The Xla (X-linked anemia) Mouse: A Transient Neonatal Anemia Caused by a Gata1 Splicing Mutation,

Abstract Abstract 3162 The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice. Disclosures: No relevant conflicts of interest to declare.

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Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

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SLC20A1 at the Interface between Macrophages and Red Blood Cells

Blood ◽

10.1182/blood-2019-123758 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 428-428

Author(s):

Federica Quattrone ◽

Riem Gawish ◽

Rui Martins ◽

Anna-Dorothea Gorki ◽

Martin Watzenboeck ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Erythropoietin Receptor ◽

Embryonic Lethality ◽

Strong Impact ◽

Severe Anemia ◽

Heme Transport ◽

Slc Transporters ◽

Erythroid Precursors

Red blood cells (RBCs) comprise 84% of our body's cells and have the essential function of transporting oxygen from the lungs to all organs. The daily production rate of RBCs is enormous and the pathways mediating this process are quite complex. One important aspect hereby is the synthesis of hemoglobin and heme, the iron-containing prosthetic group that enables hemoglobin to bind oxygen.RBCs differentiate within the erythropoietic niche, which consists of erythroid precursors and specialized macrophages. These so-called nursing macrophages provide nutrients and iron, a fundamental component of heme. The precise mechanisms of heme transport within a cell and the potential of heme transfer between cells is not completely understood. To get insights into heme transport we took advantage of the cytotoxic capacity of heme and performed a CRISPR-Cas9 loss of function screen by focusing on SLC transporters. We identified SLC20A1 as an essential protein mediating heme toxicity and verified that intracellular heme levels as well as heme-induced downstream gene inductions were reduced in the absence of Slc20a1. Based on these evidences we hypothesized that SLC20A1 might be involved in the trafficking of heme, possibly important during erythropoiesis, since mice lacking Slc20a1 exhibit embryonic lethality due to liver apoptosis and anemia (Beck L. et al., PLoS One 2013). To test the biological role of Slc20a1 in adult mice, we conditionally deleted Slc20a1 using tamoxifen in ERT2-Cre Slc20a1fl animals and observed the spontaneous development of severe anemia and splenomegaly within 2 weeks after tamoxifen administration. We further discovered that the anemia-related expansion of red pulp macrophages (RPM) consisted predominantly of non-recombined wild type cells, whereas successfully recombined (Slc20a1 deficient) cells seemed stuck in the monocytic stage. These data suggest that Slc20a1 might be involved in the differentiation and maturation of monocytes to RPMs. When we performed a specific Slc20a1 deletion in nursing macrophages (CD169 Cre), mice neither showed signs of anemia, nor experienced impaired recovery upon anemia induction following phlebotomy or induction of hemolysis. However, deleting Sc20a1 specifically in the erythroid compartment using Slc20a1fl mice crossed to erythropoietin receptor-cre (EpoR-Cre) animals, resulted in embryonic lethality. Mice died around E12.5 due to severe anemia. Analysis of E11.5 animals disclosed erythroid precursors to be arrested in the pro-erythroblast stage.These data suggest that SLC20A1 is a protein involved in heme-mediated toxicity and possibly in the trafficking of heme with strong impact on fetal and adult erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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Erythroferrone Represses Hepcidin Expression in a Mouse Model of Malaria

Blood ◽

10.1182/blood.v124.21.4022.4022 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4022-4022

Author(s):

Leon Kautz ◽

Chloe Latour ◽

Wlodarczyk Myriam ◽

Nicolas Blanchard ◽

Tomas Ganz ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Malaria Infection ◽

Time Course ◽

Conflicts Of Interest ◽

Elevated Serum ◽

Severe Anemia ◽

Wild Type ◽

Hepcidin Expression ◽

High Parasitemia

Abstract Introduction: Malaria, a mosquito-borne disease caused by a parasite, represents a major global health challenge in developing countries, resulting in over half a million deaths each year. Among the many clinical complications, the multiplication of the parasites in erythrocytes leads to a severe anemia secondary to hemolysis and increased erythrophagocytosis. Malarial anemia is also characterized by insufficient erythropoiesis to compensate for the loss of red blood cells, despite high erythropoietin (EPO) levels. Iron is an essential functional component of erythrocyte hemoglobin, therefore the production of erythrocytes requires the timely delivery of iron to erythroid precursors. The availability of iron for erythropoiesis is controlled by hepcidin-induced endocytosis and degradation of ferroportin, the iron exporter which delivers iron to plasma from absorptive enterocytes and erythrocyte-recycling macrophages. In the late phase of malarial infection, hepcidin is suppressed but the mechanism of suppression is unknown. The erythroid hormone erythroferrone (ERFE) has been recently described as an important regulator of hepcidin expression during increased erythropoietic activity. We assessed hepcidin and erythroferrone expression in mouse malaria and found that ERFE is necessary for hepcidin suppression during malaria infection. Methods: To study the regulation of hepcidin in malaria, we used the rodent malaria parasite Plasmodium berghei K173 (PbK). Mice infected with PbK develop a lethal form of malaria with a high parasitemia and severe anemia and eventually die 18 to 20 days after infection. C57BL/6 mice were challenged intraperitoneally with 106 PbK-parasitized erythrocytes. The parasitemia and the hematologic parameters, were monitored during 18 days (Table 1). Table RBC (106/µL) HGB (g/dL) HCT (%) Parasitemia (%) Controls 8.8 +/- 0.6 15.1 +/- 0.9 39.8 +/- 3.1 0 Day 7 7.7 +/- 0.9 12.6 +/- 1.5 33.8 +/- 4.0 2 +/- 1 Day 9 7.0 +/- 0.3 11.6 +/- 0.6 32.5 +/- 1.6 4 +/- 1 Day 11 5.9 +/- 0.5 9.9 +/- 0.9 27.8 +/- 1.9 4 +/- 1 Day 13 3.8 +/- 0.8 6.6 +/- 1.2 21.5 +/- 2.3 20 +/- 5 Day 16 2.0 +/- 0.7 3.9 +/- 1.2 14.7 +/- 4.5 42 +/- 11 Day 18 1.7 +/- 0.4 3.6 +/- 0.7 14.8 +/- 2.7 68 +/- 10 Results: Thirteen days after infection, mice showed a high parasitemia (20% of infected red blood cells) and significantly decreased RBC (3.8x106/µL), hemoglobin concentration (6.6 g/dL) and hematocrit (21.5%) despite elevated serum EPO levels (not shown). We examined the time course of liver hepcidin expression and serum hepcidin concentration and found that hepcidin production was profoundly reduced 11 to 18 days after infection. As expected given the increase in EPO production after infection, hepcidin suppression was accompanied by an increase in erythroferrone mRNA expression in the bone marrow and the spleen. To determine whether ERFE plays a role in hepcidin suppression during malaria infection, we studied wild-type and Erfe-deficient mice after PbK infection. Erfe-/- mice failed to adequately suppress hepcidin expression after infection with PbK compared with wild-type mice. Figure 1 Figure 1. Conclusion: Erythroferrone may be responsible for hepcidin suppression and compensatory iron acquisition during malaria infection. Funded in part by ANR (project ANR-13-BSV3-0015-01) and FRM (project DEQ2000326528) Disclosures No relevant conflicts of interest to declare.

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An Essential Role for the RNA Editor-Exonuclease Axis in Terminal Erythroid Differentiation

Blood ◽

10.1182/blood-2020-142757 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-3

Author(s):

Areum Han ◽

Alena V. Yermalovich ◽

Vanessa Lundin ◽

Daniel S. Pearson ◽

Mariam Hachimi ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Rna Degradation ◽

Erythropoietin Receptor ◽

Conditional Knockout ◽

Erythroid Progenitors

Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

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Rap-536 Induces Reticulocyte Maturation in Wild-Type Mice, Increases Survival of Beta-Thalassemic Reticulocytes, and Increases Red Blood Cells in a Mouse Model of Alpha-Thalassemia

Blood ◽

10.1182/blood-2020-137373 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Melih Acar ◽

Madhulika Jupelli ◽

Roberto A. Abbiati ◽

Harish N. Ramanathan ◽

Cristina C. Santini ◽

...

Keyword(s):

Mouse Model ◽

Red Blood Cells ◽

Blood Cells ◽

Wild Type ◽

Publicly Traded ◽

The Past ◽

Alpha Thalassemia ◽

Reticulocyte Maturation ◽

Erythroid Maturation ◽

Parameter Values

Luspatercept is a recombinant fusion protein that binds and sequesters several endogenous transforming growth factor-beta superfamily ligands, including growth differentiation factor 11, thereby diminishing Smad2/3 signaling in target cells involved in erythropoiesis. Luspatercept, and its murine analog RAP-536, have been shown to act as erythroid maturation agents via their effects on late-stage erythropoiesis by inducing erythroblast maturation, leading to increases in red blood cells (RBCs) and hemoglobin (Hb). This study demonstrated that thalassemic (th3/+) reticulocytes are unstable, and that RAP-536, in addition to its function as an erythroid maturation agent, modulates the maturation of wild-type (WT) and th3/+ reticulocytes. Furthermore, RAP-536 treatment increased RBCs and decreased bilirubin in a mouse model of alpha-thalassemia (129S-Hba-a1tm1Led/J). To examine whether acute RAP-536 treatment acts on reticulocytes and alters reticulocyte levels in blood, the blood of WT mice was analyzed 3, 12, and 24 hours, and 2, 3, 4, and 7 days after a single dose of RAP-536 (10 or 30 mg/kg) or vehicle. RAP-536 treatment increased RBCs, Hb, and hematocrit significantly at all time points, compared with vehicle. However, in mice treated with RAP-536, reticulocytes in blood decreased significantly on Days 2, 3, and 4 and returned to normal baseline levels on Day 7. Analysis of reticulocyte subpopulations in blood 3 days after RAP-536 treatment showed that the relative percentages of immature reticulocytes (CD71+ or high RNA content) within the blood reticulocyte population decreased, suggesting that reticulocytes released from the bone marrow (BM) were more mature and/or reticulocytes matured faster in blood. A quantitative pharmacology (QP) model was developed to explore which RAP-536-induced modulations of erythropoiesis in WT mice can simulate the experimental observations. The model represents erythroblast, reticulocyte, and RBC (erythrocyte) maturation stages in BM, peripheral blood, and spleen, in the presence or absence of a RAP-536 effect. The QP model consists of a system of ordinary differential equations, with homeostatic parameter values assigned from literature or experimental measures, and RAP-536-perturbed parameter values regressed by fitting the model to erythropoiesis data of RAP-536-treated WT mice. Comparison of model parameters for homeostatic versus RAP-536-perturbed states indicated that RAP-536 leads to an increase in the erythroblast-to-reticulocyte and reticulocyte-to-RBC conversion rates, the transfer of BM reticulocytes to blood, and a delayed increase in erythroblast production. To directly test whether RAP-536 treatment affects reticulocyte development in blood, comparative blood transfusion experiments were performed. Biotinylated GFP+ blood from WT mice (C57BL/6-Tg(UBC-GFP)30Scha/J) and biotinylated GFP− blood from th3/+ beta-thalassemic mice (B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) were co-transfused into GFP− WT recipient mice (C57BL/6J), which were subsequently treated with RAP-536 or vehicle. In the donor reticulocyte population, th3/+ reticulocyte percentage decreased continuously up to 3 days after transfusion, suggesting that many of the th3/+ reticulocytes were eliminated before they could form RBCs. However, compared with vehicle, RAP-536 treatment led to increased persistence of the relative percentages of th3/+ reticulocytes (Figure A). Consequently, 7 days after transfusion, when most reticulocytes have matured to RBCs, the percentage of th3/+ RBC among donor RBCs was higher with RAP-536 (Figure B). Finally, treatment of an alpha-thalassemia mouse model (129S-Hba-a1tm1Led/J) with RAP-536 10 mg/kg for 8 weeks increased RBCs and hematocrit and reduced serum bilirubin, compared with vehicle. These results suggest that RAP-536 is, as previously shown, an erythroid maturation agent, which also modulates reticulocyte maturation in blood. In WT mice, RAP-536 modulated blood reticulocyte dynamics consistent with faster maturation. RAP-536 also prolonged the persistence of th3/+ reticulocytes and maintained a higher frequency of th3/+ RBCs. These data, together with the finding that RAP-536 reduces hemolysis in an experimental alpha-thalassemia disease model, suggest that luspatercept has the potential to improve anemias associated with hemolysis and/or reticulocytosis. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. Abbiati:Bristol Myers Squibb: Current Employment. Ramanathan:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Santini:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ratushny:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Dunshee:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Suragani:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Loos:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

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Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.

Blood ◽

10.1182/blood.v114.22.2534.2534 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2534-2534

Author(s):

Akira Niwa ◽

Tomoki Fukatsu ◽

Katsutsugu Umeda ◽

Itaru Kato ◽

Hiromi Sakai ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Specific Antigen ◽

Embryonic Stem ◽

Ips Cells ◽

Oxygen Carriers ◽

Developmental Pathway ◽

Ips Cell

Abstract Abstract 2534 Poster Board II-511 Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production. Disclosures: No relevant conflicts of interest to declare.

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TET2 Mutations in Polycythemia Vera (PV) in Some Cases Follow Rather Than Precede JAK2 V617F Mutation, Are Not a Disease-Initiating Event, Affect Mainly Erythropoiesis, and Contribute to Increased Aggressivity of PV Clone.

Blood ◽

10.1182/blood.v114.22.3913.3913 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3913-3913

Author(s):

Sabina Swierczek ◽

Christine Bellanne-Chantelot ◽

Donghoon Yoon ◽

Cecile Saint-Martin ◽

Soo Jin Kim ◽

...

Keyword(s):

X Chromosome ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Jak2v617f Mutation ◽

Erythroid Progenitors ◽

Aberrant Splicing ◽

Mutational Burden ◽

Published Evidence ◽

Tet2 Mutation

Abstract Abstract 3913 Poster Board III-849 The Ten-Eleven Translocation (TET) 2 gene is a tumor suppressor gene. Its mutations of which are frequently found in PV and other myeloid malignancies. The published evidence suggests that a TET2 mutational burden is present in a higher proportion than JAK2V617F in PV stem cells; its mutations typically precede the JAK2V617F mutation, and are preferentially expressed in myeloid cells. We studied 40 PV patients. Two had known TET2 mutation, one with an intronic mutation (3954+2T>A) predicted to cause aberrant splicing, and the other with a deletion of a single nucleotide in exon 3 (3138delT) leading to the predicted truncation of the TET2 peptide. We quantitated the mutational burden of JAK2V617F and TET2 and the clonality of blood lineages using X-chromosome allelic usage ratios in the blood cells and BFU-E colonies. We also followed the mutational burden of the JAK2V617F and TET2 somatic mutations and expression of TET2 mRNA and monitored the proportion of polyclonal cells, in in vitro expanded erythroid progenitors. These data were compared to the PV patients without known TET2 mutations. Using an X-chromosome-based transcriptional clonality assay, the PV patients had predominantly clonal reticulocytes, granulocytes, platelets and, in those available, CD34-positive cells. Studies of individual BFU-E found that in two PV patients, the TET2 mutations followed rather than preceded the JAK2V617F mutation. We report that only a fraction of clonal CD34+ cells carry the TET2 mutation, and demonstrate that a small proportion of largely polyclonal T cells also carry the TET2 mutation. We report that the presence of both JAK2V617 and TET2 mutations favors accumulation of the mutated erythroid progenitors, while in similar conditions the PV JAK2V617-positive and TET2-negative cells are at a proliferative disadvantage compared to normal erythroid progenitors. We also examined the clonality of these in vitro-expanded erythroid progenitors to determine if the dormant minor populations of nonclonal hematopoietic cells are preferentially expanded along with those belonging to the PV clone. We found that some PV erythroid progenitors with JAK2V617F but no known TET2 mutations became polyclonal after in vitro erythroid expansion, while two PV patients with JAK2V617F and TET2 mutations remained clonal, suggesting that the TET2 mutated clonal progenitors retained their proliferative advantage. Lastly, compared to normal erythroid progenitors wherein TET2 mRNA increases with erythroid maturation, it decreases in PV erythroid progenitors regardless of the presence of a TET2 mutation. As predicted, the intronic TET2 mutation causing aberrant splicing had decreased TET2 expression compared to controls and other PV samples in all cells examined. However, the TET2 mRNA transcript in peripheral blood granulocytes and platelets in JAK2V617F positive PV, regardless of TET2 mutations, was significantly increased compared with normal controls. We conclude that loss-of-function TET2 mutations in the two studied PV subjects are not the PV initiating events. Our data suggest that these TET2 mutations in PV preferentially affect the erythroid lineage, contribute to increased erythroid proliferation, and cause relative inhibition of PV granulopoiesis and megakaryopoiesis. However, in aggregate, these in vitro data also suggest that the acquisition of the TET2 somatic mutations increases the aggressivity of the PV clone. Disclosures: No relevant conflicts of interest to declare.

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Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽

10.1182/blood.v118.21.344.344 ◽

2011 ◽

Vol 118 (21) ◽

pp. 344-344

Author(s):

Daniel Garcia Santos ◽

Matthias Schranzhofer ◽

José Artur Bogo Chies ◽

Prem Ponka

Keyword(s):

Red Blood Cells ◽

Heme Oxygenase ◽

Blood Cells ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Wild Type ◽

Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place 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. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

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Oxidative Stress and Increased Destruction of Red Blood Cells Contribute to the Pathophysiology of Anemia Caused By DMT1 Deficiency

Blood ◽

10.1182/blood.v124.21.4027.4027 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4027-4027 ◽

Cited By ~ 1

Author(s):

Zuzana Zidova ◽

Daniel Garcia-Santos ◽

Katarina Kapralova ◽

Pavla Koralkova ◽

Renata Mojzikova ◽

...

Keyword(s):

Oxidative Stress ◽

Red Blood Cells ◽

Blood Cells ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Heme Oxygenase 1 ◽

Wild Type ◽

Divalent Metal Transporter 1 ◽

Oxidative Defense

Abstract 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.

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Band 3 Phosphorylation Induces Irreversible Alteration of Stored Red Blood Cells

Blood ◽

10.1182/blood.v128.22.5029.5029 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5029-5029

Author(s):

Slim Azouzi ◽

Yves Colin Aronovicz ◽

Catia Pereira ◽

Marc Romana ◽

Thierry Peyrard ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Cluster Formation ◽

Immunoblot Analysis ◽

Progressive Increase ◽

Band 3 ◽

Igg Antibody ◽

Functional Studies ◽

Previous Demonstration

Abstract Introduction: Storage of red blood cells (RBCs) for transfusion purposes is accompanied by a number of morphological and biochemical changes (storage lesions) that reduce post-transfusion survival/efficacy and increase risk for adverse reactions in the recipients. The clearance of altered and older RBCs from circulation is triggered by the clustering of Band 3, an aggregate state that is recognized by a low-affinity naturally occurring IgG antibody (Nab). Considering the key role of Band 3 in the maintenance of RBC structure and survival, elucidation of functional and structural modifications of Band 3 during storage should lead to new approaches aiming to improve RBC storage and post-transfusion viability. Results: Immunoblot analysis of RBC membrane proteins using an anti-phosphotyrosine antibody showed a progressive increase in the phosphorylation status of Band 3 during RBC storage (Figure 1). In addition, using the quenching fluorescence of eosin-5-maleimide (EMA), we showed an increase of the mobile fraction of Band 3. These findings are consistent with previous demonstration that tyrosine phosphorylation of Band 3 reduces its affinity for ankyrin, leading to the release of the immobile fraction of Band 3 from the skeleton complex, and enhancement of the lateral mobility of Band 3 into the lipid bilayer. Immunoblot experiments using an antibody that specifically recognizes the clustered form of Band 3 revealed an increase of Band 3 cluster formation from the 28th day of storage. We also showed that the release of microparticles (MPs) that occurs during RBC aging increases from the 28th day of storage (Figure 2). Finally, stopped-flow-based functional studies showed a decrease of the anion exchanger activity of Band 3 from the 28th day of storage. Conclusion: Altogether, our results suggest that the 28th of storage represents a key moment for the molecular processes leading to irreversible lesions of RBCs and allow us to propose a new Band 3 phosphorylation/clustering-based mechanism of RBC aging. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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