Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis. We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage. Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation. Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis. Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR. Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. Disclosures No relevant conflicts of interest to declare.

ERYTHROFERRONE AS A NEW BIOMARKER ASSOCIATED WITH ANEMIA IN IRAQI PATIENTS WITH CKD

Background: Erythroferrone (ERFE) is a glycoprotein hormone synthesis and release by erythroblasts. Recently identified as an erythropoietic regulator and activated in response to stimulating erythropoietin (Epo). In chronic kidney diseases (CKD), anemia is a hallmark disorder due to a decrease in hyposensitive erythropoietic to the Epo; these patients recommended to use of Erythropoiesis-stimulating agents (ESAs). The aim: This study aimed to assess serum ERFE level in patients with CKD and investigate the continuing effects of long-term anemic ESA use associated with markers of erythropoiesis and iron metabolism. Methods: Sixty-five CKD patients divided in two groups, included 30 hemodialyses (HD) and 35 without hemodialysis (non-HD) CKD patients, were compared to 25 healthy voluntaries matched by gender and age enrolled in the current study. Serum ERFE level was measured by an enzyme-linked immunosorbent assay (ELISA). Results: Serum ERFE level was significantly elevated in HD patients median (IQR) about 17.25 (13.4) ng/mL, odds ratio (OR = 10.161), (AUC 0.996) greater than CKD 4(6.1) ng/ml, (OR = 6.295), (AUC = 0.984) p 0.001; also, these are positively correlated with the use of ESA in HD, and CKD (r = 1.00 and r = 0.95) respectively as compared to healthy group 2(2.1) ng/ml. Serum ERFE levels were significantly negative (p 0.05) in both CKD and HD patients related to GFR (r = -0.396, and r = -0.68), transferrin saturation (TS%) (r = -0.842, and r = -0.877), serum levels of Ferritin (r = -0.865 and r = -0.866), and Iron (r = -0.860, and r = -0.851), RBC (r = -0.841, and r = -0.843), hemoglobin (Hb) (r = -0.758, and r = -0.796). Conclusion: The present study demonstrated that elevated serum ERFE levels associated with erythropoietic activity and anemia are higher in CKD with HD and non -HD patients treated with ESA than in non-ESA patients. This study suggested using ERFE as a successful tool for erythropoietic activity inspection in CKD, especially those taking ESAs to treat anemia.

Dynamic changes in murine erythropoiesis from birth to adulthood: implications for the study of murine models of anemia

Liver, spleen, and bone marrow are 3 key erythropoietic tissues in mammals. In the mouse, the liver is the predominant site of erythropoiesis during fetal development, the spleen responds to stress erythropoiesis, and the bone marrow is involved in maintaining homeostatic erythropoiesis in adults. However, the dynamic changes and respective contributions of the erythropoietic activity of these tissues from birth to adulthood are incompletely defined. Using C57BL/6 mice, we systematically examined the age-dependent changes in liver, spleen, and bone marrow erythropoiesis following birth. In addition to bone marrow, the liver and spleen of newborn mice sustain an active erythropoietic activity that is gradually lost during first few weeks of life. While the erythropoietic activity of the liver is lost 1 week after birth, that of the spleen is maintained for 7 weeks until the erythropoietic activity of the bone marrow is sufficient to sustain steady-state adult erythropoiesis. Measurement of the red cell parameters demonstrates that these postnatal dynamic changes are reflected by varying indices of circulating red cells. While the red cell numbers, hemoglobin concentration, and hematocrit progressively increase after birth and reach steady-state levels by week 7, reticulocyte counts decrease during this time period. Mean cell volume and mean cell hemoglobin progressively decrease and reach steady state by week 3. Our findings provide comprehensive insights into developmental changes of murine erythropoiesis postnatally and have significant implications for the appropriate interpretation of findings from the variety of murine models used in the study of normal and disordered erythropoiesis.

Non Invasive Evaluation of Bone Marrow Activity in Patients with Sickle Cell Disease: Correlation with Disease Features, Genotype, Markers of Erythropoiesis, Iron Metabolism and Hydroxyurea Treatment

Background: Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by pathological polymerization of hemoglobin, increased red cell rigidity and poor microvascular blood flow with consequent tissue ischemia and infarction. Thus, hemolytic anemia, vaso-occlusion and vasculopathy are the hallmarks of its clinical presentation. The transferrin receptor (TfR) mediates the transport of iron into cells and the circulating TfR can be measured as soluble transferrin receptor (sTfR). sTfR levels are frequently used to establish the diagnosis of iron deficiency anemia, especially in the context of inflammation, but they also reflect bone marrow erythropoietic activity (BMA) and mass. Erythropoietic activity has been found to be the most important determinant of sTfR levels. In this context, we aimed to study and evaluate bone marrow activity in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) based in sTfR measurements and explore possible correlations with of key features of the disease such as: the hemolytic component, vaso-occlusive crises (VOC), acute chest syndrome, venous thrombosis, arterial thrombosis including stroke, avascular necrosis, pulmonary hypertension, hydroxyurea therapy, inflammation and renal injury. along with other biomarkers of erythropoiesis and iron metabolism such as Placental Growth Factor (PlGF), Growth Differentiation Factor-15 (GDF-15), Ferritin and Hepcidin-25. Patients and Methods: Ninety adult Caucasian patients with HbS/βthal [49 patients under hydroxyurea (HU+) treatment and 41 patients without hydroxyurea (HU-) treatment], were included in this study, while 22 apparently healthy individuals of similar age and gender served as controls. None of the patients has received any transfusions at least 6-monthes before enrollment in the study. Along with hematologic and blood chemistry parameters determination, levels of circulating sTfR, PlGF, GDF-15 and Hepcidin-25 were measured in patients with HbS/βthal and controls using RUO and IVD immunoenzymatic techniques. BMA activity was calculated from the established formula: patient-sTFR/meanControl-sTFR. Results: We found that: sTfR levels were markedly elevated in all patients with HbS/βthal compared to controls (4.8±2.2 vs. 1.0±0.2 mg/L, p<0.001), resulting in a 1.6-11.9 fold increase of BMA. No correlation was found between BMA and disease features as well as regarding hydroxyurea treatment BMA (p>0.434). BMA correlated significantly with the markers of the erythropoietic and hemolytic component such as: Hemoglobin (r=-0.434, p<0.001); Reticulocyte Production Index (r=0.645, p<0.001); LDH (r=0.570, p<0.001); Billirubin (r=0.540, p<0.001), PlGF (r=0.597, p<0.001) and Hb A levels (r=-0.493, p<0.001), while no correlation was found between BMA and Hb F levels. Furthermore, BMA values correlated significantly only with GDF-15 (r=0.466, p<0.001), while interestingly no correlation was found between BMA and Ferritin and Hepcidin-25 levels alone (r=0.101, p>0.351 and r=-0.043, p>0.710, respectively), but a negative correlation was found between BMA and Hepcidin-25/Ferritin ratio, (r=-0.330, p=0.005). Conclusions: Our findings demonstrate that all patients with HbS/βthal studied have a significantly increased degree of erythroid BMA as assessed by measurements of sTfR levels. Erythroid BMA correlated significantly with Hepcidin/Ferritin ratio, which is an index of the degree of Hepcidin expression relative to iron overload. The correlation of erythroid BMA with Hb A levels, indicate the important role of βthal genotype in HbS/βthal disease. Furthermore, BMA is not related to hydroxyurea therapy and/or iron metabolism parameters in these patients. This implicates a likely complex action of hydroxyurea, which causes intermittent cytotoxic suppression of erythroid progenitors and cell stress signaling. The latter affects erythropoiesis, leading to recruitment of erythroid progenitors with increased HbF levels, although the number of erythroid progenitors -the main source of sTfR- remains stable. Disclosures Voskaridou: Genesis: Consultancy, Research Funding; Protagonist: Research Funding; Celgene Corporation: Consultancy, Research Funding; Acceleron: Consultancy, Research Funding; Addmedica: Membership on an entity's Board of Directors or advisory committees.

Comparative analysis of hematopoietic activity of total RNA from bone marrow cells and splenocytes of rats with chronic benzene-induced anemia

Цель - оценка лечебных свойств суммарной РНК на модели хронической гипопластической анемии, выявление особенностей гемопоэтического действия суммарных РНК, выделенных из костного мозга и лимфоидных клеток селезенки. Методика. Работа выполнена на 36 белых беспородных крысах-самках массой 130-250 г. Хроническую токсическую анемию моделировали подкожным введением смеси химически чистого бензола (0,05 мл/100 г) и стерильного растительного масла. Смесь вводили трижды с интервалом в 7 сут. Контрольным животным (группа 1 в каждой серии) в эти же сроки внутривенно вводили равное по объему количество 0,9% раствора NaCl. Выполнено 2 серии экспериментов. В 1-й серии оценивали гемопоэтическую активность суммарных РНК лимфоидных клеток селезенки, во 2-й - суммарных РНК костного мозга. Препараты суммарной РНК получали из костного мозга и лимфоидных клеток селезенки интактных крыс, а также из клеток тех же органов крыс, подвергнутых кровопусканию (2% от массы тела) за 17 ч до выделения из них РНК. Суммарную РНК выделяли методом гуанидин тиоцианат-фенол-хлороформной экстракции. Для выяснения влияния суммарных РНК на состояние эритроидного ростка костного мозга в препаратах, окрашенных по Паппенгейму, оценивали количественный и качественный состав эритроидных островков. Результаты. Сравнение гемопоэтической активности суммарных РНК, выделенных из костного мозга и лимфоидных клеток селезенки здоровых животных, при их введении крысам с экспериментальной хронической бензольной анемией, показало, что различия клеточного состава лимфоидных органов-источников РНК отражается на их гемопоэтических свойствах. Суммарная РНК из лимфоидных клеток селезенки интактных животных в первую очередь способствует восстановлению количества лейкоцитов, в то время как суммарная РНК лимфоидных клеток костного мозга в начальный период регенерации не оказывает заметного влияния на лейкопоэз. Показано также, что суммарная РНК, выделенная из костного мозга, в целом обеспечивает более интенсивный процесс гемопоэза. Кроме того, обнаружена более высокая эритропоэтическая активность суммарных РНК, выделенных из клеток обоих лимфоидных органов крыс-доноров, стимулированных кровопотерей, по сравнению с таковой суммарных РНК, полученных из органов интактных крыс. Заключение. Подтверждена гемопоэтическая активность суммарной РНК костного мозга и лимфоидных клеток селезенки при гипопластической анемии. Выявлена более высокая эритропоэтическая активность суммарных РНК обоих лимфоидных органов животных после кровопускания по сравнению с их лейкопоэтической активностью. Aim. To evaluate therapeutic properties of total RNA on a model of chronic hypoplastic anemia and to elucidate features of the hemopoietic effect of total RNA isolated from bone marrow splenic lymphoid cells. Methods. Experiments were performed on 36 white mongrel rats weighing 230-250 g. Chronic toxic anemia was induced by subcutaneous injections of a mixture of chemically pure benzene (0.05 ml/100 g) and sterile vegetable oil. The mixture was injected three times with an interval of 7 days. Control animals (group 1 in each series) received intravenous injections of an equal volume of saline at the same time intervals. Two experimental series were performed. In series 1 and 2, hemopoietic activity of total RNAs from splenic lymphoid cells and from bone marrow, respectively, was evaluated. Total RNA was obtained from splenic lymphoid cells and from bone marrow of intact rats and rats subjected to hemorrhage (2% of body weight) 17 h prior to the RNA isolation. Total RNA was isolated by guanidine-thiocyanate-phenol-chloroform extraction. The effect of total RNA on the erythroid lineage in the bone marrow was evaluated by the quantitative and qualitative composition of erythroid islets in Pappenheim-stained preparations. Results. Comparing the hematopoietic activity of total RNA isolated from bone marrow and splenic lymphoid cells of healthy animals and administered to rats with experimental chronic benzene anemia showed that the difference in cell composition of the lymphoid organs, sources of the RNAs, impacted on the RNA hemopoietic properties. The total RNA from splenic lymphoid cells of intact animals contributed primarily to the recovery of leukocyte count while the RNA from the bone marrow of intact rats did not significantly influence leukopoiesis during the early regeneration period. It was also shown that the total RNA extracted from the bone marrow generally provided a more intensive hemopoiesis. In addition, the erythropoietic activity of total RNA isolated from the cells of both lymphoid organs of the donor rats stimulated by blood loss was higher than that of the total RNA isolated from intact rats. Conclusion. The total RNA isolated from bone marrow and lymphoid cells spleen exerted a hemopoietic effect in hypoplastic anemia. The erythropoietic activity of total RNA from both lymphoid organs of animals after hemorrhage was higher than the RNA leukopoietic activity.

Decreased Iron Utilization Leads to Dysregulation of Iron Homeostasis in Aplastic Anemia

Aplastic Anemia (AA) arises due to specific failure of bone marrow precursor cells or stem cells to produce adequate amount of mature hematopoietic cells. Erythropoiesis utilizes majority of iron in humans. Erythropoietic activity is fine-tuned with iron regulation since they are both intrinsically regulated. Hepcidin, the main iron regulator is the target of erythropoietic regulators like GDF15, TWSG1 and erythroferrone. Even though the dysregulation of iron balance is well understood in beta thalassemia where ineffective erythropoiesis is the main cause, regulation of iron is poorly elucidated in bone marrow failure where erythropoiesis is minimal. In this study, we analysed several iron regulatory molecules in a cohort of patients with AA and tried to understand iron regulation in bone marrow failure. The current study investigated 77 patients with newly or recently diagnosed AA and had received less than ten transfusions from the year 2016 to 2018. Their haematological parameters were documented. Serum hepcidin was assessed using ELISA kit (DRG Diagnostics) and serum ferritin and soluble transferrin receptor (sTfR) was measured by chemiluminescent immunoassay. DNA was extracted from peripheral blood and telomere length was measured using quantitative real-time PCR (qPCR) using relative quantitation method. The association between different iron regulators were analysed using SPSS Software. The median age of AA patients was 38 (5-72) years and the number of males and females were 49 (64%) and 28 (36%) respectively. Patients were grouped based on severity into non severe AA (NSAA) (n=17, 22%), Severe AA (SAA) (n=51, 66%) and Very Severe AA (VSAA) (n=9, 12%). Out of 77 AA patients, 50 had not received any red cell transfusion in the last one month prior to the sample collection; twenty seven had received transfusions ranging from 1 to 9. The mean haemoglobin was 7.6±1.87g/dL, with a median WBC count of 2900 (100-8400)/cu.mm and a median platelet count of 9000 (2000-66000)/ul. Serum ferritin levels ranged from 12.8 to 5728ng/ml (Median- 1208ng/ml). The median soluble transferrin receptor (sTfR) levels were 1.29mg/L (0.52-6.34). Fifty four patients (54/77, 70%) had sTfR values less than the lower limit of normal (Normal range 1.8-4.6 mg/L). Median serum hepcidin was 40.66ng/ml (1.15-81.2). Serum ferritin levels positively correlated with serum hepcidin (r=0.61, p=0.000) whereas it correlated negatively with sTfR (r=-0.408, p=0.000). No significant association was observed between the number of transfusions and ferritin levels (r=0.087, p=0.453). There was no significant difference in the ferritin levels when patients were grouped according to severity (Median Ferritin, Range - 1208, 12.8-2984ng/ml in NSAA; 1099, 61.8-5728ng/ml in SAA; 1288, 268-3824; p= 0.83). Significantly higher hepcidin levels were observed in VSAA as compared to NSAA (Figure.1, p= 0.014). There was no significant association observed between response and biochemical parameters. A quartile analysis based on ferritin values was carried out to study the effect of iron overload on haematological parameters and iron regulators (Table.1). With increasing ferritin values, hepcidin levels significantly increased (p=0.001) whereas sTfR levels were reduced (p=0.002). In AA, decrease in erythropoietic activity as evidenced by reduced sTfR levels leads to minimal iron utilization and accumulation of iron in the tissues as ferritin. This stimulates the production of hepcidin and further exacerbates iron loading and leads to dysregulation of iron homeostasis. Increased iron in the marrow can lead to oxidative stress and may further be detrimental to the limited intrinsic haematopoiesis. Hence better understanding of iron regulation in AA may advance to effective therapeutic options. Disclosures No relevant conflicts of interest to declare.