Search

Background: Ferritin, the central iron storage protein, has attracted attention as a biomarker of severe COVID-19. Few studies have investigated regulators of iron metabolism in the context of COVID-19. The aim was to evaluate biomarkers for iron metabolism in the acute phase response to community-acquired pneumonia (CAP) caused by SARS-CoV-2 compared to CAP caused by bacteria or influenza virus in hospitalized patients.Methods: A cross-sectional study of 164 patients from the Surviving Pneumonia Cohort recruited between January 8, 2019 and May 26, 2020. Blood samples were collected at admission and analyzed for levels of C-reactive protein (CRP), ferritin, soluble transferrin receptor, erythroferrone, and hepcidin.Results: Median (IQR) hepcidin was higher in SARS-CoV-2 with 143.8 (100.7-180.7) ng/mL compared to bacterial and influenza infection with 78.8 (40.1-125.4) and 53.5 (25.2-125.8) ng/mL, respectively. The median ferritin level was more than 2-fold higher in patients with SARS-CoV-2 compared to the other etiologies (pConclusion: Higher levels of hepcidin and lower levels of erythroferrone despite lower CRP levels among patients with SARS-CoV-2 compared to those infected with bacteria indicate alterations in iron metabolism in patients with SARS-CoV-2 infection.

Purpose: We investigated whether hepcidin and erythroferrone (ERFE) could complement the Athlete Biological Passport (ABP) in indirectly detecting a 130 mL packed red blood cells (RBCs) autologous blood transfusion. Endurance performance was evaluated.Methods: Forty-eight healthy men (n = 24) and women (n = 24) participated. Baseline samples were collected weekly followed by randomization to a blood transfusion (BT, n = 24) or control group (CON, n = 24). Only the BT group donated 450 mL whole blood from which 130 mL RBCs was reinfused four weeks later. Blood samples were collected 3, 7, 14, 21 and 28 days after donation, and 3, 6, and 24 hours and 2, 3, and 6 days following reinfusion. In the CON group samples were collected with the same frequency. Endurance performance was evaluated by a 650-kCal time trial (n = 13) before and one and six days after reinfusion.Results: A time×treatment effect existed (P < 0.05) for hepcidin and ERFE. Hepcidin was increased (P < 0.01) ~110 and 89% six and 24 hours after reinfusion. Using an individual approach (99% specificity, e.g. allowing 1:100 false-positive), sensitivities, i.e. true positives, of 30% and 61% was found for hepcidin and ERFE, respectively. For the ABP, the most sensitive marker was Off-hr score ([Hb] (g·L-1) - 60 × √RET%) (P < 0.05) with a maximal sensitivity of ~58% and ~ 9% following donation and reinfusion, respectively. Combining the findings for hepcidin, ERFE and the ABP yielded a sensitivity across all time-points of 83% following reinfusion in BT. Endurance performance increased 24 hours (+6.4%, P < 0.01) and six days following reinfusion (+5.8%, P < 0.01).Conclusions: Hepcidin and ERFE may serve as biomarkers in an anti-doping context following an ergogenic, small-volume blood transfusion.

Background In ineffective erythropoiesis, hepcidin synthesis is suppressed by erythroid regulators, namely erythroferrone and growth differentiation factor-15. For the first time, the hypothesis that iron overload in megaloblastic anemia may be related to ineffective erythropoiesis is explored by describing the kinetics of hepcidin, erythroferrone, and growth differentiation factor-15 levels in a patient diagnosed with megaloblastic anemia associated with iron overload. Case presentation An 81-year-old Caucasian male was admitted for fatigue. He had type-2 diabetes previously treated with metformin, ischemic cardiac insufficiency, and stage-3 chronic kidney disease. Vitiligo was observed on both hands. Biological tests revealed normocytic non-regenerative anemia associated with hemolysis, thrombocytopenia, and elevated sideremia, ferritin, and transferrin saturation levels. Megaloblastic anemia was confirmed with undetectable blood vitamin B12 and typical cytological findings like hyper-segmented neutrophils in blood and megaloblasts in bone marrow. The patient received vitamin B12 supplementation. At 3 months, biological parameters reached normal values. Hepcidin kinetics from diagnosis to 3 months inversely correlated with those of erythroferrone and growth differentiation factor-15. Conclusions This case suggests that iron-overload mechanisms of dyserythropoietic anemias may apply to megaloblastic anemias.

Soha R Youssef,1 Esraa H Hassan,2 Caroline S Morad,3 Adel A Elazab Elged,1,4 Rasha A El-Gamal1 1Clinical Pathology Department, Ain Shams University, Cairo, Egypt; 2Regional Blood Transfusion Center, Cairo, Egypt; 3Internal Medicine and Rheumatology Department, Ain Shams University, Cairo, Egypt; 4Galala University, Suez, EgyptCorrespondence: Rasha A El-GamalFaculty of Medicine, Ain Shams University, Abbasseya, Cairo, EgyptTel +20 1145405005Email rashaelgamal@med.asu.edu.egPurpose: Erythroferrone (ERFE) is well acknowledged for its inhibitory function on hepcidin synthesis in the liver during stress erythropoiesis, thereby ensuring sufficient iron supply to bone marrow erythroblasts. Hepcidin plays an indispensable role in the pathogenesis of anemia of chronic disease (ACD). Thus, ERFE was suggested to protect against ACD in various diseases. Rheumatoid arthritis (RA) is commonly involved with ACD and high hepcidin levels, with a further increase of the latter in active states. The present study is a case-control study that aimed to determine the pattern of ERFE expression in RA patients with concomitant ACD and study its relationship with hepcidin, erythropoietin (EPO) and disease activity.Patients and Methods: Fifty-five RA patients with ACD were categorized into active and inactive RA using the disease activity score (DAS28); 15 healthy subjects were included as control subjects. ERFE was measured for patients and control subjects using quantitative real-time polymerase chain reaction, in addition to testing for CBC, ESR, CRP, iron profile parameters and hepcidin. EPO was assessed for patients of both active and inactive RA groups.Results: ERFE and hepcidin showed the highest levels in active RA; ERFE values were similar in control subjects and inactive RA patients, while hepcidin was significantly higher in inactive RA than control subjects. Patients with high ERFE levels had higher RBC, Hct, MCV, hepcidin and EPO levels. Stepwise regression analysis has identified DAS28 and disease duration as the best predictors of ERFE values, whereas ERFE and hepcidin were independent predictors of disease activity.Conclusion: We introduce ERFE as a novel marker of RA activity. Although the inhibitory effect of ERFE on hepcidin is not evident, our results still indicate that ERFE may have a beneficial erythropoietic effect in the context of ACD in RA disease activity.Keywords: erythroferrone, rheumatoid arthritis, anemia of chronic disease, hepcidin, iron, DAS28

BACKGROUND: The developing fetus requires adequate iron and produces its own hormones to regulate this process. Erythroferrone (ERFE) is a recently identified iron regulatory hormone, and normative data on ERFE concentrations and relations between iron status and other iron regulatory hormones at birth are needed. OBJECTIVES: The objective of this study was to characterize cord ERFE concentrations at birth and assess interrelations between ERFE, iron regulatory hormones, and iron status biomarkers in 2 cohorts of newborns at higher risk of neonatal anemia. METHODS: Umbilical cord ERFE concentrations were measured in extant serum samples collected from neonates born to women carrying multiples (age: 21–43 y; n = 127) or teens (age: 14–19 y; n = 164). Relations between cord blood ERFE and other markers of iron status or erythropoiesis in cord blood were assessed by linear regression and mediation analysis. RESULTS: Cord ERFE was detectable in all newborns delivered between 30 and 42 weeks of gestation, and mean concentration at birth was 0.73 ng/mL (95% CI: 0.63, 0.85 ng/mL). Cord ERFE was on average 0.25 ng/mL lower in newborns of black as opposed to white ancestry (P = 0.04). Cord ERFE was significantly associated with transferrin receptor (β: 1.17, P 25% of variance explained). CONCLUSIONS: Neonates born to teens and women carrying multiples were able to produce ERFE in response to neonatal cord iron status and erythropoietic demand. ERFE, however, did not capture significant variance in newborn iron or Hb concentrations. In these newborns, cord hepcidin and the hepcidin:EPO ratio explained the most variance in iron status indicators at birth.

Current markers of iron deficiency (ID) such as ferritin and hemoglobin have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a 4-wk baseline, a 4-wk intervention at either sea level or altitude, and a 4-wk follow-up. Participants (n=39) were randomly assigned to 20 IU·kg body wt-1 rHuEPO or placebo injections every second day for 3 wk during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE (P ≤ 0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE (P < 0.05) and hepcidin levels (P < 0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared to altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin (P < 0.05) and ERFE (P ≤ 0.001) parallel with increases in hematocrit (P < 0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found (R2 = 0.13, P < 0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an anti-doping context.Clinical trial registration: Clinical trials identifier, NCT04227665

BACKGROUND: Maintaining adequate iron status during pregnancy is important for the mother and her developing fetus. Iron homeostasis is influenced by 3 regulatory hormones: erythropoietin (EPO), hepcidin, and erythroferrone (ERFE). To date, normative data on ERFE across pregnancy and its relations to other hormones and iron status indicators are limited. OBJECTIVES: The objective of this study was to characterize maternal ERFE across pregnancy and at delivery and evaluate the utility of hepcidin, ERFE, and EPO in identifying women with increased iron needs. METHODS: ERFE was measured in extant serum samples collected from 2 longitudinal cohorts composed of women carrying multiple fetuses (n = 79) and pregnant adolescents (n = 218) at midgestation (∼26 wk) and delivery (∼39 wk). Receiver operating characteristic curves were generated to characterize the predictive ability of serum ERFE, hepcidin, and EPO and their ratios to identify women at increased risk of iron deficiency and anemia. RESULTS: In these pregnant women, mean ERFE was 0.48 ng/mL at both ∼25 wk of gestation and at delivery. ERFE was positively associated with EPO at midgestation (β = 0.14, P = 0.002, n = 202) and delivery (β = 0.12, P

The large amount of iron required for hemoglobin synthesis keeps iron homeostasis and erythropoiesis inter-connected, both iron levels being affected by increased erythropoiesis, and erythropoiesis regulated by serum iron. The connection between these 2 processes is maintained even when erythropoiesis is ineffective. In the last years great advances in the understanding of the mechanisms of this crosstalk have been achieved thanks to the discovery of 2 essential players: hepcidin, the master regulator of iron homeostasis, and erythroferrone, the long sought erythroid regulator. In addition, how circulating transferrin-bound iron contributes to the crosstalk between the 2 systems has started to be unraveled.

Vadadustat is an investigational hypoxia-inducible factor prolyl hydroxylase inhibitor that increases endogenous erythropoietin production and has been shown to decrease hepcidin levels, ameliorate iron restriction, and increase hemoglobin concentrations in anemic patients with chronic kidney disease (CKD). In studies of physiological responses to other erythropoietic stimuli, erythropoietin induced erythroblast secretion of erythroferrone (ERFE), which acts on the liver to suppress hepcidin production and mobilize iron for erythropoiesis. We therefore investigated whether vadadustat effects on erythropoiesis and iron metabolism are dependent on ERFE. Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Erythroferrone (ERFE) is the main erythroid regulator of hepcidin, the homeostatic hormone controlling plasma iron levels and total body iron. When the release of erythropoietin from the kidney stimulates the production of new red blood cells, it also increases the synthesis of ERFE in bone marrow erythroblasts. Increased ERFE then suppresses hepcidin synthesis, thereby mobilizing cellular iron stores for use in heme and hemoglobin synthesis. Recent mechanistic studies have shown that ERFE suppresses hepcidin transcription by inhibiting bone morphogenetic protein signaling in hepatocytes. In ineffective erythropoiesis, pathological overproduction of ERFE by an expanded population of erythroblasts suppresses hepcidin and causes iron overload, even in non-transfused patients. ERFE may be a useful biomarker of ineffective erythropoiesis and an attractive target for treating its systemic effects.

Introduction To clarify mechanisms of ineffective erythropoiesis on iron metabolism, studies on erythroid factors that regulating hepcidin suppression have been carried out. The aim of the current study is to identify associations between erythropoiesis and iron homeostasis parameters in β-thalassemias. Materials and methods This study consisted of 83 subjects: 21 thalassemia major (TM), 20 thalassemia intermedia (TI), 20 thalassemia trait (TT), and 22 healthy subjects (HS). Erythroferrone (ERFE), hepcidin, growth differentiation factor-15 (GDF15), erythropoietin (EPO), and iron status parameters were measured. Results Our results showed that TM and TI patients had higher hepcidin than the TT and control groups. The hepcidin/ferritin in TM patients was significantly lower than the other groups. GDF15 in TM and TI patients was significantly higher than in the TT and control groups. Also, TI group had significantly higher ERFE concentration and EPO activity when compared with the TM, TT, and HS groups. EPO activity showed positive correlation with ERFE and GDF15 concentrations. We could not find any correlation between ERFE and hepcidin concentrations. Conclusions ERFE may be one of the parameters used to demonstrate erythropoietic activity level in thalassemias. More detailed studies are needed to clarify the role of ERFE in iron metabolism in the patients with thalassemias.

Part 1: New crossroads. The first part describes two new concepts in an attempt to unravel novel pathophysiological features of hereditary anemias. Chapter 2 reviews the pathophysiological aspects of pyruvate kinase (PK) deficiency, focusing on the interplay between PK-activity and reticulocyte maturation, stretching the importance of adequate oxidative control for normal mitophagy, which forms the fulfilling step of reticulocyte maturation. We propose late-stage ineffective erythropoiesis as a key pathophysiological concept in pyruvate kinase deficiency. Chapter 3 and 4 focusses on the erythropoietin (EPO)-fibroblast growth factor 23 (FGF23) signaling pathway in hereditary anemias. This, only briefly, previously described link between EPO and FGF23 may add to the understanding of (insufficient) EPO upregulation or responsiveness in hereditary anemias, as well as the bone mineralization disorders complicating these diseases. We speculate on a potential role for this pathway, not directly linked to erythroferrone (ERFE), as a target for treatment with the opportunity to ameliorate ineffective erythropoiesis and development of disease complications. Part 2: The challenge called heterogeneity. This part includes three articles that discuss unique disease characteristics and unique phenotypic appearances. Sickle cell disease earned a unique place among the hemolytic anemias as an important part of hemolysis does not occur extravascular (e.g. in the spleen or liver), but in the (micro)circulation itself. The relative balance between intra- and extravascular hemolysis seems to determine one’s risk of vasculopathic disease complications. In chapter 5 we introduce a ratio of two laboratory markers that represents the contribution of intravascular hemolysis to total hemolysis in an individual sickle cell disease patient, and this simple ratio was clearly associated with pulmonary hypertension and early death. Chapter 6 describes the unique monocyte transcriptome of sickle cell disease patients, sketching the balance of both pro- and anti-inflammatory pathways. In this chapter, we highlight the leading role of heme-oxygenase 1, an enzyme that breaks down heme, in determining this balance. Chapter 7 describes our attempt to untangle the relation between genotypes and phenotypes in hereditary spherocytosis. Which turned out to be an unproductive attempt. This led us speculating on the importance of other (genetic and non-genetic) modifying factors in determining phenotypic profiles. Part 3: About iron. This part focusses on the interplay between iron and erythropoiesis. Chapter 8 functions as a bridge between part 2 and part 3, as it reports on disease- specific patterns of the EPO-ERFE-hepcidin signaling pathway regulating iron. However, with the notion that broad inter-individual variety exists in each disease. Chapter 9 reflects on old, previously unpublished iron absorption and ferrokinetic data. This data provides an answer on the question what happens with intestinally absorbed iron in an already saturated portal circulation. And finally, chapter 10 describes the results of the PPI shine again study, a phase III randomized, placebo-controlled trial in a cross-over design investigating the efficacy of proton pump inhibitors in the prevention and treatment of iron-loading in patients with non-transfusion-dependent hereditary anemias.

Part 1: New crossroads. The first part describes two new concepts in an attempt to unravel novel pathophysiological features of hereditary anemias. Chapter 2 reviews the pathophysiological aspects of pyruvate kinase (PK) deficiency, focusing on the interplay between PK-activity and reticulocyte maturation, stretching the importance of adequate oxidative control for normal mitophagy, which forms the fulfilling step of reticulocyte maturation. We propose late-stage ineffective erythropoiesis as a key pathophysiological concept in pyruvate kinase deficiency. Chapter 3 and 4 focusses on the erythropoietin (EPO)-fibroblast growth factor 23 (FGF23) signaling pathway in hereditary anemias. This, only briefly, previously described link between EPO and FGF23 may add to the understanding of (insufficient) EPO upregulation or responsiveness in hereditary anemias, as well as the bone mineralization disorders complicating these diseases. We speculate on a potential role for this pathway, not directly linked to erythroferrone (ERFE), as a target for treatment with the opportunity to ameliorate ineffective erythropoiesis and development of disease complications. Part 2: The challenge called heterogeneity. This part includes three articles that discuss unique disease characteristics and unique phenotypic appearances. Sickle cell disease earned a unique place among the hemolytic anemias as an important part of hemolysis does not occur extravascular (e.g. in the spleen or liver), but in the (micro)circulation itself. The relative balance between intra- and extravascular hemolysis seems to determine one’s risk of vasculopathic disease complications. In chapter 5 we introduce a ratio of two laboratory markers that represents the contribution of intravascular hemolysis to total hemolysis in an individual sickle cell disease patient, and this simple ratio was clearly associated with pulmonary hypertension and early death. Chapter 6 describes the unique monocyte transcriptome of sickle cell disease patients, sketching the balance of both pro- and anti-inflammatory pathways. In this chapter, we highlight the leading role of heme-oxygenase 1, an enzyme that breaks down heme, in determining this balance. Chapter 7 describes our attempt to untangle the relation between genotypes and phenotypes in hereditary spherocytosis. Which turned out to be an unproductive attempt. This led us speculating on the importance of other (genetic and non-genetic) modifying factors in determining phenotypic profiles. Part 3: About iron. This part focusses on the interplay between iron and erythropoiesis. Chapter 8 functions as a bridge between part 2 and part 3, as it reports on disease- specific patterns of the EPO-ERFE-hepcidin signaling pathway regulating iron. However, with the notion that broad inter-individual variety exists in each disease. Chapter 9 reflects on old, previously unpublished iron absorption and ferrokinetic data. This data provides an answer on the question what happens with intestinally absorbed iron in an already saturated portal circulation. And finally, chapter 10 describes the results of the PPI shine again study, a phase III randomized, placebo-controlled trial in a cross-over design investigating the efficacy of proton pump inhibitors in the prevention and treatment of iron-loading in patients with non-transfusion-dependent hereditary anemias.

Iron is necessary for essential processes in every cell of the body, but the erythropoietic compartment is a privileged iron consumer. In fact, as a necessary component of hemoglobin and myoglobin, iron assures oxygen distribution; therefore, a considerable amount of iron is required daily for hemoglobin synthesis and erythroid cell proliferation. Therefore, a tight link exists between iron metabolism and erythropoiesis. The liver-derived hormone hepcidin, which controls iron homeostasis via its interaction with the iron exporter ferroportin, coordinates erythropoietic activity and iron homeostasis. When erythropoiesis is enhanced, iron availability to the erythron is mainly ensured by inhibiting hepcidin expression, thereby increasing ferroportin-mediated iron export from both duodenal absorptive cells and reticuloendothelial cells that process old and/or damaged red blood cells. Erythroferrone, a factor produced and secreted by erythroid precursors in response to erythropoietin, has been identified and characterized as a suppressor of hepcidin synthesis to allow iron mobilization and facilitate erythropoiesis.

Hanudel et al. investigated the effects of hypoxia-inducible factor-prolyl hydroxylase domain inhibitors (HIF-PHIs) on iron metabolism in a chronic kidney disease (CKD) mouse model and showed that vadadustat, an HIF-PHI, exerted beneficial effects on anemia and iron disorders independently of erythroferrone. Vadadustat also inhibited the progression of CKD and the CKD-associated increase of plasma fibroblast growth factor 23 in CKD mice. This study provides new insights into the action of HIF-PIHs in CKD.

Erythroferrone (ERFE) is a hormone produced by erythroblasts in the bone marrow in response to erythropoietin controlling iron storage release through its actions on hepcidin, which acts on hepatocytes to suppress expression of the hormone hepcidin. Erythroferrone now considered is one of potential clinical biomarkers for assessing erythropoiesis activity in patients with blood disorders regarding to iron imbalance. Since discovery of in 2014 by Dr. Leon Kautz and colleagues and till now no more enough studies in Erythroferrone among human, most studies are conducted in animals. In this review we briefly address the Role of Erythroferrone hormone as erythroid regulator of hepcidin and iron metabolism during thalassemia and in iron deficiency anemia. Studies in this review were identified through a search using the following electronic databases: PubMed, Academia, Scopus, Google Scholar, and another open database source. Conclusion: Most of studies concluded that, in people with thalassemia and iron deficiency anemia, erythroferrone levels in the blood are higher than in people without thalassemia and iron deficiency anemia. Knowing the mechanisms of erythroferrone as erythroid regulator of hepcidin and iron metabolism during thalassemia and in iron deficiency anemia important in the diagnosis and treatment for both conditions. The erythroferrone hormone may act as potential factor in physiological hepcidin suppressor in cases with iron deficiency anemia and thalassemia disease and play a key role in treatment process among those patients in status of iron deficiency or iron overload. However, till now few studies of the function of ERFE in humans because is recently discovered and remains to be investigated and most studies are conducted among animals.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow–derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

Iron is the most abundant transition metal in the human body and an essential element required for growth and survival. Our understanding of the molecular control of iron metabolism has increased dramatically over the past 20 years due to the discovery of hepcidin, which regulates the uptake of dietary iron and its mobilization from macrophages and hepatic stores. Anemia and iron deficiency are common in chronic kidney disease. The pathogenesis of anemia of chronic kidney disease is multifactorial. Correction of anemia requires two main treatment strategies: increased stimulation of erythropoiesis, and maintenance of an adequate iron supply to the bone marrow. However, there are still many uncertainties in regard to iron metabolism in patients with chronic kidney disease and in renal replacement therapy. The aim of this review was to summarize the current knowledge on iron metabolism in this population, including new biomarkers of iron status. There is an area of uncertainty regarding diagnostic utility of both erythroferrone (ERFE) and hepcidin in end-stage renal disease (ESRD) patients. Higher concentration of hepcidin in oligoanuric patients may reflect decreased renal clearance. Furthermore, the hepcidin-lowering effect of ERFE in ESRD patients treated with erythropoiesis-stimulating agents (ESAs) may be blunted by underlying inflammation and concomitant iron treatment. Thus, future studies should validate the use of ERFE as a biomarker of erythropoiesis and predictor of response to iron and ESA therapy in dialysis-dependent patients.

Spontaneous abortion is a common, detrimental outcome of pregnancy, and can be induced by a variety of factors, including pathophysiological conditions and socioeconomic circumstances. Despite numerous studies examining the aetiology of spontaneous abortion, there is limited understanding of the disordered iron transportation between mother and fetus through the placenta. Recently, erythroferrone (ERFE) was recognized as a novel negative regulator of hepcidin that can elevate nutritional iron absorption and macrophagic iron egress for enhanced erythropoiesis. However, its diagnostic significance in different disease conditions associated with iron remains poorly understood. In the current study, we discovered disordered maternal iron homeostasis in women who had spontaneous abortions during early pregnancy, as characterized by increased serum iron and hepcidin levels, and conversely, reduced serum ERFE levels, compared to healthy control individuals and women with normal pregnancy. Comprehensive statistical analyses revealed the correlation between different variables and pregnancy status, signifying the pronounced diagnostic value of an increased ratio of serum hepcidin and ERFE (HE ratio) in recognizing adverse pregnancy status. In contrast to previous non-selective discrete surrogates, such as iron, hepcidin and ferritin, the HE ratio may otherwise stand for a novel and more representative hallmark for early spontaneous abortion.

Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic processes, biochemical characterization of the protein is lacking. Here, we show that multiple protein modifications are critical for myonectin secretion and multimerization. Abolishing N-linked glycosylation by tunicamycin, glucosamine supplementation, or glutamine substitutions of all four potential Asn glycosylation sites blocked myonectin secretion. Mass spectrometry confirmed that Asn-229 and Asn-281 were glycosylated, and substituting both Asn sites with Gln prevented myonectin secretion. Although Asn-319 is not identified as glycosylated, Gln substitution caused protein misfolding and retention in the endoplasmic reticulum. Of the four conserved cysteines, Cys-273 and Cys-278 were required for proper protein folding; Ala substitution of either site inhibited protein secretion. In contrast, Ala substitutions of Cys-142, Cys-194, or both markedly enhanced protein secretion, suggesting endoplasmic reticulum retention that facilitates myonectin oligomer assembly. Secreted myonectin consists of trimers, hexamers, and high-molecular weight (HMW) oligomers. The formation of higher-order structures via intermolecular disulfide bonds depended on Cys-142 and Cys-194; while the C142A mutant formed almost exclusively trimers, the C194A mutant was impaired in HMW oligomer formation. Most Pro residues within the short collagen domain of myonectin were also hydroxylated, a modification that stabilized the collagen triple helix. Inhibiting Pro hydroxylation or deleting the collagen domain markedly reduced the rate of protein secretion. Together, our results reveal key determinants that are important for myonectin folding, secretion, and multimeric assembly and provide a basis for future structure–function studies.

Background This study aimed to determine associations among short- and long-acting erythropoiesis stimulating agents (ESAs), changes in serum fibroblast growth factor 23 (FGF23) and biomarkers of iron metabolism. Methods Among 108 patients on hemodialysis (HD), 44 received every 2 weeks or monthly doses of continuous erythropoiesis receptor activator (CERA), 31 received weekly doses of darbepoetin-α, 24 received three doses per week of epoetin-β and 9 were not treated with an ESA. Intact and C-terminal FGF23 and transferrin saturation (TSAT), ferritin, erythroferrone and hepcidin 25 were measured in blood samples collected before the HD session at the end of the dialysis week (baseline, Day 0) and on Days 3, 5, 7 and 14 thereafter. Results Levels of ferritin, hepcidin 25 and erythroferrone as well as TSAT were significantly decreased or elevated in patients treated with CERA compared with other types of ESAs. Levels of C-terminal FGF23 increased in all groups during the observation period. Levels of intact FGF23 and ratios of intact FGF23 to C-terminal FGF23 gradually decreased between Days 3 and 7 in the CERA but not in the other groups. Multivariate models associated changes in hepcidin 25 and phosphate with those of intact FGF23. Conclusion The long-acting ESA CERA might influence levels of intact FGF23 by increasing FGF23 cleavage in patients on HD in association with prolonged hepcidin 25 suppression.

Background Erythroferrone (ERFE) is an erythroid hormone putatively involved in stress erythropoiesis. Its regional clearance and circulating form in humans, as well as levels in normal health and coronary disease remain unclear. Methods To establish a reference interval, ERFE was measured in 155 healthy volunteers using the Intrinsic LifeSciences ELISA. To identify trans-organ gradients in ERFE, regional blood sampling was undertaken in patients (n = 13) undergoing clinically indicated cardiac catheterisation. The Intrinsic ELISA was assessed for reproducibility, stability, linearity and possible cross-reactivity, interference and anticoagulant effects. Circulating forms of ERFE were evaluated by HPLC. Results In healthy individuals, the median concentration of ERFE was 0.51 ng/mL (IQR: 0.12–1.25), with men (n = 78) having higher levels than women (n = 77) (0.67 vs 0.32 ng/mL, p = 0.0001). ERFE concentrations in trans-organ sampling revealed no clear organ of clearance or production. Samples with high endogenous ERFE levels were suppressed by haemoglobin (≥2 g/L), bilirubin (≥200 µmol/L), lipaemia (>1 g/L), and freeze thawing (≥2 cycles), but this was not observed with low ERFE concentrations. Endogenous ERFE immunoreactivity was 46% higher in EDTA plasma compared with serum and lithium heparin plasma. On SE-HPLC, ERFE eluted as intact and cleaved forms. Conclusion We provide a useful reference range for ERFE in EDTA plasma. We found no specific site of secretion or clearance. The Intrinsic ELISA performed adequately but is limited by interference and stability when endogenous levels are high. Circulating forms are multiple and complex.

Recently, erythropoietin (EPO) was identified as regulator of fibroblast growth factor 23 (FGF23). Proteolytic cleavage of biologically active intact FGF23 (iFGF23) results in the formation of C-terminal fragments (cFGF23). An increase in cFGF23 relative to iFGF23 suppresses FGF receptor signaling by competitive inhibition. EPO lowers the i:cFGF23 ratio, thereby overcoming iFGF23-mediated suppression of erythropoiesis. We investigated EPO-FGF23 signaling and levels of erythroferrone (ERFE) in 90 patients with hereditary hemolytic anemia (www.trialregister.nl [NL5189]). We show, for the first time, the importance of EPO-FGF23 signaling in hereditary hemolytic anemia: there was a clear correlation between total FGF23 and EPO levels (r = +0.64; 95% confidence interval [CI], 0.09-0.89), which persisted after adjustment for iron load, inflammation, and kidney function. There was no correlation between iFGF23 and EPO. Data are consistent with a low i:cFGF23 ratio. Therefore, as expected, we report a correlation between EPO and ERFE in a diverse set of hereditary hemolytic anemias (r = +0.47; 95% CI, 0.14-0.69). There was no association between ERFE and total FGF23 or iFGF23, which suggests that ERFE does not contribute to the connection between FGF23 and EPO. These findings open a new area of research and might provide potentially new druggable targets with the opportunity to ameliorate ineffective erythropoiesis and the development of disease complications in hereditary hemolytic anemias.

Hypoferremia results as an acute phase response to infection and inflammation aiming to reduce iron availability to pathogens. Activation of toll-like receptors (TLRs), the key sensors of the innate immune system, induces hypoferremia mainly through the rise of the iron hormone hepcidin. Conversely, stimulation of erythropoiesis suppresses hepcidin expression via induction of the erythropoietin-responsive hormone erythroferrone. Iron deficiency stimulates transcription of the osteocyte-secreted protein FGF23. Here we hypothesized that induction of FGF23 in response to TLR4 activation is a potent contributor to hypoferremia and, thus, impairment of its activity may alleviate hypoferremia induced by lipopolysaccharide (LPS), a TLR 4 agonist. We used the C-terminal tail of FGF23 to impair endogenous full-length FGF23 signaling in wild-type mice, and investigated its impact on hypoferremia. Our data show that FGF23 is induced as early as pro-inflammatory cytokines in response to LPS, followed by upregulation of hepcidin and downregulation of erythropoietin (Epo) expression in addition to decreased serum iron and transferrin saturation. Further, LPS-induced hepatic and circulating hepcidin were significantly reduced by FGF23 signaling disruption. Accordingly, iron sequestration in liver and spleen caused by TLR4 activation was completely abrogated by FGF23 signaling inhibition, resulting in alleviation of serum iron and transferrin saturation deficit. Taken together, our studies highlight for the first time that inhibition of FGF23 signaling alleviates LPS-induced acute hypoferremia.

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

Sickle cell disease (SCD) has a distinct pattern of transfusional iron overload (IO) when compared to transfusion-dependent β-thalassaemia major (TDT). We conducted a single institution prospective study to evaluate plasma biomarkers of iron regulation and inflammation in patients with SCD with IO (SCD IO cases, n = 22) and without IO (SCD non-IO cases, n = 11), and non-SCD controls (n = 13). Hepcidin was found to be inappropriately low, as evidenced by a significantly higher median hepcidin/ferritin ratio in non-SCD controls compared to SCD IO cases (0·3 vs. 0·02, P < 0·0001) and SCD non-IO cases (0·3 vs. 0·02, P < 0·0001), suggesting that certain inhibitory mechanism (s) work to suppress hepcidin in SCD. As opposed to the SCD non-IO state, where hepcidin shows a strong significant positive correlation with ferritin (Spearman ρ = 0·7, P = 0·02), this correlation was lost when IO occurs (Spearman ρ = -0·2, P = 0·4). Although a direct non-linear correlation between erythroferrone (ERFE) and hepcidin did not reach statistical significance both in the IO (Spearman ρ = -0·4, P = 0·08) and non-IO state (Spearman ρ = -0·6, P = 0·07), patients with highest ERFE had low hepcidin levels, suggesting that ERFE contributes to hepcidin regulation in some patients. Our results suggest a multifactorial mechanism of hepcidin regulation in SCD.

Iron is biologically essential, but also potentially toxic; as such it is tightly controlled at cell and systemic levels to prevent both deficiency and overload. Iron regulatory proteins post-transcriptionally control genes encoding proteins that modulate iron uptake, recycling and storage and are themselves regulated by iron. The master regulator of systemic iron homeostasis is the liver peptide hepcidin, which controls serum iron through degradation of ferroportin in iron-absorptive enterocytes and iron-recycling macrophages. This review emphasizes the most recent findings in iron biology, deregulation of the hepcidin-ferroportin axis in iron disorders and how research results have an impact on clinical disorders. Insufficient hepcidin production is central to iron overload while hepcidin excess leads to iron restriction. Mutations of hemochro-matosis genes result in iron excess by downregulating the liver BMP-SMAD signaling pathway or by causing hepcidin-resistance. In iron-loading anemias, such as β-thalassemia, enhanced albeit ineffective ery-thropoiesis releases erythroferrone, which sequesters BMP receptor ligands, thereby inhibiting hepcidin. In iron-refractory, iron-deficiency ane-mia mutations of the hepcidin inhibitor TMPRSS6 upregulate the BMP-SMAD pathway. Interleukin-6 in acute and chronic inflammation increases hepcidin levels, causing iron-restricted erythropoiesis and ane-mia of inflammation in the presence of iron-replete macrophages. Our improved understanding of iron homeostasis and its regulation is having an impact on the established schedules of oral iron treatment and the choice of oral versus intravenous iron in the management of iron deficiency. Moreover it is leading to the development of targeted therapies for iron overload and inflammation, mainly centered on the manipulation of the hepcidin-ferroportin axis.

Since iron overload is the commonest cause of morbidity and mortality in β thalassemia major (β-TM), it represents one major target in therapeutic management of the disease. The recently discovered erythroid regulator, erythroferrone (ERFE), governed by high levels of erythropoietin, was found to suppress hepcidin expression, thus increasing iron availability for developing erythroid progenitors. We aimed to investigate ERFE levels in Egyptian β-TM patients as an attempt to understand its role in the prediction of iron overload states. Our study included 70 β-TM patients, divided into two subgroups according to the degree of iron overload, and 30 sex and age-matched healthy subjects. ERFE gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and serum hepcidin was measured using enzyme-linked immunosorbent assay (ELISA) technique. Both ERFE gene expression levels and transferrin saturation (TS%) values were able to discriminate among cases with different degrees of iron overload, in contrast to hepcidin. TS% was acknowledged as the best predictor of iron overload (AUC 0.893) in comparison with serum hepcidin and ERFE gene levels (AUC 0.807 and 0.677, respectively), and ERFE gene expression was an independent predictor for the estimated TS%. In conclusion, we suggest that using the ERFE gene expression, combined with serum hepcidin estimation, can substantiate the role of estimated TS% as a promising tool in screening for iron overload in β-TM patients.

This review focuses on discoveries regarding doping with recombinant human erythropoietin and other erythropoiesis-stimulating agents (ESAs), reported since 2017. While ESAs are known to increase the total mass of hemoglobin (Hbmass), V ˙ O 2 m a x and maximal power output unresolved questions have remained with respect to their effects on endurance performance. ESAs are detectable by electrophoresis or isoelectric focusing and immunoblotting of urine (or blood) samples. The methods have undergone some improvements. Novel erythropoietic substances will become available in the near future. Analytical methods are already available for the detection of Epo mimetic peptides, hypoxia-inducible factor stabilizers, and activin type II receptor ligand traps. The ‘athlete biological passport’ is an indirect instrument in antidoping efforts. Additional hematologic parameters such as hepcidin and erythroferrone may strengthen the athlete biological passport approach.

Simple Summary Intense physical activity contributes to an increased consumption of oxygen transported by red blood cells. The red blood cells’ differentiation and proliferation process is mainly stimulated by erythropoietin (EPO) and erythroferrone (ERFE), which are novel markers of erythroid activity. The purpose of this study was to assess the level of concentration of these hormones in athletes’ blood. Seventy-three clinically healthy men took part in this study. Participants were divided into groups according to their physical activity, as assessed by the questionnaire survey. The first group included 39 athletes, the second group included 18 men with moderate physical activity, and the third—16 men with a sedentary lifestyle. Men with a high level of weekly physical activity had significantly different concentrations of ERFE and EPO than men with insufficient weekly physical activity. Higher endogenic ERFE and EPO levels are indicators of increased erythropoiesis in the period of intensified physical activity. The results obtained suggest the important role of endogenic EPO in the process of adaptation to intense physical activity. Abstract Intense physical activity contributes to an increased demand for red blood cells, which transport oxygen to working muscles. The purpose of this study was to assess the concentration of erythroferrone (ERFE), the novel marker of erythroid activity in athletes, during the beginning of their training season. The study group consisted of 39 athletes aged 23.24 ± 3.77 years. The study was carried out during the athletes’ preparatory period of the training cycle. The control group consisted of 34 healthy men aged 22.33 ± 2.77 years. The erythropoietic activity was evaluated by determining athletes’ concentrations of erythropoietin (EPO) and erythroferrone (ERFE). The level of physical activity was assessed using the International Physical Activity Questionnaire (IPAQ). In the athletes’ group, we observed higher concentrations of EPO (Me = 12.65 mIU/mL) and ERFE (40.00 pg/mL) compared to the control group (EPO: Me = 5.74 mIU/ml, p = 0.001; ERFE: Me = 25.50 pg/mL, p = 0.0034). The average intensity of physical exercise significantly differentiated the participants as far as EPO and ERFE concentrations. These results suggest that intense physical activity, at least at the beginning of the training season, may stimulate EPO production, which increases ERFE release. This seems to be an adaptative mechanism that provides adequate iron for enhanced erythropoiesis.

Sickle cell anemia and β-thalassemia intermedia are very different genetically determined hemoglobinopathies predisposing to pulmonary hypertension. The etiologies responsible for the associated development of pulmonary hypertension in both diseases are multi-factorial with extensive mechanistic contributors described. Both sickle cell anemia and β-thalassemia intermedia present with intra and extravascular hemolysis. And because sickle cell anemia and β-thalassemia intermedia share features of extravascular hemolysis, macrophage iron excess and anemia we sought to characterize the common features of the pulmonary hypertension phenotype, cardiac mechanics, and function as well as lung and right ventricular metabolism. Within the concept of iron, we have defined a unique pulmonary vascular iron accumulation in lungs of sickle cell anemia pulmonary hypertension patients at autopsy. This observation is unlike findings in idiopathic or other forms of pulmonary arterial hypertension. In this study, we hypothesized that a common pathophysiology would characterize the pulmonary hypertension phenotype in sickle cell anemia and β-thalassemia intermedia murine models. However, unlike sickle cell anemia, β-thalassemia is also a disease of dyserythropoiesis, with increased iron absorption and cellular iron extrusion. This process is mediated by high erythroferrone and low hepcidin levels as well as dysregulated iron transport due transferrin saturation, so there may be differences as well. Herein we describe common and divergent features of pulmonary hypertension in aged Berk-ss (sickle cell anemia) and Hbb th/3+ (intermediate β-thalassemia) mice and suggest translational utility as proof-of-concept models to study pulmonary hypertension therapeutics specific to genetic anemias.

BackgroundIn pediatric kidney transplant recipients, anemia is common and oftentimes multifactorial. Hemoglobin concentrations may be affected by traditional factors, such as kidney function and iron status, as well as novel parameters, such as fibroblast growth factor 23 (FGF23).MethodsHere, we evaluated associations among erythropoietic, iron-related, and FGF23 parameters in a cohort of pediatric kidney transplant recipients, hypothesizing that multiple factors are associated with hemoglobin concentrations.ResultsIn a cross-sectional analysis of 59 pediatric kidney transplant recipients (median (interquartile range) age 16.3 (13.5, 18.6) years, median estimated glomerular filtration rate (eGFR) 67 (54, 87) ml/min/1.73 m2), the median age-related hemoglobin standard deviation score (SDS) was -2.1 (-3.3, -1.1). Hemoglobin SDS was positively associated with eGFR and calcium, and was inversely associated with erythropoietin (EPO), mycophenolate dose, and total, but not intact, FGF23. In multivariable analysis, total FGF23 remained inversely associated with hemoglobin SDS, independent of eGFR, iron parameters, EPO, and inflammatory markers, suggesting a novel FGF23-hemoglobin association in pediatric kidney transplant patients. In a subset of patients with repeat measurements, only delta hepcidin was inversely associated with delta hemoglobin SDS. Also, delta EPO positively correlated with delta erythroferrone (ERFE), and delta ERFE inversely correlated with delta hepcidin, suggesting a possible physiologic role for the EPO-ERFE-hepcidin axis in the setting of chronic kidney disease (CKD).ConclusionOur study provides further insight into factors potentially associated with erythropoiesis in pediatric kidney transplant recipients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Iron is essential for all living organisms. It is strictly controlled by iron transporters, transferrin receptors, ferroportin and hepcidin. Erythroferrone (ERFE) is an iron-regulatory hormone which is highly expressed in erythroblasts by erythropoietin (EPO) stimulation and osteoblasts independently of EPO by sequestering bone morphogenetic proteins and inhibiting hepatic hepcidin expression. Although the hepcidin suppressive function of ERFE is known, its receptors still require investigation. Here, we aim to identify ERFE receptors on the HepG2 and Huh7 cells responsible for ERFE. Recombinant ERFE (rERFE) was first produced in HEK293 cells transfected with pcDNA3.1 + ERFE, then purified and detected by Western blot. The liver cells were treated with an rERFE-rich medium of transfected HEK293 cells and a purified rERFE-supplemented medium at various time points, and hepcidin gene (Hamp1) expression was determined using qRT-PCR. The results show that 37-kD rERFE was expressed in HEK293 cells. Hamp1 was suppressed at 3 h and 6 h in Huh7 cells after rERFE treatments (p &lt, 0.05), then restored to the original levels. Hamp1 was activated after treatment with purified rERFE for 24 h and 48 h. Together, these results reveal that ERFE suppressed Hamp1 expression in liver cells, possibly acting on membrane ERFE receptor, which in Huh7 cells was more sensitive to the ERFE concentrate.

Objective Hepatitis C virus (HCV) eradication is associated with decreased serum ferritin and increased serum low-density lipoprotein-cholesterol (LDL-C) levels, although the mechanisms underlying these changes remain unclear. This study aimed to identify the mechanisms underlying the changes in iron and lipid metabolism after HCV eradication. Methods We retrospectively investigated iron and lipid metabolism changes in 22 patients with chronic hepatitis or compensated liver cirrhosis with HCV genotype 1b infection after HCV eradication. We measured the serum erythroferrone (ERFE) levels to assess the association with these metabolic changes. Patients were administered ledipasvir 90 mg and sofosbuvir 400 mg once daily for 12 weeks and were observed for 12 more weeks to evaluate the sustained virological response. Results Half of the patients were men. At baseline, the serum ferritin and ERFE levels were elevated, while the serum LDL-C levels were within the normal range. All patients achieved a sustained virological response at 24 weeks; furthermore, the serum ferritin and ERFE levels were significantly decreased, and the serum LDL-C levels were significantly increased at 24 weeks from baseline (p

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron, the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2), however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection, liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein, however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.

Objective: The aim of this study was to evaluate non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) levels and other parameters of iron metabolism in children undergoing therapy for acute leukemia or after hematopoietic cell transplantation (HCT), in the context of iron overload. Patients: A total number of 85 children were prospectively included into four groups: controls, acute leukemia de novo, acute leukemia after intensive treatment, and after HCT. Methods: The following iron metabolism parameters were analyzed: (1) parameters measuring functional and storage iron pools: NTBI, LPI, iron, transferrin, total iron-binding capacity, ferritin, ferritin heavy and light chains, (2) proteins regulating iron absorption and its release from tissue stores: hepcidin, soluble hemojuvelin, soluble ferroportin-1, (3) proteins regulating the erythropoietic activity of bone marrow: erythroferrone, erythropoietin, soluble transferrin receptor. Results: Intensive treatment of leukemia in children was associated with the presence of serum NTBI and LPI, which was the highest in the HCT group followed by the acute leukemia after treatment and de novo groups. In patients after HCT, the most significant changes were found in NTBI, LPI, iron, ferritin, hepcidin, and ferroportin-1 levels. Conclusions: The occurrence of NTBI and LPI in the circulation and the intensification of disturbances in iron metabolism were associated with the intensity of the anti-leukemic treatment.

Hematopoietic cell transplantation (HCT) brings important alterations in erythropoiesis and iron metabolism. Hepcidin, which regulates iron metabolism, increases in iron overload or inflammation and decreases with iron deficiency or activated erythropoiesis. Erythroferrone (ERFE) is the erythroid regulator of hepcidin. We investigated erythropoiesis and iron metabolism after allogeneic HCT in 70 patients randomized between erythropoietin (EPO) treatment or no EPO, by serially measuring hepcidin, ERFE, CRP (inflammation), soluble transferrin receptor (sTfR, erythropoiesis), serum iron and transferrin saturation (Tsat; iron for erythropoiesis) and ferritin (iron stores). We identified biological and clinical factors associated with serum hepcidin and ERFE levels. Serum ERFE correlated overall with sTfR and reticulocytes and inversely with hepcidin. Erythroferrone paralleled sTfR levels, dropping during conditioning and recovering with engraftment. Inversely, hepcidin peaked after conditioning and decreased during engraftment. Erythroferrone and hepcidin were not significantly different with or without EPO. Multivariate analyses showed that the major determinant of ERFE was erythropoiesis (sTfR, reticulocytes or serum Epo). Pretransplant hepcidin was associated with previous RBC transfusions and ferritin. After transplantation, the major determinants of hepcidin were iron status (ferritin at all time points and Tsat at day 56) and erythropoiesis (sTfR or reticulocytes or ERFE), while the impact of inflammation was less clear and clinical parameters had no detectable influence. Hepcidin remained significantly higher in patients with high compared to low pretransplant ferritin. After allogeneic HCT with or without EPO therapy, significant alterations of hepcidin occur between pretransplant and day 180, in correlation with iron status and inversely with erythroid ERFE.

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.

Red blood cell transfusions (RBCT) remain the cornerstone of supportive care in lower-risk myelodysplastic syndrome (LRMDS) [1]. Transfusion dependency in LRMDS patients is associated with inferior outcomes, mainly attributed to severe bone marrow failure [2]. However, iron toxicity, due to frequent RBCT or ineffective erythropoiesis, may be an additional negative prognostic factor [3,4,5,6]. Recently, much progress has been made in unraveling the iron metabolism. The peptide hormone hepcidin is the key regulator by inhibiting iron uptake through degradation of ferroportin, a cellular iron exporter [7]. Erythroferrone and GDF15, produced by erythroblasts, inhibit hepcidin production, which leads to increased uptake and cellular release of iron for the purpose of erythropoiesis [8]. The pathophysiology of iron metabolism in MDS is still not completely understood. Exceedingly high reactive oxygen species (ROS) levels are associated with iron toxicity, disease development, and progression in MDS patients [9,10,11,12]. Malondialdehyde (MDA), resulting from lipid peroxidation of polyunsaturated fatty acids, is a biomarker of oxidative stress [10, 12]. Currently, little is known about the prognostic impact of ROS in MDS patients. The aim of this study is twofold: (1) describe iron and oxidative stress parameters over time in LRMDS patients and (2) to assess their effect on overall and progression-free survival. The EUMDS registry prospectively collects observational data on newly diagnosed LRMDS patients from 148 centers in 16 countries in Europe and Israel as of January 2008. All patients provided informed consent. Clinical data were collected at baseline and at each six-monthly follow-up visit. Serum samples were collected prospectively at each visit from 256 patients included in six participating countries. Conventional iron parameters were measured with routine assays. We additionally analyzed hepcidin, growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), non-transferrin bound iron (NTBI), labile plasma iron (LPI), and MDA. Subjects were prospectively followed until death, loss to follow-up, or withdrawal of consent. All iron parameters were measured centrally at the department of Laboratory Medicine of the Radboudumc, Nijmegen, The Netherlands. Serum samples were collected just prior to transfusion in transfusion-dependent patients and stored at −80 °C. Details on the assays and reference ranges of hepcidin, GDF15, sTfR, NTBI, LPI, and MDA are provided in the supplement. The Spearman rank test was used to evaluate correlations between iron parameters. We stratified the results by transfusion dependency per visit and the presence of ring sideroblasts. When evaluating temporal changes in iron parameters, with linear quantile mixed models, we excluded patients from the timepoint they received iron chelation therapy. Overall survival (OS) was defined as the time from MDS diagnosis to death or, in case of progression-free survival, to date of progression or death; patients still alive at the end of follow-up were censored. Time-dependent Kaplan–Meier curves and cox proportional hazards models were used. In total, 256 consecutive patients, were included in this study. Over five six-monthly visits, 1040 samples were collected. Table 1 describes the patient characteristics. Most patients without ring sideroblasts were transfusion-independent at diagnosis (nonRS-TI; 55.9%), 18.8% with ring sideroblasts were transfusion-independent (RS-TI), 18.4% without ring sideroblasts were transfusion-dependent (nonRS-TD), and 7% with ring sideroblasts were transfusion-dependent patients (RS-TD). The median follow-up time was 6.6 years (95% CI 5.9–7.0). LPI was positively correlated with transferrin saturation (TSAT) (r = 0.15, p < 0.001, Fig. S1). LPI values increased exponentially at TSAT values above 80%. This effect was most pronounced in the transfusion-dependent groups, but also observed in the RS-TI group. MDA was weakly correlated with NTBI (r = 0.09, p = 0.069) and negatively correlated with hemoglobin level (r = −0.1, p = 0.033). GDF15 and hepcidin were negatively correlated in the RS-TI and nonRS-TD group and significantly negatively correlated in the RS-TD group (r = −0.34, p = 0.007, Fig. S2). Serum ferritin levels were elevated in all subgroups with a mean value of 858 µg/L at visit 5. The highest serum ferritin levels were observed in the RS-TD group (mean value at visit 5: 2092 µg/L, Table S1). Serum ferritin increased significantly per visit in the RS-TD group (beta 454.46 µg/L; 95% CI 334.65–574.27), but not in the other groups (Table S2). All subgroups, except for the nonRS-TI, had elevated TSAT levels. TSAT levels were most markedly increased in the RS-TD group with a mean TSAT of 88% at visit 5 (Table S1). In both transfusion-dependent groups the median increase per visit was significant (Table S2). LPI was elevated in the RS-TD group exclusively with a mean value of 0.59 µmol/L at visit 5 (Table S1). NTBI was elevated in all subgroups, with the highest values in the RS-TD group (Table S1). The increase in median NTBI level was significant in both transfusion-dependent groups (Table S2). Hepcidin levels were markedly elevated in the nonRS-TD group. Interestingly, hepcidin levels were lower in the RS-TD group, probably reflecting ineffective erythropoiesis, likewise supported by lower hepcidin/ferritin ratios in RS groups (Table S1). Median hepcidin levels increased over time in the transfusion-dependent subgroups only (Table S2). GDF15 levels, analyzed in the light of its potential role in hepcidin suppression, were increased in all subgroups (Table S1). The RS subgroups had higher GDF15 levels compared to the nonRS groups, reflecting increased erythropoiesis. Mean sTfR levels were within the reference range in all subgroups except for the RS-TI group, which showed elevated levels, reflecting...

Recently, a new erythroid regulator, erythroferrone (ERFE), which downregulates hepatic hepcidin production, has been identified. However, the relationship between ERFE and abnormal iron metabolism in MDS is unclear. In this study, we examined the level of ERFE mRNA during ex vivo erythroid differentiation using cord blood CD34+ cells and we further analyzed whether ERFE could be produced by MDS cells using a public database (GSE58831). ERFE mRNA was increased during normal erythroid differentiation. An analysis of GSE58831 indicated that ERFE expression in bone marrow (BM) MDS cells was higher than that in healthy volunteer (HV)-derived BM cells. ERFE expression significantly and positively correlated with the expression of erythropoietin (EPO) receptors (EPO-R), ALAS2 (5'-Aminolevulinate Synthase 2), STEAP3 (STEAP family member 3) and the presence of ring sideroblasts or the SF3B1 mutation. These results suggest that EPO-R+ MDS cells with ring sideroblasts or an SF3B1 mutation produce high levels of ERFE that may be associated with a reduction in hepcidin.

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation., Haematologica, 105 (4), ISSN:0390-6078, ISSN:1592-8721

Thalassemia is a common inherited monogenic disease. It is characterized by chronic hemolysis, ineffective erythropoiesis (IE) and iron overload. Despite advances in transfusion practices and chelation therapy, still many limitations in delivering these standard therapies exist. Challenges of currently available standard care and advances in understanding the underlying pathophysiological mechanisms in thalassemia stimulated research towards development of novel therapeutic targets. Agents reducing IE as Jak 2 inhibitors and Activin II receptor traps are promising and are currently in clinical trials. Other approaches targeting iron dysregulation as mini-hepcidins, exogenous transferrin and erythroferrone inhibitors are in preclinical studies. Gene therapy, a rapidly evolving field, has exhibited remarkable progress in recent years. Studies have focused on β or γ-globin addition, over expression of endogenous γ-globin-activating transcription factors, silencing of γ-globin repressors and genome editing of β-globin mutations or γ-globin repressors. In this article we provide an overview of emerging recent trends in treatment of thalassemia targeting IE, iron dysregulation and novel curative treatments as gene therapy and gene editing.

We recently described myonectin (also known as erythroferrone) as a novel skeletal muscle–derived myokine with metabolic functions. Here, we use a genetic mouse model to determine myonectin’s requirement for metabolic homeostasis. Female myonectin-deficient mice had larger gonadal fat pads and developed mild insulin resistance when fed a high-fat diet (HFD) and had reduced food intake during refeeding after an unfed period but were otherwise indistinguishable from wild-type littermates. Male mice lacking myonectin, however, had reduced physical activity when fed ad libitum and in the postprandial state but not during the unfed period. When stressed with an HFD, myonectin-knockout male mice had significantly elevated VLDL–triglyceride (TG) and strikingly impaired lipid clearance from circulation following an oral lipid load. Fat distribution between adipose and liver was also altered in myonectin-deficient male mice fed an HFD. Greater fat storage resulted in significantly enlarged adipocytes and was associated with increased postprandial lipoprotein lipase activity in adipose tissue. Parallel to this was a striking reduction in liver steatosis due to significantly reduced TG accumulation. Liver metabolite profiling revealed additional significant changes in bile acids and 1-carbon metabolism pathways. Combined, our data affirm the physiologic importance of myonectin in regulating local and systemic lipid metabolism.—Little, H. C., Rodriguez, S., Lei, X., Tan, S. Y., Stewart, A. N., Sahagun, A., Sarver, D. C., Wong, G. W. Myonectin deletion promotes adipose fat storage and reduces liver steatosis.

Erythropoiesis is the predominant consumer of iron in humans and other vertebrates. By decreasing the transcription of the gene encoding the iron-regulatory hormone hepcidin, erythropoietic activity stimulates iron absorption, as well as the release of iron from recycling macrophages and from stores in hepatocytes. The main erythroid regulator of hepcidin is erythroferrone (ERFE), synthesized and secreted by erythroblasts in the marrow and extramedullary sites. The production of ERFE is induced by erythropoietin (EPO) and is also proportional to the total number of responsive erythroblasts. ERFE acts on hepatocytes to suppress the production of hepcidin, through an as yet unknown mechanism that involves the bone morphogenetic protein pathway. By suppressing hepcidin, ERFE facilitates iron delivery during stress erythropoiesis but also contributes to iron overload in anemias with ineffective erythropoiesis. Although most of these mechanisms have been defined in mouse models, studies to date indicate that the pathophysiology of ERFE is similar in humans. ERFE antagonists and mimics may prove useful for the prevention and treatment of iron disorders.

β-thalassaemia is a rare genetic condition caused by mutations in the β-globin gene that result in severe iron-loading anaemia, maintained by a detrimental state of ineffective erythropoiesis (IE). The role of multiple mechanisms involved in the pathophysiology of the disease has been recently unravelled. The unbalanced production of α-globin is a major source of oxidative stress and membrane damage in red blood cells (RBC). In addition, IE is tightly linked to iron metabolism dysregulation, and the relevance of new players of this pathway, i.e., hepcidin, erythroferrone, matriptase-2, among others, has emerged. Advances have been made in understanding the balance between proliferation and maturation of erythroid precursors and the role of specific factors in this process, such as members of the TGF-β superfamily, and their downstream effectors, or the transcription factor GATA1. The increasing understanding of IE allowed for the development of a broad set of potential therapeutic options beyond the current standard of care. Many candidates of disease-modifying drugs are currently under clinical investigation, targeting the regulation of iron metabolism, the production of foetal haemoglobin, the maturation process, or the energetic balance and membrane stability of RBC. Overall, they provide tools and evidence for multiple and synergistic approaches that are effectively moving clinical research in β-thalassaemia from bench to bedside.

Background:Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis.Methods:To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β–thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone.Results:We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss.Conclusions:Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.Funding:YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.