ALBERT

Beschreibung: Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts. "Unshielded" heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme. So far, virtually no information about the expression of HO-1 in erythroblasts has been produced. However, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1-mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3. Our data indicates that HO-1 expression is increased in the liver, spleen and kidney of β-thalassemic mice compared to wild-type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses have increased levels of HO-1 at mRNA and protein levels as well as a decrease in phosphorylated eIF2-α levels. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release. To investigate the contribution of HO-1 to the pathology associated with β-thalassemia, wild-type and thalassemic (th3/+) mice were injected with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO-1 inhibitor) during a 4-week period, 3 times a week. Our results show that β-thalassemic mice injected with SnPP display a decrease in the spleen index, hemoglobin levels, red blood cell counts, reticulocyte counts and liver iron content when compared to PBS injected β-thalassemic mice. Additionally, HO-1 inhibition reduced ineffective erytropoiesis in β-thalassemia mice. Our results indicate that β-thalassemic erythroblasts have inappropriately high levels of "free" heme that is continuously degraded by HO-1. Further research is needed to determine whether iron liberated from heme by HO-1 is directly responsible for the damage of β-thalassemic erythroblasts. 1Garcia-Santos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 2269-77, 2014. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 2104 Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient's cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient's bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient's results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient's urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient's GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells. Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Thalassemias are a heterogeneous group of red blood cell (RBC) disorders ranging from a clinically severe phenotype requiring lifesaving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. In β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. Moreover, in β-thalassemia an erythroid-specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts. Physiologically, heme can be degraded only via heme oxygenases (HO). Circulating erythrocytes contain the majority of heme destined for catabolism; this process takes place primarily in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. Recently, we have provided unequivocal evidence that this enzyme is present in erythroid progenitors as well as their differentiated progenies.1 "Unshielded" heme is toxic, but this toxicity will likely be augmented, if HO1 releases iron from heme. We hypothesize that in β-thalassemic erythroblasts HO1-mediated release of iron from heme is the major culprit responsible for cellular damage. Additionally, it has been shown that prevention of heme-derived iron release from splenic and hepatic macrophages improves β-thalassemia phenotype2. Therefore, suppression of HO1-mediated heme catabolism from senescent RBC could be beneficial in reversing thalassemic phenotype. To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3; we obtained these mice from Dr. Stefano Rivella. Our data indicates that HO1 expression is increased in the liver of β-thalassemic mice as compared to wild type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells from β-thalassemic fetuses increased HO1 mRNA and protein levels to a higher degree than in their wild type counterparts. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release from the tetrapyrrole macrocycle. To investigate the contribution of HO1 to the pathology associated with β-thalassemia, wild type and thalassemic (th3/+) mice were injected intraperitoneally with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO inhibitor) during a 4-weeks, 3-times a week. Our results show that β-thalassemic mice injected with SnPP have increased hemoglobin levels and red blood cell counts, and display a decrease in the spleen index, reticulocyte counts and liver iron content when compared to PBS-injected β-thalassemic mice. Furthermore, while hepcidin levels remain unchanged, liver ferroportin expression decreases in SnPP-injected β-thalassemic mice. Our results indicate that β-thalassemic erythroblasts have high levels of HO1, which would be expected to degrade any "free" heme. Further research is needed to determine whether iron liberated from heme by HO1 is directly responsible for the damage of β-thalassemic erythroblasts. 1GarciaSantos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 226977, 2014. 2Nai A, et al. Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia. Blood 119 (21): 5021, 2012. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by macrocytic anemia, reticulocytopenia and reduced number of erythroblasts in the bone marrow. Etiology and symptoms of DBA are closely associated with mutations of genes coding for 12 ribosomal proteins. Approximately 40% of patients are dependent on regular transfusions. In chronically transfused DBA patients, severe iron overload with tissue hemosiderosis with the need for iron chelation develops and has a substantial impact on morbidity and mortality of DBA patients. Liver is the most affected organ, but extra-hepatic iron accumulation, although less well documented, appears to occur early and at high frequency. Despite these facts a detailed analysis of iron metabolism in DBA is missing. We therefore aimed to determine selected markers of erythropoid activity and iron metabolism including the levels of the key regulator of iron homeostasis, hepcidin, in 14 DBA patients from the Czech National DBA Registry. Ten patients receive regular transfusions, two are treated with steroids and two are in the remission. None of the patients had signs of an inflammatory process at the time of the examination. Following markers of iron metabolism were analysed: iron, total iron binding capacity, transferrin saturation, ferritin and soluble transferrin receptor (TfR). Serum hepcidin was measured by proteomic analysis. Levels of rowth differentiation factor 15 (GDF 15), aputative marker of erythroid activity, were measured by ELISA. Pearls’ staining was used for the liver iron detection. In all transfusion dependent patients, low levels of soluble TfR and reduced number of erythroblasts in the bone marrow confirm severally suppressed erythropoiesis which corresponded with dramatically elevated serum erythropoietin (EPO) levels. Analysis of iron parameters showed increased transferrin saturation and high ferritin levels. The two patients who are currently in the remission had near normal blood counts and iron parameters. Liver biopsy on 5 selected patients revealed excessive iron accumulation in both liver hepatocytes and macrophages which can be attributed to increased iron uptake and non-effective erythrocytes-derived iron recycling, respectively. Measurement of hepcidin levels showed significant elevation in DBA cohort in comparison with healthy controls (p〉0.0005). The highest hepcidin was detected for transfusion dependent patients, the lowest (near normal) for patients in remission. As erythropoiesis is known to produce a signal for hepcidin suppression, patients in remission with restored erythropoietic activity are likely able to attenuate hepcidin expression and increase the pool of iron available for improved erythropoiesis. On the other hand repeated blood transfusions contribute to the suppression of erythropoiesis thus likely relieving hepcidin synthesis and worsening iron recycling. We also found that elevated hepcidin positively correlated with ferritin levels. No correlation was detected with EPO or GDF15, the putative negative regulator of hepcidin production. These results indicate that EPO is an indirect suppressor of hepcidin synthesis and that GDF15 possibly plays a hepcidin-regulatory role in the disease states associated with ineffective erythropoiesis but not in DBA. In conclusion we propose that in DBA patients iron overload develops as a consequence of the combination of repeated transfusions and absent or reduced erythropoiesis in the bone marrow. At the systemic level the increased hepcidin promotes iron retention in macrophages and thus is responsible for impaired iron recycling. Acknowledgments Grant support: Ministry of Health, Czech Republic, grant No. NT11059 to DP and NT/13587 to VD, Czech Grant Agency, grant No. P305/11/1745, and Internal Grant of Palacky University Olomouc (LF_2013_010). We thank dr. Thomas Ganz for validation of our HPLC-MS-based hepcidin measurements. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Inactivating mutations in divalent metal transporter 1 (DMT1) are associated with a severe defect in erythroid iron utilization and cause moderate to severe hypochromic microcytic anemia in human patients and two rodent models. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes the suppression of colony-forming capacity of erythroid progenitors. Using in vitro cultures of fetal liver cells we were able to recapitulate this in vivo defect. We confirmed abnormal pattern of erythroid differentiation and increased apoptosis (2.5-times) of DMT1-mutant erythroblasts when compared to wild-type (wt) fetal liver erythroblats. Determination of 2’,7’-Dichlorofluorescein diacetate-dependent intensity of fluorescence, which is proportional to the concentration of reactive oxygen species (ROS), revealed elevated levels of ROS in DMT1-mutant erythroblats when compared to wt erythroblast. This result suggests that oxidative stress contributes to the apoptosis in DMT1-mutant cells. We also observed that the defective erythroid differentiation of DMT1-mutant erythroblasts is marked by a blunted induction of heme oxygenase-1, an enzyme that co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells (Garcia-Santos et al., Blood, 2014, 123 (14): 2269-77). In further studies we focused on mature red blood cells (RBC), because it is known that nutritional iron deficiency and certain types of congenital hypochromic anemia are associated with increased levels of ROS and shortened life span of RBC that can be at least partially attributed to a programmed cell death of erythrocytes, so called eryptosis (Lang et al., Int J Biochem Cell Biol, 2012, 44 (8): 1236-43). Using labeling with carboxyfluorescein diacetate succinimidyl ester, we observed an accelerated clearance of DMT1-mutant RBC from circulating blood when compared to wild-type RBC. In vitro, DMT1-mutant RBC exposed to hyperosmotic shock or glucose depletion showed significantly increased levels of phosphatidylserine on the membrane detected by Annexin V binding. Together, these results confirmed eryptosis of DMT1-mutant RBC. As eryptosis is proposed to be triggered via activation of Ca2+ cation channels, we next measured the concentration of cytosolic Ca2+ using Fluo3/AM fluorescent dye and found significantly elevated content of intracellular Ca2+ in DMT1-mutant RBC when compared to wt RBC. In addition, DMT1-mutant RBC had higher levels of ROS than wt RBC despite significantly increased activity of anti-oxidative defense enzymes; glutathione peroxidase (1.6-times), catalase (1.9-times) and methemoglobin reductase (1.9-times). This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBC is not sufficient to eliminate ROS effectively. Furthermore, DMT1-mutant RBC also showed accelerated anaerobic glycolysis as detected by increased activities of hexokinase (2.5-times), pyruvate kinase (2.4-times), glucose-phosphate isomerase (3.2-times). This result together with reduced ATP/ADP (1.6-times) ratio in DMT1-mutant RBC when compared to wt RBC suggests an increased demand for ATP in DMT1-mutant erythrocytes. In conclusion we propose that increased oxidative stress and accelerated destruction of RBC contribute to the pathophysiology of anemia caused by DMT1-deficiency. Grant support: Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic, grant No. NT13587, Education for Competitiveness Operational Program, CZ.1.07/2.3.00/20.0164, Internal Grant of Palacky University Olomouc, LF_2014_011 and in part by the Canadian Institutes of Health Research (D.G-S., P.P.). Disclosures No relevant conflicts of interest to declare.

Beschreibung: Divalent metal transporter 1 (DMT1, also known as NRAMP2 and SLC11A2) is a transmembrane protein important for intestinal iron (Fe2+) absorption and erythroid iron utilization. Homozygous or compound heterozygous mutations in DMT1 are associated with moderate to severe hypochromic microcytic anemia in human patients and a mouse model - mk/mk mice. We have previously reported that DMT1 deficiency leads to an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast which also showed increased rate of apoptosis. For human samples we observed suppression of colony-forming capacity of erythroid progenitors that can be corrected by the addition of iron saturated chelate Fe-SIH. Later we proved this result also for mk/mk progenitors and showed reduced number of mk/mk CFU-E (164±25 vs. 283±50) and BFU-E (9±4 vs. 22±5) colonies in comparison to the colonies of wild-type (wt) mice and improvement of the colony growth with Fe-SIH. In our following studies we focused on mature erythrocytes, the last stage of erythroid differentiation that has not been analyzed yet. We first determined the in vivo half-life of red blood cells (RBC). Isolated RBCs from mk/mk mice and wt controls were in vitro labeled with CFSE fluorescent dye and injected into the wt mice. The intensity of RBCs fluorescence was measured on the 1st, 7th, 10th, 14th, 19th, 26th and 30th day following the injection. We observed an accelerated clearance of CFSE-labeled mk/mk RBCs from circulating blood when compared to wt RBCs, which indicates increased destruction of DMT1-mutant erythrocytes in vivo. It is known, that mature RBCs retain the ability to undergo stress-induced death (eryptosis), characterized by their shrinkage, membrane blebbing and phosphatidylserine surface exposure. This process may be triggered by iron deficiency. To determine the involvement of eryptosis in mk/mk RBCs clearance, RBCs were exposed to different stress conditions in vitro. A significantly increased number of Annexin V-positive RBCs was detected for mk/mk RBCs when compared to wt RBCs after 5 and 7 hour exposure to hyperosmotic shock (400mM sucrose) and glucose depletion, respectively. These results indicate shortened life span of DMT1-mutant erythrocytes and their reduced ability to cope with stress. To unravel the possible underlying mechanisms we focus on two processes important for RBC survival; anti-oxidative defense and anaerobic glycolysis. We observed 1.5 to 2-fold higher activity of glutathione peroxidase, catalase and methemoglobin reductase and elevated levels of methemoglobin in mk/mk RBCs in comparison to wt RBCs, indicating increased oxidative stress in mk/mk RBCs. Increased activity of hexokinase (2.5 times) and pyruvatkinase (2.4 times) together with reduced ratio of ATP/ADP in mk/mk mice compared with wt mice (from 2.89±0.56 μmol/L to 1.71±0.49 μmol/L) shows enhanced demand for glycolytically derived ATP to maintain the stability of RBC membrane in mk/mk mice. Our analyzes suggest that DMT1 deficiency negatively affects metabolism and life span of mature erythrocytes; two other aspects of defective erythropoiesis contributing to the pathophysiology of the disease. Grant support Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic Grant No. NT11208 and Internal Grant of Palacky University Olomouc (LF_2013_010). Disclosures: No relevant conflicts of interest to declare.