TMPRSS6-LRX. This GalNAc-conjugated, TMPRSS6 ASO was evaluated in a placebo-controlled, double-blind, randomized, single-center Phase 1 clinical study enrolling healthy volunteers. During an 8-week period, placebo or IONIS-TMPRSS6-LRX was administered subcutaneously four times (Weeks 1, 4, 6 and 8) at doses of 20, 40 or 60 mg. At doses of 20 and 40 mg, mean (±SEM) levels of serum iron were reduced 34±10% and 49±7% on week 10 (The 60 mg treatment cohort is ongoing). Consistent with the reduction of plasma iron, the mean (+/-SEM) percent transferrin saturation was reduced from baseline levels of 28±3% and 30±1% to 14±2% and 13±2%, for 20 and 40 mg groups, respectively, at Week 10. Furthermore, plasma hepcidin levels were increased from 2.1±0.6 and 2.5±0.6 nM to 2.7±0.6 and 6.7±0.9 nM, respectively. During this time period, there were small reductions in Hgb (-9±2%), reticulocyte Hgb (-13±2%) at the 40 mg dose. There were no serious adverse events in the study and the treatment-emergent adverse events were generally mild. In summary, IONIS-TMPRSS6-LRX, a novel antisense oligonucleotide targeting TMPRSS6, effectively reduces plasma iron levels and has the potential as a therapeutic for patients with beta-thalassemia and related disorders. The safety profile of IONIS-TMPRSS6-LRX supports further development. DisclosuresMcCaleb:Ionis Pharmaceuticals, INC: Employment, Equity Ownership. Lickliter:Nucleus Network: Employment. Dibble:Ionis Pharmaceuticals, INC: Employment. Schneider:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Guo:Ionis Pharmaceuticals, Inc: Employment. Hughes:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Geary:Ionis Pharmaceuticals, INC: Employment, Other: shareholders. Monia:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights.

Hepcidin Predicts Decrease in Erythroid Expansion in Sickle Cell Disease Patients Treated with Inhaled Steroids

Sickle cell disease (SCD) is a recessively inherited hemoglobin disorder; the most common and severe form is a consequence of homozygous βS mutation. High concentration of hemoglobin S damages RBC membranes, leading to hemolysis, vaso-occlusion, and inflammation, which together result in anemia, recurrent painful crises, and multiple end-organ damage (i.e. brain, kidney, lung, and bone). Anemia in SCD is multi-factorial. Causes include hemolysis, ineffective erythropoiesis, impaired iron utilization, insufficient erythropoietin responsiveness, and low oxygen affinity [Sherwood Blood 1986]. RBC transfusions are used to ameliorate symptoms and complications but indications are not clearly defined for adults. We postulate that impaired iron utilization in SCD is a consequence of complex regulation of hepcidin, the main hormone regulating circulating and systemic iron. We aim to explore correlation between hepcidin, inflammation and erythropoiesis to enhance our understanding of iron metabolism, hepcidin regulation, and pathophysiology of anemia in SCD. Ultimately this may yield more specific indications for RBC transfusion, and pave the way for novel therapeutic approaches.High hepcidin results in the sequestration of iron within macrophages and prevents further iron absorption in the gastrointestinal tract. Hepcidin expression is enhanced by iron and inflammation and suppressed by hypoxia and erythropoiesis. Distinguishing the relative contribution of these competing effects on hepcidin in SCD is complicated. For example, adult SCD patients in steady state have significantly lower hepcidin relative to heterozygous controls and do not correlate with markers of inflammation and erythropoiesis [Kroot Haematologica 2009]. In a randomized, placebo-controlled trial, inhaled steroids (mometasone) were administered to non-asthmatic SCD patients [Glassberg Am J Hematol 2017]. The results demonstrate improved pain scores and reduced hemolysis in the treated group although the mechanism of this effect is not known. In a subsequent analysis, markers of inflammation are suppressed in the treated group, correlating with macrophage activation. We thus evaluate hepcidin expression in stored serum samples from this trial of SCD patients treated with inhaled steroids or placebo. Samples before and after treatment were analyzed. Serum hepcidin quantification was performed with Hepcidin-25 ELISA kit (Intrinsic Lifesciences LLC). In addition, we evaluate if and how hepcidin levels correlate with pro-inflammatory markers measured with olink inflammation assay.Serum hepcidin concentration is not different in steroid- vs. placebo-treated SCD patients before or after treatment and no significant difference is observed in the hepcidin ratio after:before treatment between the two groups (Fig 1a). However, in the steroid group, hepcidin concentrations are increased in a higher proportion of patients although the difference does not reach statistical significance (Fig 1b). We thus hypothesize that hepcidin levels may be a marker of increased erythropoiesis relative to inflammation in SCD. Both IL-6 (Fig 2a) and IL-10 (Fig 2b), cytokines known to induce hepcidin expression during inflammation, decrease after treatment with steroids relative to placebo, and neither correlates with hepcidin concentration after treatment (Table I). Hepcidin is however inversely correlated with adenosine deaminase (ADA) and hemoglobin S and directly correlative with fibroblast growth factor 23 (FGF23), monocyte chemoattractant protein 1 (MCP1), and hemoglobin F in both placebo- and steroid-treated groups (Table I), potentially suggesting that hepcidin inversely correlates with expanded erythropoiesis. We thus use the newly available human ELISA kit for erythroferrone (ERFE) (Intrinsic Lifesciences LLC), a recently identified erythroid regulator of hepcidin [Kautz Nat Gen 2014], to evaluate correlation with hepcidin. Our results demonstrate that serum ERFE concentration decreases in steroid- (Fig 3a) but not placebo-treated SCD patients (Fig 3b), and no correlation with hepcidin is observed. Taken together, these data for the first time demonstrate a decrease in erythropoiesis and erythroid-regulation-induced hepcidin suppression in inhaled-steroid-treated SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evidence for an Alternative Mechanism Suppressing Hepcidin during the Recovery from Hemorrhage-Induced Anemia

IntroductionThe liver-produced hormone hepcidin regulates the body iron stores. Its expression is induced by iron and inflammatory cytokines but repressed by the erythroid regulator erythroferrone (ERFE) when erythropoietic activity intensifies during anemia. Although Erfe-deficient mice fail to appropriately suppress hepcidin during the first 24h following hemorrhage, these mice still recover from anemia with a few days delay suggesting that another mechanism compensates for the absence of ERFE. We therefore decided to study the kinetic of hepcidin during the recovery from anemia induced by bleeding in Erfe-deficient mice.Material and methodsSix week-old C57BL/6 WT and Erfe-deficient mice were phlebotomized (500 μL) and analyzed 1, 2, 3, 4, 5 and 6 days after phlebotomy until full recovery.ResultsLiver hepcidin mRNA expression was suppressed 5-fold one to five days after phlebotomy in WT mice. In contrast with the sustained inhibition of hepcidin, serum ERFE concentration progressively decreased after 24 hours to reach its baseline at day 4. Interestingly, although hepcidin levels were unchanged after 24 hours, Erfe-deficient exhibited significantly reduced hepcidin levels after 48 hours. Hepcidin mRNA and protein levels were comparable to those of WT mice 2 to 5 days after phlebotomy. Interestingly, the repression of hepcidin occurred without any change in phosphorylation of the effectors Smadd1/5/8 and in hepatic expression of the BMP/SMAD target genes Atoh8, Smad7 and Id1. Similarly, mRNA expression of the proposed negative regulators of hepcidin Gdf15, Twsg1 and Gdf11 was not increased in the spleen and the bone marrow of phlebotomized mice compared to control mice. Finally, disruption of the erythroid compartment by irradiation or injection of carboplatin prevented the suppression of hepcidin in WT and Erfe-deficient mice.ConclusionAn alternative mechanism regulates hepcidin independently of iron and ERFE during stress erythropoiesis. Our data suggest that a second yet unknown erythroid regulator of hepcidin may exist. DisclosuresNo relevant conflicts of interest to declare.

Novel Insights Into Systemic Iron Regulation

Iron, an essential element in mammals, is absorbed by duodenal enterocytes, enters the circulation through the iron exporter ferroportin, (FPN), circulates bound to transferrin and is uptaken through Transferrin Receptor 1. If in excess, iron is stored in macrophages and hepatocytes and released when needed. To maintain systemic iron homeostasis and to avoid the formation of “non transferrin bound iron” (NTBI), a highly reactive form which causes organ damage, the liver synthetizes hepcidin that, binding FPN, blocks iron export to the circulation. Hepcidin integrates signals from body iron, erythropoiesis and inflammatory cytokines. Defective hepcidin production causes iron overload and organ failure in Hereditary Hemochromatosis and Thalassemia; hepcidin excess leads to anemia in Iron Refractory iron Deficiency Anemia (IRIDA) and Anemia of Inflammation (AI).In hepatocytes hepcidin is under the control of the BMP-SMAD pathway, which is activated in a paracrine manner by BMP2 and BMP6 produced by liver sinusoidal endothelial cells. BMP2 maintains hepcidin basal levels, while BMP6 controls its expression in response to iron. The two ligands have different affinity for BMP type I receptors ALK2 and ALK3, suggesting two distinct branches of the hepcidin activation pathway. This possibility is consistent with the non-redundant function of BMP2 and BMP6, the different iron phenotype of hepatocyte-conditional ALK2 and ALK3 KO mice and the residual ability of BMP6 to activate hepcidin in hemochromatosis mice. Moreover ALK2, but not ALK3, is inhibited by the immunophilin FKBP12 in the absence of ligands. The BMP pathway activation depends upon the coreceptor hemojuvelin (HJV), the MHC class I protein HFE and the second transferrin receptor (TFR2). Mutations of all these proteins lead to decreased hepcidin expression in hemochromatosis.Hepcidin expression is inhibited in iron deficiency, hypoxia and when erythropoiesis is increased. Inhibitors are the liver transmembrane serine protease TMPRSS6, whose genetic inactivation causes IRIDA, and the erythroid hormone erythroferrone (ERFE), which is released by erythropoietin-stimulated erythroblasts. The mechanism of hepcidin inhibition by ERFE is unclear; still to allow ERFE function the BMP-SMAD pathway has not to be hyperactive. Intriguingly, both iron deficiency and erythropoiesis require epigenetic modifications at the hepcidin locus with HDAC3-dependent reversible loss of H3K9ac and H3K4me3.Hepcidin also acts as an antimicrobial peptide since its expression, increased by proinflammatory cytokines, such as IL6 through JAK2-STAT3 signaling, restricts iron availability for microbial growth. This first-line of defense against infections negatively influences erythropoiesis since chronic hepcidin activation causes AI. Despite persistent JAK2-STAT3 activation, inhibition of the BMP-SMAD pathway reduces hepcidin activation in AI experimental rodent models, suggesting that hepcidin activation in inflammation requires a functional BMP-SMAD pathway. Independently from hepcidin, inflammation also reduces FPN mRNA levels, favoring macrophage iron sequestration.The identification of hepcidin-ferroportin axis molecular players has translational implications. In primary and secondary iron overload hepcidin agonists (hepcidin peptides or mimics, agents that inhibit the hepcidin inhibitor TMPRSS6 and likely the ALK2-inhibitor FKBP12) and ferroportin inhibitors are potentially useful to prevent iron overload and/or to favor iron redistribution to macrophages. In case of AI, hepcidin antagonists (including anti-hepcidin, anti-HJV and anti-BMP6 monoclonal antibodies, L-enantiomeric oligonucleotides targeting hepcidin, siRNA against hepcidin, non-anticoagulant heparins, the ALK2 inhibitor momelotinib) might improve erythropoiesis increasing iron availability.The effect of some agents that have now entered the clinical phase will become apparent in the coming years. DisclosuresCamaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Inhibiting the Immunophilin FKBP12: A Potential Therapeutic Approach to Iron Overload/Low Hepcidin Disorders

IntroductionHepcidin negatively regulates body iron by binding and degrading the iron exporter ferroportin. Its expression is mainly controlled by the liver BMP-SMAD pathway whose activation requires BMP ligands (BMP2 and BMP6), constitutively active BMP type II receptors and the type I receptors ALK2 and ALK3. The co-receptor hemojuvelin (HJV) further potentiates the signaling. Mutations in key genes of the pathway impair hepcidin synthesis in Hereditary Hemochromatosis (HH), one of the most severe form being due to HJV mutations. As current therapies are symptomatic and do not correct hepcidin deficiency, alternative targeted therapies to increase hepcidin production are needed.We have demonstrated that the immunophilin FKBP12 binds ALK2 in hepatoma cells to prevent uncontrolled activation of the BMP pathway in the absence of ligands. We also demonstrated that FKBP12 sequestration by immunosuppressive drugs, such as FK506 (tacrolimus, TAC) or rapamycin, upregulate hepcidin in vitro. The role of FKBP12 is maintained in vivo since acute TAC treatment of wild type (WT) mice increases hepcidin expression (Colucci et al., Blood 2017).The aim of this study is to investigate whether pharmacologic inactivation of FKBP12, by TAC or antisense oligonucleotides (ASO), upregulates hepcidin for therapeutic purposes.MethodsPrimary hepatocytes isolated from WT, Hjv and Tfr2 KO mice (sv129/j background) were treated with increasing concentrations of TAC. Hepcidin and Id1 expression was investigated by qRT-PCR. Nine-weeks-old Hjv KO male mice were treated for 28 days with TAC (0.37 mg/h) delivered through surgically implanted mini-osmotic pumps. Six-weeks-old WT mice, were treated twice a week for 6 weeks with 50 mg/kg of Fkbp12 or control ASO. Mice were sacrificed and analyzed for iron, CBC, erythropoiesis and liver expression of hepcidin and BMP-SMAD target genes.ResultsTAC treatment of primary HCs from Hjv KO (Colucci et al., Blood 2018) and Tfr2 KO mice upregulates hepcidin as in WT mice, suggesting that HJV and TFR2 are dispensable for FKBP12-dependent hepcidin regulation and providing the proof of principle for FKBP12 targeting in HH. First, we explored a drug repurposing approach in the severe Hjv KO mice by chronic subcutaneous delivery of suboptimal, non-immunosuppressive TAC doses. Treatment of Hjv KO mice with TAC upregulates hepcidin via BMP-SMAD pathway activation, as assessed by Id1 and Smad7 upregulation. Since TAC also inhibits calcineurin upon FKBP12 sequestration and to avoid potential off-targets effect, FKBP12 was inactivated by ASO. Fkbp12 ASO treatment of WT mice decreases Fkbp12 expression by about 70-80% in liver, spleen and kidney, but not in the bone marrow. Fkbp12 ASO-treated mice exhibit microcytic anemia, decreased serum iron and upregulation of liver BMP-SMAD target genes. However, hepcidin remains inappropriately high considering the low serum iron. Fkbp12 ASO-treated mice show increased spleen immature erythroid precursors and increased expression of erythroferrone (Erfe). This is due to the unexpected effect of FKBP12 on spleen erythropoiesis and likely explains the lack of hepcidin upregulation.ConclusionsChronic TAC treatment in Hjv KO mice, which causes FKBP12 sequestration, improves hepcidin expression via BMP-SMAD pathway and favors spleen iron retention, suggesting that this “drug repurposing approach” may be beneficial for all iron overload disorders exhibiting low hepcidin.ASO-Fkbp12 in WT mice efficiently downregulates hepatic Fkbp12, upregulates the BMP-SMAD pathway but leaves hepcidin unchanged compared to control mice. We hypothesize that this result reflects the concomitant hepcidin inhibition due to decreased serum iron and increased Erfe expression. The latter is a consequence of spleen Fkbp12 reduction in ASO-treated mice, suggesting that a hepatocyte targeting Fkbp12 ASO is required for therapeutic purposes.Since Fkbp12 binds and inhibits ALK2 (Colucci et al., Blood 2017), our in vitro and in vivo data suggest that HJV and TFR2 functionally interact with ALK3 but not with ALK2. DisclosuresAghajan:Ionis Pharmaceuticals, Inc: Employment. Guo:Ionis Pharmaceuticals, Inc: Employment. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe.Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate >2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation.Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone.Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis. DisclosuresJasuja:Pfizer: Employment. Sawant:Pfizer: Employment. Pittman:Pfizer: Employment. Quan:Pfizer: Employment.

Iron homeostasis in pregnancy and spontaneous abortion.

During pregnancy, iron requirements are increased to support maternal erythropoietic expansion and fetal growth and development. To meet these requirements, dietary iron absorption increases, and available iron stores are mobilized. These adjustments are thought to be in large part mediated by the iron-regulatory hormone hepcidin, which controls the concentrations of ferroportin, the sole exporter of iron into the extracellular fluid and blood plasma. Hepcidin regulation of iron availability during healthy and abnormal pregnancies is not well understood. In our cross-sectional study, we compared hepcidin, iron and hematological parameters between nonpregnant control women, healthy pregnant women in the first and second trimester, and women with spontaneous abortion in the first trimester. We found that in healthy pregnancy, hepcidin increased in the first trimester compared with nonpregnant women, but then decreased during the second trimester. The second trimester hepcidin levels decreased despite stable serum iron concentrations, suggesting active suppression of hepcidin, presumably to enhance iron availability as iron demand increases. In women with spontaneous abortion during the first trimester, hepcidin, serum iron, and ferritin concentrations were all increased compared with the first trimester healthy pregnancy. Although the specific mechanisms remain to be determined, our findings demonstrate that maternal hepcidin is regulated by signals related to the progression of pregnancy, and that pregnancy loss is associated with profound changes in maternal iron metabolism. These observations highlight the existence of fetoplacental signals that modulate maternal iron homeostasis.

Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice.

Erythropoietin (EPO) acts on erythroid progenitor cells to promote their survival and differentiation to mature erythrocytes. Along with this canonical role, EPO is also reported to modulate energy metabolism, resulting in improved glucose tolerance and insulin sensitivity. EPO also stimulates the production of the hormone erythroferrone (ERFE) which acts to suppress hepcidin production, thus increasing dietary iron absorption and mobilizing stored iron for use in erythropoiesis. ERFE (initially termed myonectin) was also reported have an effect on systemic lipid metabolism by promoting the clearance of nonesterifed fatty acids (NEFA) from circulation. As increased levels of circulating NEFA blunt insulin sensitivity and impair glucose tolerance, ERFE‐induced clearance of NEFA after EPO administration would have a beneficial effect on glucose metabolism. The aim of this study was to determine if the known metabolic effect of EPO treatment on glucose homeostasis is mediated by ERFE, produced in response to EPO. Mice lacking Erfe did not differ from wild‐type mice in blood lipid parameters, blood glucose, and glucose or insulin tolerance at baseline or after chronic EPO treatment. Additionally, hepcidin suppression and the response of erythrocyte parameters to chronic EPO treatment were unaffected by the absence of Erfe. These findings suggest that the known beneficial effects of EPO on glucose metabolism are not attributable to an accompanying increase in ERFE production, and that Erfe is dispensable for normal glucose homeostasis. Furthermore, our data indicate that ERFE‐independent mechanisms can suppress hepcidin in response to chronically elevated EPO levels.