Suppression Of Erythropoiesis Research Articles

Intestinal inflammation modulates expression of the iron-regulating hormone hepcidin depending on erythropoietic activity and the commensal microbiota.

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

Вплив продукту декарбоксилювання L-аргініну на морфофункціональні показники еритрону за умов експериментального цукрового діабету у щурів

This paper reports the results of a study of the impact of the agmatine treatment on erythrocyte resistance to acid hemolytic, reticulocyte count and reticulocyte production index, erythrocyte surface architectonics in streptozotocin-induced diabetic rats. Our results indicate that treatment of diabetic rats with agmatine causes a suppression of erythropoiesis and increases the resistance of erythrocytes against HCl-induced hemolysis. It was shown a 10% increase in the number of young red blood cells and 35% reduction in the number of structurally transformed erythrocytes that are capable of restoring normal biconcave disc shape under physiological condition. Our data demonstrate an improvement of morphofunctional state of red blood cells in diabetes and reflect the positive effect of agmatine treatment on erythrone due to the glucose-lowering action.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice.

Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts.

Hepcidin-dependent and hepcidin-independent regulation of erythropoiesis in a mouse model of anemia of chronic inflammation.

Increased hepcidin antimicrobial peptide correlates with hypoferremia and anemia in various disease states, but its requirement for anemia of inflammation has not been adequately demonstrated. Anemia of inflammation is usually described as normocytic and normochromic, while diseases associated with over expression of hepcidin, alone, are often microcytic and hypochromic. These differences in erythrocyte parameters suggest anemia in many inflammatory states may not be fully explained by hepcidin-mediated iron sequestration. We used turpentine-induced sterile abscesses to model chronic inflammation in mice with targeted disruption of Hepcidin 1 [Hepc1 (-/-)] or its positive regulator, Interleukin-6 [IL-6 (-/-)], to determine whether these genes are required for features characteristic of anemia of inflammation. Although hemoglobin levels did not decline in Hepc1 (-/-) mice with sterile abscesses, erythrocyte numbers were significantly reduced compared to untreated Hepc1 (-/-) mice. In contrast, both hemoglobin concentration and erythrocyte number declined significantly in wild type and IL-6 (-/-) mice with sterile abscesses. Both Hepc1 (-/-) and IL-6 (-/-) mice had increased erythrocyte mean cell volume and mean cell hemoglobin following sterile abscesses, while wild types had no change. Thus, IL-6 (-/-) mice with sterile abscesses exhibit an intermediate phenotype between wild type and Hepc1 (-/-). Our results demonstrate the requirement of Hepc1 for the development of anemia in this rodent model. Simultaneously, our results demonstrate hepcidin-independent effects of inflammation on the suppression of erythropoiesis. Our results suggest chronic anemia associated with inflammation may benefit from interventions protecting erythrocyte number in addition to anti-hepcidin interventions aimed at enhancing iron availability.

A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin

AbstractAnemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus–treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus–treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia.

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

AbstractAnemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Interferon-γ suppresses activin A/NF-E2 induction of erythroid gene expression through the NF-κB/c-Jun pathway

Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-β superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.

Methylation Status of Immune Response Genes Promoters in Cell-Free DNA Differs in Hemodialyzed Patients with Diabetic Nephropathy According to the Intensity of Anemia Therapy

Background: Anemia is a major complication of end-stage renal disease. Hemodialysis itself is regarded as a stimulus activating inflammation. Pro-inflammatory cytokines are able to suppress erythropoiesis. In this pilot study, we analyzed the changes in methylation status of promoters of immune response genes in cell-free DNA to detect the differences between diabetic subjects (n = 18) with different therapeutic doses of recombinant erythropoietin. Methods: The extent of promoter methylation of 24 genes in plasma cell-free DNA was examined before and after hemodialysis using EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity (Qiagen). Results: The patients with higher methylation status of gene sequences IL13RA1, IL15, EDG3 and INHA in interdialytic interval were significantly overrepresented in the group with none or mild anemia therapy. Conclusion: The results are in agreement with the fact that IL13 and IL15 are known inhibitors of erythropoiesis and with considered immunomodulatory role of cell-free DNA.

The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels

SummaryATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

Red Blood Cell Alloimmunization and Hemolytic Disease of the Fetus and Newborn: New Approaches Using Animal Models

Women may become alloimmunized to foreign red blood cell (RBC) antigens after exposure during transfusion or pregnancy/delivery. These alloantibodies can be detrimental to developing fetuses/neonates, with six in every 1000 pregnancies being affected by maternal alloantibodies. Despite being described more than 80 years ago, hemolytic disease of the fetus and newborn (HDFN) has few preventive or therapeutic options. Outside of antigen avoidance, the most effective maternal preventive therapy to date is Rh immune globulin. However, it targets just one of the more than 50 antigens implicated in HDFN, and its mechanism of action remains unknown. Furthermore, treatment options for affected fetuses are limited largely to intrauterine transfusions of antigen negative RBCs. With a decrease in HDFN cases due to Rh(D), KEL has emerged as one of the most widely implicated antigens. We now describe a transgenic murine model in which the human KEL antigen is expressed on murine RBCs, with clinical significance in both pregnancy and transfusion settings. Transgenic animals were generated with expression of the human KEL2 antigen (referred to herein as KEL) on their RBCs using a β-globin promoter. KEL expression was evaluated on fetal liver and bone marrow erythroid precursors, mature RBCs, and organs. C57BL/6 or MuMT females were bred with transgenic heterozygous KEL males. Maternal serum was collected during pregnancy and after delivery, with anti-KEL glycoprotein responses measured by flow cytometric crossmatch. Newborn pups were evaluated for KEL positivity, degree of anemia, and presence of maternal anti-KEL. Additionally, the effect of anti-KEL alloantibodies on fetal RBC precursors was evaluated at multiple stages of gestation. Lastly, females alloimmunized through pregnancy were transfused with incompatible KEL RBCs labeled with a lipophilic dye, and females alloimmunized through transfusion were bred with KEL males. Transgenic mice have RBC specific expression of the KEL glycoprotein, with antigen detected on early and late RBC precursors in the fetal liver and bone marrow but not on any tested organ. Fetal/maternal blood exchange was maximally detected after delivery, with the majority of mothers developing anti-KEL glycoprotein antibodies (IgM and all IgG subclasses) within weeks of delivery. All IgG subclasses of anti-KEL crossed the placenta and bound to RBC precursors and mature RBCs in developing KEL positive pups; KEL negative pups had positive indirect antiglobulin tests. Maternal anti-KEL glycoprotein titers increased with antigen exposure, with fewer KEL positive pups born during successive pregnancies to alloimmunized C57BL/6 but not MuMT females. Affected KEL positive pups were anemic, and some were stillborn and hydropic-appearing. As has been described in humans, maternal anti-KEL alloantibodies suppressed the development of fetal KEL RBC precursors at all levels of maturation. Additionally, newborn pups had a relative reticulocytopenia, with reticulocyte recovery within a week after birth. Multiparous alloimmunized females exhibited rapid clearance of transfused incompatible KEL RBCs, and females alloimmunized through KEL RBC transfusion also had pups affected by HDFN. To the best of our knowledge, this is the first animal model of HDFN in which pregnancy stimulates alloantibodies to a paternally derived RBC antigen, with these alloantibodies being clinically significant in both pregnancy and transfusion settings. The anti-KEL glycoprotein alloantibodies generated in this model have similar effects on murine RBCs as anti-KEL1 alloantibodies have on human RBCs. Such effects include suppression of erythropoiesis, fetal anemia, and even hydrops fetalis. This model thus provides a platform to study not only the induction, placental transfer, and consequences of maternal RBC alloantibodies, but also to investigate potential preventive therapies (including “KEL” immune globulin). Long-term translational goals of this work include minimizing the dangers of RBC alloantibodies to developing fetuses and neonates, through the development of targeted maternal immunomodulatory therapies. Disclosures:No relevant conflicts of interest to declare.

Erythropoiesis and Iron Metabolism In Diamond-Blackfan Anemia

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. AcknowledgmentsGrant 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.

Inhibition Of Erythropoiesis By Iron Overload Is Mediated Through TGFβ Signaling

Several diseases, such as β-thalassemia and myelodysplastic syndrome, are characterized by iron overload and dysfunction erythropoiesis. Multiple retrospective studies suggest that iron overload is associated with increased disease burden and shortened survival in patients. It is general consensus that iron overload has an inhibitory effect on erythroid differentiation, but direct evidence is lacking and the mechanisms underlying this phenomenon are incompletely understood. Excess iron is associated with the generation of reactive oxygen species (ROS) that result in cellular damage. We hypothesized that iron overload leads to the stimulation of myelosuppressive pathways that result in decreased erythropoiesis and resulting cytopenias. We first evaluated the effect of exogenous iron on the growth of primary human CD34+ stem cells and demonstrated that iron overload leads to decreased erythroid colony formation in vitro. Furthermore, in our in vivo experiments, we administered a total of 130 mg iron dextran IP over 20 days to C57BL/6 mice and compared these mice with PBS injected age and gender matched controls (n=5/group). We demonstrate that iron injection results in fewer circulating reticulocytes (200 vs. 310 x 109 cells/L; P=0.001) and hemoglobin (13.8 vs. 15 g/dL; P=0.0004). Using anti-CD44 and TER119 as flow cytometric markers, our data reveals a reduction in bone marrow erythroid burden (total erythroid precursors 9.6 vs. 15.3% in controls; P=0.04) with a disproportionately greater effect on orthochromatophilic erythroblasts (4.4 vs. 6.9%; P=0.02). As expected, bone marrow-derived erythroid precursors from iron loaded mice have more ROS (MFI 1497 vs. 958; P=0.05) as measured by flow cytometry, again with a disproportionately greater effect on orthochromatophilic erythroblasts (MFI 671 vs. 403; P=0.01). No effect on cell cycle was observed. To evaluate the mechanism behind iron induced suppression of erythropoiesis, we conducted a functional screen with variety of cytokine inhibitors. We observed that a specific inhibitor of the TGFβ receptor I kinase led to reversal of iron induced suppression of erythroid colonies from primary CD34+ cells in vitro. The ability of TGFβ kinase inhibitor, LY-215, in reversing iron mediated suppression was confirmed in variety of hematopoietic cell lines, resulting in the inhibition of iron induced apoptosis in these cells. Taken together, our data demonstrates for the first time that iron overload has direct suppressive effects on erythropoiesis. These effects are reversed by specific inhibitors of the TGFβ receptor I kinase and thus provide a preclinical rationale for these inhibitors in anemias associated with iron overload. Disclosures:No relevant conflicts of interest to declare.

Anti-erythropoietin antibody levels and its association with anaemia in different strains of semi-immune mice infected with Plasmodium berghei ANKA

BackgroundMalaria anaemia is still a major public health problem and its pathogenesis still unclear. Interestingly, the progression of anaemia is at relatively low parasitaemia with some mortality in the semi-immune individuals in the endemic areas despite adequate erythropoietin (EPO) synthesis. A recent study has shown that treatment with exogenous anti-erythropoietin (anti-EPO) antibodies (Ab) of infected mice gives protection against malaria infection, suggesting an important role for anti-EPO Ab in malaria. The objective of the study was to evaluate anti-EPO antibody levels in anaemic condition of different strains of semi-immune mice with malaria.MethodologySemi-immune status was attained in four mice strains (Balb/c, B6, CBA and NZW) by repeated infections with 104Plasmodium berghei ANKA, and treatment with chloroquine/pyrimethamine. ELISA was used to measure anti-EPO Ab, transferrin and EPO while inflammatory cytokines measurement was done using bead-based multiplex assay kit.ResultsThe mean anti-EPO Ab levels in the mice strains [Optical Density (OD) values at 450 nm: Balb/c (2.1); B6 (1.3); CBA (1.4) and NZW (1.7)] differed (p = 0.045), and were significantly higher when compared with uninfected controls, p < 0.0001, and mean anti-EPO Ab levels in the mice strains at recovery [OD values at 450 nm: Balb/c (1.8); B6 (1.1); CBA (1.5) and NZW (1.0) also differed (p = 0.0004). Interestingly, EPO levels were significantly high in NZW and low in Balb/c mice (p < 0.05), with those of B6 and CBA of intermediary values. Again, NZW were highly parasitaemic (20.7%) and the other strains (Balb/c, B6 and CBA) ranged between 2.2-2.8% (p = 0.015). Anti-EPO Ab correlated positively with extent of Hb loss (r = 0.5861; p = 0.003). Correlation of anti-EPO antibody with EPO was significant only in Balb/c mice (r = −0.83; p = 0.01). Significant levels of IL6 and IFNγ (p < 0.0001), both known to be associated with erythropoiesis suppression were observed in the Balb/c. Transferrin was significantly lower in Balb/c (p < 0.0001) when compared with the other mice strains (B6, CBA and NZW).ConclusionThis is the first ever report in estimating endogenous anti-EPO antibodies in malaria anaemia. The data presented here suggest that anti-EPO Ab is produced at infection and is associated with Hb loss. Host factors appear to influence anti-EPO antibody levels in the different strains of mice.

Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with β-thalassemia major: a longitudinal study

Abstractβ-thalassemia major causes ineffective erythropoiesis and chronic anemia and is associated with iron overload due to both transfused iron and increased iron absorption, the latter mediated by suppression of the iron-regulatory hormone hepcidin. We sought to determine whether, in β-thalassemia major, transfusion-mediated inhibition of erythropoiesis dynamically affects hepcidin. We recruited 31 chronically transfused patients with β-thalassemia major and collected samples immediately before and 4 to 8 days after transfusion. Pretransfusion hepcidin was positively correlated with hemoglobin and ferritin and inversely with erythropoiesis. The hepcidin-ferritin ratio indicated hepcidin was relatively suppressed given the degree of iron loading. Posttransfusion, hemoglobin and hepcidin increased, and erythropoietin and growth differentiation factor-15 decreased. By multiple regression, pre- and posttransfusion hepcidin concentrations were both associated positively with hemoglobin, inversely with erythropoiesis, and positively with ferritin. Although men and women had similar pretransfusion hemoglobin, men had significantly increased erythropoiesis and lower hepcidin, received a lower transfusion volume per liter blood volume, and experienced a smaller posttransfusion reduction in erythropoiesis and hepcidin rise. Age of blood was not associated with posttransfusion hemoglobin or ferritin change. Hepcidin levels in patients with β-thalassemia major dynamically reflect competing influences from erythropoiesis, anemia, and iron overload. Measurement of these indices could assist clinical monitoring.

Nitrogen monoxide inhibits haem synthesis in mouse reticulocytes

AI (anaemia of inflammation) often manifests in patients with chronic immune activation due to cancer, chronic infections, autoimmune disorders, rheumatoid arthritis and other diseases. The pathogenesis of AI is complex and involves cytokine-mediated inhibition of erythropoiesis, insufficient erythropoietin production and diminished sensitivity of erythroid progenitors to this hormone, and retention of iron in haemoglobin-processing macrophages. NO (nitric oxide) is a gaseous molecule produced by activated macrophages that has been identified as having numerous effects on iron metabolism. In the present study, we explore the possibility that NO affects iron metabolism in reticulocytes and our results suggest that NO may also contribute to AI. We treated reticulocytes with the NO donor SNP (sodium nitroprusside). The results indicate that NO inhibits haem synthesis dramatically and rapidly at the level of erythroid-specific 5-aminolaevulinic acid synthase 2, which catalyses the first step of haem synthesis in erythroid cells. We also show that NO leads to the inhibition of iron uptake via the Tf (transferrin)-Tf receptor pathway. In addition, NO also causes an increase in eIF2α (eukaryotic initiation factor 2α) phosphorylation levels and decreases globin translation. The profound impairment of haem synthesis, iron uptake and globin translation in reticulocytes by NO raises the possibility that this gas may also contribute to AI.

Changes in the Hematopoietic System and Blood Under the Influence of Heavy Metal Salts Can Be Reduced with Vitamin E.

The aim of our work was to study the blood parameters and bone marrow morphological changes in rats exposed to increased amounts of heavy metal salts and the effect of vitamin E. Investigation of bone marrow structural features and blood parameters was performed in sexually mature Wistar male rats (n=84). Exposure to increased amounts of heavy metal salts led to the inhibition of erythropoiesis and leukopoiesis, as well as a synchronized increase in the number of megakaryocytes which was clearly reflected in the blood: the number of erythrocytes, leukocytes and Hb decreased, and the number of platelets increased. These changes in the blood and bone marrow were less pronounced when vitamin E was used as an adjuster. When increased amounts of HMS enter the rats` bodies, suppression of erythropoiesis and leukocytopoiesis occurs while thrombocytopoiesis increases. These changes depend on the period of intake of heavy metal salts. The adjustment of vitamin E reduces the severity of the cytotoxic effect of heavy metals and improves readaptation in the recovery period.

The Mean Corpuscular Volume and Hydroxyurea in Brazilian Patients with Sickle Cell Anemia: A Surrogate Marker of Compliance

The suppression of erythropoiesis by Hydroxyurea (HU) therapy is associated with increase in mean corpuscular volume, in addition to the increase in Hb F. Monitoring the mean corpuscular volume values and the presence of macrocytosis are effective tools of adherence to the treatment with HU in patients with sickle cell anemia. The aim of this study is to monitor the mean corpuscular volume values after starting treatment with HU to determine if macrocytosis can be used as a surrogate marker of compliance with therapy. We conducted a prospective cohort study over one year with measurements of blood counts and mean corpuscular volume after starting therapy with HU in 95 patients with sickle cell anemia who were regularly followed in our ambulatory outpatient unit. In one-year of successful use of HU the mean value of the mean corpuscular volume increased significantly. The Andersen and Gill model demonstrated that the increase of one unit of MCV implies a 5% reduction in the risk of visiting the emergency room. Monitoring mean corpuscular volume values after prescribing HU alerts the provider of noncompliance in order to counsel the patient in question for better adherence to the use of HU that could improve the quality of care and to reduce morbidity and the frequency of acute pain crises and associated healthcare costs.

Stromal cell-mediated inhibition of erythropoiesis can be attenuated by Sotatercept (ACE-011), an activin receptor type II ligand trap

Red cell production is primarily determined by the action of erythropoietin. Additional erythropoiesis-regulatory factors include molecules and cellular interactions occurring within the bone marrow (BM) microenvironment. Sotatercept (ACE-011) is an activin receptor ligand trap that binds several members of the TGF-β superfamily. Treatment with ACE-011 reverses bone loss and reduces the degree of osteoporosis, but it is accompanied by elevated hemoglobin and hematocrit levels. The mechanisms underlying the beneficial effects of ACE-011 on red cell production remain unknown. This study explores the means by which ACE-011 promotes erythropoiesis. We showed that ACE-011 does not directly affect erythroid differentiation of human CD34(+) cells invitro. We next tested whether ACE-011 acts indirectly by affecting BM accessory cells. Conditioned media produced by BM stromal cells (SCs) inhibited erythroid differentiation of CD34(+) cells while maintained their ability to proliferate. However, conditioned media from SCs treated with ACE-011 partially restored erythropoiesis, coinciding with changes in the molecular and secretory profile of SCs, including the expression and secretion of erythropoiesis-modulatory factors. We conclude that inhibitory factors produced by BM SCs invitro might control erythropoiesis invivo and that agents that reverse these microenvironmental signals could provide an approach to attenuate anemia in clinical conditions.

Stromal Cells-Mediated Inhibition of Erythropoiesis Can Be Counteracted by Sotatercept (ACE-011), an Activin Receptor Type II (ActRIIA) Fusion Protein

AbstractAbstract 1254Sotatercept (ACE-011) is an activin type IIA receptor (ActRIIA) fusion protein which antagonizes activin and is currently being evaluated in clinical trials for the treatment of cancer-related bone loss and anemia. Administration of sotatercept has been associated with an increase in the hemoglobin (Hb) and hematocrit (Ht) levels in preclinical and clinical studies, yet the mechanisms underlying the effects of this drug on erythropoiesis remain unknown. We, therefore, addressed the potential mechanisms underlying the effects of sotatercept on erythroid lineage by investigating its direct and indirect influences on in vitro erythropoiesis.The direct effects of sotatercept on the ability of human primary CD34+ cells to form erythroid (Ery) colonies and to generate Ery cells in liquid cultures was examined. Neither the number, the size nor the morphology of BFU and CFU-E colonies were affected by sotatercept treatment. Likewise, the percentage of CD36+/Glycophorin (GPA)+ cells generated in liquid cultures treated with sotatercept was similar to that observed in control cultures. We further showed that treatment with activin induced Ery differentiation of K562 erythroleukemic cells and this effect was counteracted by sotatercept, which is in agreement with its function as activin trap. By contrast, activin alone or in combination with sotatercept did not stimulate Ery differentiation of primary CD34+ cells, suggesting that sotatercept does not affect in vitro erythropoiesis directly or through its interaction with activin.Since stromal cells (SC) are both a major source of activin and important players in the regulation of hematopoiesis we next examined if sotatercept might influence the ability of BM-derived SC to affect erythropoiesis. Conditioned media (CM) produced by a heterogeneous population of SC inhibited Ery differentiation of CD34+ cells (i.e. the percentage of CD36+/GPA+ cells was 5-fold lower in cultures containing SC-CM as compared to control) and skewed their phenotype towards the myeloid lineage. SC-mediated inhibition of erythropoiesis was associated with the maintenance of a CD34+ cells phenotype (i.e. twice as many cells were CD34+in cultures differentiated in the presence of SC-CM as compared to control) and with the abrogation of the Ery gene signature, including a drastic suppression of most Hb and glycophorin encoding genes as well as genes involved in Hb stabilization and heme metabolism.When identical experiments were performed in the presence of CM produced by SC treated with sotatercept, the ability of SC-derived CM to inhibit erythropoiesis was significantly diminished. CD34+ cells differentiated in media conditioned by sotatercept-treated SC, generated 1.7-fold more CD36+/GPA+ Ery progenitors (p value ≤ 0.005) and 1.3-fold more mature GPA+ eryrthroblasts (p value ≤ 0.05) as compared to those generated in the presence of CM from untreated SC. In addition, they formed 1.4 times greater number of BFU-E colonies than that observed in the presence of CM derived from untreated SC. Importantly, CM from sotatercept-treated SC did not affect other hematopoietic lineages (e.g. expression of CD33, CD14 or CD61). These observations indicated that treatment with sotatercept modulated SC function to create a milieu of soluble factors which are more permissive for erythropoiesis.To indentify these potential factors we performed gene expression profiling of SC cultured in the presence or absence of sotatercept. The number of genes significantly affected by the drug was relatively small, but included genes encoding proteins which are known for their erythropoiesis-stimulatory roles (e.g. Angiotensin II, SDF1, BMP6 and IGFBP2), proteins which are potent inhibitors erythropoiesis (e.g. VEGF) and proteins with less well defined effects on erythroid lineage (e.g. BMP2, Oncostatin and Il-6). We validated these findings by measuring the levels of these secreted proteins, as well as of activin levels in the SC-CM, and demonstrated that sotatercept modifies the secretory profile of SC.We propose that SC-derived negative signals may represent an important component which contributes to the regulation of erythropoiesis. The stimulatory effects of sotatercept on red cell production, therefore, appear to be a consequence of its ability to counteract such inhibitory signals and to induce the production of a variety of Ery-promoting factors by the BM microenvironment. Disclosures:Iancu-Rubin:Celgene: Research Funding. Hoffman:Celgene: Research Funding.