Six weeks of dynamic apnoeic training stimulates erythropoiesis but does not increase splenic volume

Purpose This study examined the influence of dynamic apnoea training on splenic volume and haematological responses in non-breath-hold divers (BHD). Methods Eight non-BHD performed ten maximal dynamic apnoeas, four times a week for six weeks. Splenic volumes were assessed ultrasonically, and blood samples were drawn for full blood count analysis, erythropoietin, iron, ferritin, albumin, protein and osmolality at baseline, 24 h post the completion of each week’s training sessions and seven days post the completion of the training programme. Additionally, blood samples were drawn for haematology at 30, 90, and 180 min post session one, twelve and twenty-four. Results Erythropoietin was only higher than baseline (6.62 ± 3.03 mlU/mL) post session one, at 90 (9.20 ± 1.88 mlU/mL, p = 0.048) and 180 min (9.04 ± 2.35 mlU/mL, p = 0.046). Iron increased from baseline (18 ± 3 µmol/L) post week five (23 ± 2 µmol/L, p = 0.033) and six (21 ± 6 µmol/L; p = 0.041), whereas ferritin was observed to be lower than baseline (111 ± 82 µg/L) post week five (95 ± 75 µg/L; p = 0.016), six (84 ± 74 µg/L; p = 0.012) and one week post-training (81 ± 63 µg/L; p = 0.008). Reticulocytes increased from baseline (57 ± 12 × 109/L) post week one (72 ± 17 × 109/L, p = 0.037) and six (71 ± 17 × 109/L, p = 0.021) while no changes were recorded in erythrocytes (p = 0.336), haemoglobin (p = 0.124) and splenic volumes (p = 0.357). Conclusions Six weeks of dynamic apnoeic training increase reticulocytes without altering mature erythrocyte concentration and splenic volume.

PSVIII-4 Bone marrow architectonics of turkeys hybrid cross «Converter»

The aim of this research was to study bone marrow architectonics and the peculiarities of hematopoiesis of turkeys cross «Converter». To study the hemopoiesis at the age of 50 days, the samples of the red bone marrow from the femur were selected. Samples were prepared according to standard methods for histological and cytological examination. Four lineages of hematopoiesis (erythrocytic, monocytic, granulocytic and platelet) were isolated from the bone marrow of turkeys. The erythrocytic lineage was represented by erythroblasts, rubricytes and mature erythrocytes. In the process of maturation, the erythroblast initially uniformly decreased, turning into rubricyte. Then the cytoplasm and nucleus enlarged and acquired an oval shape. A mature erythrocyte was formed. Hematopoiesis of eosinophils and basophils in turkeys was similar to hematopoiesis in mammals. They also formed from myeloblasts. At the stage of promyelocyte, granularity of the cytoplasm appeared. At the stage of myelocyte, the number of granules increased, and the granularity acquired a typical shape and color. Then the nucleus acquired a rod-shaped form, and after it was segmented into 2–5 segments. Heterophiles were divided into two types (with rounded and elongated granules). Heterophile granules were about 2 times bigger than eosinophils, and cytoplasm granularity was significantly sparser. Monocytes in turkeys had a rounded cytoplasm, in which small granules were sometimes present, and the nucleus was shifted to the periphery. Mature monocytes differed from promonocytes in a smaller nuclear-cytoplasmic ratio. Platelets had a centrally located nucleus and an oval cytoplasm with granules inside. Prothrombocytes were approximately 1.5 times larger than mature platelets. The lymphoid lineage is not represented, which indicates an extramedullary lymphopoiesis. A significant difference was the absence of megakaryocytes and the development of platelets from thromboblasts. Granulocytic and monocytic lineages were similar to those in mammals, and erythroid one was represented only by nucleated erythrocytes.

Complete Spatial Mapping of Steady-State Erythropoiesis

Lack of markers to image the different progenitors has limited analyses of interactions between HSPC and their offspring. To overcome this we analyzed the expression of 250+ cell surface molecules for which antibodies are commonly available in all hematopoietic progenitors. We found 76 differentially expressed markers in at least one HSPC showing medium to bright fluorescence suggesting that many commonly used cell surface markers can be used to prospectively image HSPC in the bone marrow. We focused in erythropoiesis as it has not been possible to image step-wise red blood cell maturation in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BM BFU-E activity was restricted to Pre-MegE and CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image step-wise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells in the BM (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in the murine sternal BM and interrogate the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell in a mouse sternum. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulation. We found that Pre-MegE and Pre-CFU-E are closer to each other than predicted from random (average Pre-CFU-E to Pre-MegE distance= 92.3 µm vs. 156.7 µm random, p=0.028) but never adjacent indicating that Pre-CFU-E migrate away from Pre-MegE (0% of Pre-CFU-E adjacent to PreMegE). We also found that CFU-E were not adjacent to pre CFU-E, instead 7-8 CFU-E align to form "CFU-E strings" along the central BM (74% of CFU-E found in strings vs. 17.5% in random p<0.0001). Early erythroblasts form elongated clusters (66% early erythroblasts found within 10µm of another vs 10% in random p<0.0001) that emerge from these CFU-E strings like buds on a tree branch. Each of these early erythroblasts buds is enveloped by a large cluster of late erythroblasts, a reticulocyte cluster, and a mature erythrocyte cluster (68% of early erythroblasts buds form this 4-cluster structure vs.0% of random, P<0.0001). A recent study suggested that BM endothelial cells regulate erythroid progenitors via SCF. We found that the CFU-E strings sit on top of central BM sinusoids (average CFU-E to sinusoid distance= 0.8µm observed vs. 8.562μm random; P<0.0001) but are selectively depleted from arterioles (average CFU-E to arterioles distance=176 µm observed vs. 90.98 µm random, P<0.0001). In contrast downstream erythroid cells are farther away from sinusoids (average Early-Erythroblast to sinusoid distance=5.995 µm vs.8.224 µm random, P<0.0001; Late-erythroblast: 6.552 µm vs.9.053 µm random, P<0.0001; Reticulocytes: 6.013 µm vs.9.844 µm random, P<0.0001; Erythrocytes: 6.520 µm vs.8.986 µm random, P<0.0001). These suggest a model in which CFU-E progenitors are selectively recruited to sinusoids where they self-renew to generate strings of progenitors from which immature erythroblasts arise and mature while migrating away from the sinusoid. In summary we have found 76 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image all steps of red blood cell maturation; demonstrated that erythropoiesis takes place in highly organized 4-cluster structures emerging from strings of sinusoidal CFU-E, and demonstrated that sinusoids are the exclusive site of erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Regulation of Mature Erythrocyte Proteasomal Machinery Under Hypoxia

Proteasome machinery is a conserved cellular component to maintain normal protein homeostasis. Hypoxia is well known to control hypoxia inducible factor levels by proteasomal machinery in nucleated cells. However, the specific targets and regulation of proteasomal machinery in non-nucleated mature erythrocytes under hypoxia remain poorly understood. To determine if hypoxia regulates erythrocyte proteasomal machinery, we conducted Western blot to detect total ubiquitinated and K48 specific ubiquitinated proteins on the erythrocyte membrane in both human and mice with or without sickle cell disease (SCD), a hemolytic genetic disease with a high mortality, morbidity and frequently facing hypoxia. We found that ubiquitinated, especially K48 specific ubiquitinated proteins were significantly accumulated in both SCD Berkeley mice and humans compared to WT mice and normal controls, indicating that proteasomal machinery is impaired in SCD. Next, to determine specific ubiquitinated proteins accumulated on the membrane of human sickle erythrocytes (sRBC), we conducted immunoprecipitation of sRBC membrane proteins with total ubiquitin antibody followed by an robust and nonbiased proteomic profiling. We found significant accumulation of several categories of ubiquitinated proteins on human mature sRBC membrane, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), reactive oxygen species (ROS) related enzyme (catalase), components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Mechanistically, we revealed that the impaired proteasomal machinery found in mature sRBC was due to the blockage of trafficking of p97 bound ubiquitinated-proteins from membrane to cytosolic proteasome. As such, inhibition of p97 by CB-5083 or proteasome by MG132 led to further induction of hypoxia-induced ubiquitinated membrane proteins and sickling in cultured human sRBC. Given the fact that sphingosine-1-phosphate (S1P) contributes to sickling by binding with deoxygenated sickle Hb (deoxy-HbS), triggering deoxy-HbS anchoring membrane and releasing glycolytic enzymes, we immediately hypothesized that S1P may be involved in proteasomal machinery by regulating trafficking of p97-bound ubiquitinated proteins from membrane to cytosol in sRBC. To test this intriguing possibility, we generated SCD/Sphk1-/- mouse. Intriguingly, we found that the genetic deletion of SphK1 attenuated impaired proteasomal machinery in sRBC with less accumulation of p97 and ubiquitinated proteins on sRBC membrane, indicating that elevated S1P is detrimental in sRBC by inducing accumulation of p97 and ubiquitinated proteins on the membrane. Moreover, to determine if S1P-regulated p97 trafficking from membrane to the cytosol is unique to sRBC, we exposed wild type and SphK1-/- mice to 8% hypoxia up to 72 hours. In contrast to sRBC, we found that genetic deletion of SphK1 abolished p97 trafficking from membrane to cytosol in normal erythrocytes under hypoxia. Finally, we conducted in vitro proof-of-principle genetic studies to determine if S1P directly involves in translocation of membrane anchored p97 to cytosol using inverted ghost membrane (IGM). We demonstrated that S1P treatment induces deoxy-HbA translocation from cytosol to membrane and in turn restoring p97 release from membrane to the cytosol in IGM isolated from SphK1-/- mice only under hypoxia but not normoxia. Thus, we have provided both human and mouse genetic evidence supporting a working model: in normal individuals under hypoxia, S1P is a key factor regulating the efficient proteasomal machinery by binding deoxy-HbA, promoting deoxy-HbA anchoring membrane and in turn triggering release of p97-ubiqutinated proteins to cytosol for its degradation. With mutation in HbS, S1P promotes deoxy-HbS anchoring the membrane and forms polymers, thus blocks membrane bound p97 release and impairs proteasomal machinery in sRBC. Overall, our findings identified that S1P is a missing key component of proteasome machinery by its differential mechanism regulating p97 trafficking from membrane to cytosol in normal erythrocyte physiology under hypoxia and the pathophysiology of SCD that open up new and differential therapies for the SCD and normal individuals facing hypoxia. Disclosures No relevant conflicts of interest to declare.

The Influence of Erythrocyte Maturity on Ion Transport and Membrane Lipid Composition in the Rat

Significant relationships between ion transport and membrane lipid composition (cholesterol, total phospholipids and sphingomyelins) were found in erythrocytes of salt hypertensive Dahl rats. In these animals mean cellular hemoglobin content correlated negatively with Na+-K+ pump activity and Na+ leak but positively with Na+-K+ cotransport activity. Immature erythrocytes exhibit lower mean cellular hemoglobin content (MCHC) than mature ones. The aim of the present study was to find a relationship between erythrocyte maturity, membrane lipid composition and ion transport activity in Wistar rats aged three months which were subjected to repeated hemorrhage (blood loss 2 ml/day for 6 days) to enrich circulating erythrocytes with immature forms. Immature and mature erythrocyte fractions in control and hemorrhaged rats were separated by repeated centrifugation. Hemorrhaged rats had increased number of reticulocytes but reduced hematocrit and MCHC compared to control rats. Immature erythrocytes of hemorrhaged rats differed from mature ones of control animals by elevated Na+-K+ pump activity, reduced Na+-K+ cotransport activity and increased Rb+ leak. These ion transport changes in immature erythrocytes were accompanied by higher concentration of total phospholipids in their cell membranes. Membrane phospholipid content correlated positively with Na+-K+ pump activity and cation leaks but negatively with Na+-K+ cotransport activity. Moreover, they were also negatively related with MCHC which correlated negatively with Na+-K+ pump activity and Rb+ leak but positively with Na+-K+ cotransport activity. Thus certain abnormalities of erythrocyte ion transport and membrane lipid composition detected in hypertensive animals might be caused by higher incidence of immature cells.

Unsupervised Discovery of Early Markers of Erythroid Maturation in Human Donor Marrow

Antigenic expression during erythroid maturation is not well understood. Despite the large number of surface antigens currently described for leukocytes, only a few (e.g. CD36, CD45, CD71, CD117, and CD235a) have been well characterized on erythroid cells. Here, we apply novel bioinformatic tools to select antigens with high potential for identifying successive stages of erythroid maturation in an unsupervised and unbiased manner from a high dimensional dataset. In brief, flow cytometry was performed on 3 normal donor bone marrows using Becton-Dickinson lyoplates containing a total of 275 unique antibodies and 8 gating reagents (CD15, CD19, CD34, CD38, CD45, CD71, CD117, and CD123). Each of the 275 independent data files from the same donor was aligned using the gating reagents and a weighted nearest neighbor algorithm in order to synthesize a flow data set with 283 antigens and 50,000 events. We applied a modified SPADE algorithm to generate a maturational path for cells of the erythroid lineage starting with CD34+/CD38- progenitors through the mature erythrocyte stage. A non-parametric Kruskal–Wallis test was used to identify and rank antigens that show differential expression along the erythroid maturational sequence. To discern antigens having the greatest discrimination for early vs. late erythroid maturation, we identified 15 antigens common to the top 25 most differentially expressed antigens from each donor sample. As expected, our method correctly identified the three erythroid gating reagents (CD45, CD71 and CD117) as well as previously a previously described erythroid associated antigen (CD36). A few of the identified antigens have been described are less common but also known to be differentially expressed on erythroid cells (e.g. integrin members CD29, CD44, and CD49d), however several additional novel antigens were also identified with strong differential expression including CD46, CD58, CD81, CD98, CD99, CD164, CD220 and CD321 (Figure 1). Of note, CD49d, CD98 and CD164 are of particular interest as they appear to be gradually lost during maturation from the early normoblast through the mature erythrocyte stages. This is in contrast with the other antigens that show high expression on pronormoblasts—with rapid decline during the early normoblast stage to the level of erythrocytes. We examined selected antigens from the synthetic data of all 3 samples using manual gating, and interestingly CD99 retained expression among the erythroid cells to late stage normoblasts in 2 of the 3 samples, suggesting some element of phenotypic variability among normal individuals for this antigen. Additionally, several antigens appear useful in distinguishing erythroid lineage cells from non-erythroid precursors including beta-2 microglobulin, CD50, and HLA-ABC which all showed ubiquitous expression in non-erythroid precursors with low to absent expression among cells of the erythroid lineage. In summary, using a novel unbiased method for large-scale antigen discovery, we have identified multiple novel antigens that are differentially down regulated with progressive erythroid maturation and appear useful in further delineating erythroid progenitor maturation. Additional work is underway to correlate these antigens with our morphologic understanding of erythroid maturation. Further work in characterizing these changes in myeloid stem cell disorders is on going and may be of diagnostic utility in the diagnosis of myelodysplastic syndromes. Figure 1 [Red=High Expression, Blue=Low Expression] Figure 1. [Red=High Expression, Blue=Low Expression] Disclosures No relevant conflicts of interest to declare.

Dynamic Changes Of DNA Methylation and a Functional Role For TET2 DNA Dioxygenase In Human Erythroid Differentiation

Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. This process contains eight distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. Unlike most cell types, an important feature of erythropoiesis is that following each of the three or four mitoses that occur during terminal erythroid differentiation, the daughter cells are distinctly different from the parent cell from which they are derived. Thus, erythropoiesis is a complex process that requires tight regulation. The most extensively studied regulators of erythroid differentiation include the EPO/EPOR system and two major transcription factors, GATA1 and KLF1. In contrast to the well-established roles of growth factors, cytokines and transcription factors in regulating erythropoiesis, the regulation of erythropoiesis by other mechanisms is much less understood. In the present study, we explore the changes in DNA methylation during human terminal erythroid differentiation and DNA methylation/demethylation in human erythropoiesis. The methylation status of DNA influences many biologic processes. It has been recently reported that global demethylation occurs during both murine and human erythropoiesis. However, the dynamics of DNA methylation changes, the underlying molecular mechanism(s), and the function of DNA demethylation in erythropoiesis are not clear. To address these issues, we performed next-generation bisulfite sequencing on highly purified human erythroblasts at distinct differentiation stages. We show that while there is a global hypomethylation as terminal erythropoiesis proceeds, stage-specific analysis revealed that a significant proportion of differential methylation includes gains of methylation. Moreover, genes that presented with DNA methylation changes could be categorized into 3 groups based on the dynamics of their methylation changes. As Ten-eleven-translocation proteins (TETs) have been implicated in DNA demethylation by converting 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), we attempted to explore the role of TETs in DNA demethylation and terminal erythroid differentiation. We show that 5hmc is progressively increased during human terminal erythroid differentiation. Importantly, knockdown of TET2 by shRNA in human CD34+ cells impaired the production of 5hmc as well as terminal erythroid differentiation. Our findings demonstrate the complexity of DNA methylation dynamics and identify a functional role for TET2 in human erythroid differentiation. These findings provide new and novel insights into the mechanistic understanding of normal and disordered erythropoiesis. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, we suggest that these finding also provide novel insights into these diseases. Disclosures: No relevant conflicts of interest to declare.

Genetic Loss of Tmprss6 Increases Effective Erythropoiesis in a Mouse Model of β-Thalassemia

Abstract 482 β-thalassemia is a disorder of ineffective erythropoiesis in which oxidative damage caused by unpaired α-globin chains leads to erythroid apoptosis, increased proliferation of erythroid precursors, and impaired erythroid differentiation. Patients develop systemic iron overload that is caused by red blood cell transfusions and by insufficient inhibition of gastrointestinal iron absorption by the iron regulatory hormone hepcidin. Previously we reported that homozygous genetic loss of Tmprss6, a hepatic transmembrane serine protease that inhibits hepcidin expression by the liver, led to hepcidin elevation and systemic iron deficiency in Hbbth3/+ mice, a model of β-thalassemia intermedia. Interestingly, we also found that while maintaining similar hemoglobin levels, Hbbth3/+mice with homozygous loss of Tmprss6 showed a significant reduction in splenomegaly and marked improvement in peripheral red blood cell (RBC) morphology. Here, we investigated the effects of genetic loss of Tmprss6 on erythropoiesis in Hbbth3/+ mice. In mice of different Tmprss6-Hbb genotypes, we used flow cytometry to quantify the proportion of total bone marrow cells of the erythroid lineage by measuring expression of TER119, an antigen expressed from the pro-erythroblast through the mature erythrocyte stage. Additionally, within the TER119+ population, we quantified the different erythroblast subpopulations by analyzing the intensity of forward scatter and CD44 expression. Compared to wild type (Tmprss6+/+Hbb+/+) controls, Hbbth3/+ mice with 2 wild-type Tmprss6 alleles (Tmprss6+/+Hbbth3/+) showed a significant increase in the proportion of total erythroid cells in the bone marrow, significant increases in the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population, and a significant decrease in the proportion of mature RBCs, resulting in a marrow profile consistent with ineffective erythropoiesis. In Hbbth3/+ mice with homozygous Tmprss6 disruption (Tmprss6−/−Hbbth3/+), the proportion of immature erythroid precursors (basophilic and polychromatic erythroblasts) within the erythroid population remained significantly elevated; however, the proportion of total erythroid cells in the bone marrow was no longer increased. Compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a significant increase in the proportion of mature RBCs; this was accompanied by a reduction in reactive oxygen species (ROS) production (as assessed by the indicator CM-H2DCFDA) and apoptotic cells (as assessed by annexin V binding) within both the orthochromatic erythroblast/reticulocyte and mature red cell subpopulations. Additionally, compared to Tmprss6+/+Hbbth3/+ mice, Tmprss6−/−Hbbth3/+ mice showed a marked reduction in α-globin precipitates in membrane fractions prepared from peripheral RBCs. Interestingly, when normalized to α-globin mRNA expression, bone marrow mRNA encoding α-hemoglobin stabilizing protein (AHSP), an α-globin chaperone, was significantly higher in Tmprss6−/−Hbbth3/+ mice compared to Tmprss6+/+Hbbth3/+ mice, compatible with the known stabilization of AHSP mRNA under low iron conditions. Together, these findings suggest a model in which genetic loss of Tmprss6 in Hbbth3/+ mice leads to a systemically iron-deficient state in which reduced iron availability to erythroid precursors leads to stabilization of free α-globin chains, a reduction in both ROS formation and erythroid apoptosis, and ultimately more effective erythropoiesis. In the context of previous findings, these results indicate that hepcidin-elevating strategies based on pharmacological inhibition of Tmprss6 might alter the clinical phenotype of β-thalassemia not only by reducing systemic iron loading but also by altering erythroid maturation. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.