Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co-signaling effects

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.     

IL-7 induces myelopoiesis and erythropoiesis

IL-7 administration to mice was previously reported to increase the mobilization of progenitor cells from marrow to peripheral sites. We now report that IL-7 increases the number of mature myeloid and monocytic cells in spleen and peripheral blood. This effect required T cells, and we show that IL-7 treatment in vivo induced GM-CSF and IL-3 production by T cells with memory phenotype. However, additional myelopoietic cytokines were shown to be involved because mice deficient in both GM-CSF and IL-3 also responded to IL-7 with increased myelopoiesis. Candidate cytokines included IFN-gamma and Flt3 ligand, which were also produced in response to IL-7. Because IFN-gamma-deficient mice also increased myelopoiesis, it was suggested that IL-7 induced production of redundant myelopoietic cytokines. In support of this hypothesis, we found that the supernatant from IL-7-treated, purified T cells contained myelopoietic activity that required a combination of Abs against GM-CSF, IL-3, and anti-Flt3 ligand to achieve maximum neutralization. IL-7 administration increased the number of splenic erythroid cells in either normal, Rag1 or GM-CSF-IL-3-deficient mice, suggesting that IL-7 might directly act on erythroid progenitors. In support of this theory, we detected a percentage of TER-119(+) erythroid cells that expressed the IL-7Ralpha-chain and common gamma-chain. Bone marrow cells expressing IL-7R and B220 generated erythroid colonies in vitro in response to IL-7, erythropoietin, and stem cell factor. This study demonstrates that IL-7 can promote nonlymphoid hemopoiesis and production of cytokines active in the host defense system in vivo, supporting its possible clinical utility.     

Severe Leukopenia and Dysregulated Erythropoiesis in SCID Mice Persistently Infected with the Parvovirus Minute Virus of Mice

Parvovirus minute virus of mice strain i (MVMi) infects committed granulocyte-macrophage CFU and erythroid burst-forming unit (CFU-GM and BFU-E, respectively) and pluripotent (CFU-S) mouse hematopoietic progenitors in vitro. To study the effects of MVMi infection on mouse hemopoiesis in the absence of a specific immune response, adult SCID mice were inoculated by the natural intranasal route of infection and monitored for hematopoietic and viral multiplication parameters. Infected animals developed a very severe viral-dose-dependent leukopenia by 30 days postinfection (d.p.i.) that led to death within 100 days, even though the number of circulating platelets and erythrocytes remained unaltered throughout the disease. In the bone marrow of every lethally inoculated mouse, a deep suppression of CFU-GM and BFU-E clonogenic progenitors occurring during the 20- to 35-d.p.i. interval corresponded with the maximal MVMi production, as determined by the accumulation of virus DNA replicative intermediates and the yield of infectious virus. Viral productive infection was limited to a small subset of primitive cells expressing the major replicative viral antigen (NS-1 protein), the numbers of which declined with the disease. However, the infection induced a sharp and lasting unbalance of the marrow hemopoiesis, denoted by a marked depletion of granulomacrophagic cells (GR-1⁺ and MAC-1⁺) concomitant with a twofold absolute increase in erythroid cells (TER-119⁺). A stimulated definitive erythropoiesis in the infected mice was further evidenced by a 12-fold increase per femur of recognizable proerythroblasts, a quantitative apoptosis confined to uninfected TER-119⁺ cells, as well as by a 4-fold elevation in the number of circulating reticulocytes. Therefore, MVMi targets and suppresses primitive hemopoietic progenitors leading to a very severe leukopenia, but compensatory mechanisms are mounted specifically by the erythroid lineage that maintain an effective erythropoiesis. The results show that infection of SCID mice with the parvovirus MVMi causes a novel dysregulation of murine hemopoiesis in vivo.     

Activation of extracellular signal-regulated kinases ERK1 and ERK2 induces Bcl-xL up-regulation via inhibition of caspase activities in erythropoietin signaling

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.     

Innate immune activation during Salmonella infection initiates extramedullary erythropoiesis and splenomegaly

Systemic Salmonella infection commonly induces prolonged splenomegaly in murine or human hosts. Although this increase in splenic cellularity is often assumed to be due to the recruitment and expansion of leukocytes, the actual cause of splenomegaly remains unclear. We monitored spleen cell populations during Salmonella infection and found that the most prominent increase is found in the erythroid compartment. At the peak of infection, the majority of spleen cells are immature CD71(-)Ter119(+) reticulocytes, indicating that massive erythropoiesis occurs in response to Salmonella infection. Indeed, this increase in RBC precursors corresponded with marked elevation of serum erythropoietin (EPO). Furthermore, the increase in RBC precursors and EPO production required innate immune signaling mediated by Myd88/TRIF. Neutralization of EPO substantially reduced the immature RBC population in the spleen and allowed a modest increase in host control of infection. These data indicate that early innate immunity to Salmonella initiates marked splenic erythropoiesis and may hinder bacterial clearance.     

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.     

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells. Bovine liver erythroid cells were cultured on monolayers of human bone marrow endothelial cells previously treated with EPO and IGF-IL-3. There was a significant reduction of thymidine incorporation into both erythroid and endothelial cells in cultures pre-treated with IGF-IL-3 and EPO. Endothelial cell culture supernatants separated by ultrafiltration and ultracentrifugation from cells treated with EPO and IL-3 significantly reduced thymidine incorporation into erythroid cells as compared to identical fractions obtained from the media of cells cultured with EPO alone. These results suggest that endothelial cells treated simultaneously with EPO and IL-3 have a negative effect on erythroid cell production.     

人红细胞生成素的分离、纯化及鉴定

 用自制的苯基-琼脂糖CL-4B和羟基邻灰石等层析材料,从再生障碍性贫血病人尿中分离、纯化制得了红细胞生成素(EPO)。用多血小鼠红细胞~(56)Fe参入法测定该制品在体内的生物活力。用小鼠与人骨髓红系祖细胞培养法测其在体外的生物活力。实验结果说明,我们自制的EPO制品,不仅能用于动物,也能用于人骨贿红系祖细胞的培养。用Azocoll法测该制品中蛋白水解酶活力为阴性。     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.     

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.     

Regulation of programmed death in erythroid progenitor cells by erythropoietin: effects of calcium and of protein and RNA syntheses.

Erythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well-characterized model of glucocorticoid-treated thymocytes, activation of a Ca2+/Mg(2+)-sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+ and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus-infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]i in FVA cells. Decreasing [Ca2+]i by extracellular Ca2+ chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]i with the calcium ionophore 4-bromo-A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]i were much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]i also inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However, no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO-derived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+ and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types.     

Vanadate elevates fetal hemoglobin in human erythroid precursors by inhibiting cell maturation

Increased fetal hemoglobin (HbF) in erythroid precursors of patients with beta-hemoglobinopathies (sickle cell anemia and beta-thalassemia), in which adult hemoglobin synthesis is defective, ameliorates the clinical symptoms of the underlying diseases. The production of erythroid precursors depends on the action of erythropoietin (EPO), which prevents their apoptosis and stimulates their proliferation. EPO binds to its surface receptor, induces its homodimerization, and initiates a cascade of phosphorylation and dephosphorylation of a series of proteins by kinases and phosphatases, respectively. Vanadate inhibits various phosphatases, including those that are involved in the EPO pathway, thereby intensifying the signal. In this study, we investigated the effect of vanadate on the proliferation and maturation of human erythroid precursors in culture. When vanadate was added to cells derived from normal donors, cell maturation was delayed, as indicated by cell morphology, cell growth kinetics, the rate of appearance of hemoglobin-containing cells, and the expression of surface antigens (CD117, CD71, and glycophorin A). Analysis by high-performance liquid chromatography and flow cytometry of the hemoglobin profile of vanadate-treated normal cells revealed a higher proportion of HbF than was found in untreated cells. When vanadate was added to cells derived from patients with beta-thalassemia, a significant increase in HbF was observed. The results suggest that intensification of the EPO signal by vanadate results in maturation arrest and increased HbF production. Thus, inhibitors that are more specific and less toxic than vanadate may present a novel option for elevating HbF in patients with beta-hemoglobinopathies, as well as for intensifying the EPO response in other forms of anemia.     

The C-terminal peptide of thrombospondin-4 stimulates erythroid cell proliferation

Erythropoietin (EPO) stimulates the production of small erythroid cell stimulating factors (molecular weight <5 kDa) in cultures of bone marrow endothelial cells. We identified a fragment of thrombospondin-4 (TSP-4) as an EPO-stimulated protein in endothelial cell lysates. Pre-incubation of the low molecular weight fractions from supernatants of EPO-treated umbilical cord endothelial cells (HUVEC) with antibodies against the C-terminal residues of TSP-1,2 and TSP-4 decreased the erythroid cell stimulating activity. The C-terminal TSP-1 section corresponding to a molecular weight lower than 6 kDa has the integrin-associated protein binding motif VVM. The corresponding TSP-4 fragment, lacking the three residue sequence VVM, has a distinctive acidic peptide comprising the last 21 amino acids (C21) with the characteristics of an amphipathic helix. C21 stimulated thymidine incorporation into bovine erythroid cells, increased cell numbers in cultures of cord blood CD36+ erythroid precursors and skin fibroblasts, and decreased HUVEC proliferation. SC21, a homologous peptide of identical amino acid composition but with interchanged residues, was non-amphipathic and had no erythroid cell stimulating activity.     

Thrombospondin 1, produced by endothelial cells under the action of erythropoietin, stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of insulin-like growth factor binding protein 3

The nature of erythropoietin (EPO)-dependent, erythroid cell regulatory factors secreted by endothelial cells is largely unknown. The production of thrombospondin 1 (TSP-1) and insulin-like growth factor binding protein 3 (IGFBP-3) is increased in cultures of human umbilical vein endothelial cells (HUVEC) incubated with erythropoietin (EPO). Simultaneous incubation of HUVEC with EPO and interleukin 3 (IL-3) resulted in a decreased production, suggesting that both TSP-1 and IGFBP-3 belong to the EPO- and IL-3-dependent erythroid regulatory factors previously described in cultures of bone marrow endothelial cells. TSP-1 and TSP-1 derived synthetic peptides based on the CD36 and CD47 binding sites of TSPs increased thymidine incorporation into bovine erythroid cells of fetal liver. IGBBP-3 inhibited thymidine incorporation in the same cells. Preincubation of erythroid cells with TSP-1 eliminated the inhibitory activity of IGFBP-3. We suggest that EPO-dependent, endothelial-derived TSP-1 may play a positive role in red cell production by acting directly on erythroid cells, stimulating DNA synthesis and preventing the inhibitory action of IGFBP-3.     

Erythropoietin as an antiapoptotic, tissue-protective cytokine

Erythropoietin (EPO) increases the number of circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors. In addition to this proerythroid action, results of recent studies show that systemically administered EPO is protective in vivo, in several animal models of neuronal injury. In vitro, EPO prevents neuronal apoptosis induced by a variety of stimuli. This review summarizes the neuroprotective actions of EPO and discusses the underlying mechanisms in terms of signal transduction pathways involved. The understanding of these mechanisms will help differentiate the neuroprotective actions of EPO from its role in the bone marrow.     

Nicotinic acetylcholine receptors alpha4beta2 and alpha7 regulate myelo- and erythropoiesis within the bone marrow

Nicotine consumed upon smoking affects numerous physiological processes through nicotinic acetylcholine receptors, which mediate cholinergic regulation by the neuronal and endogenous acetylcholine. Consequently, nicotinic receptors are expressed in many non-excitable tissues including the blood. In spite of the documented effect of nicotine on hematopoiesis, little is known about the expression and role of nicotinic receptors in the course of blood cell differentiation. The aim of the present study was to investigate whether and how nicotinic receptors are involved in the development of myeloid and erythroid cells within the bone marrow. The presence of nicotinic receptors containing alpha4(beta2) and alpha7 subunits in the bone marrow cells of C57Bl/6 mice was shown by the binding of [125I]-alpha-bungarotoxin or [3H]-Epibatidine and by flow cytometry with subunit-specific antibodies or fluorescein-labeled alpha-cobratoxin. Both TER119+ (erythroid) and CD16+CD43med (myeloid) progenitor cells bound more alpha4-specific antibodies than their mature forms, while the binding of alpha-cobratoxin and alpha7-specific antibodies was also high in mature cells. According to morphological analysis, either the absence of alpha7-containing nicotinic receptors in knockout mice or their desensitization in mice chronically treated with nicotine decreased the number of myeloid and erythroid progenitors and junior cells. In contrast, the absence of beta2-containing receptors favored myelocyte generation and erythroid cell maturation. It is concluded that the development of both myeloid and erythroid cell lineages is regulated by endogenous cholinergic ligands and can be affected by nicotine through alpha7- and alpha4beta2-containing nicotinic receptors, which play different roles in the course of the cell maturation.     

IL-7-mediated protection of pro and pre-B cells from the adverse effects of corticosterone

The studies herein demonstrate that Interleukin-7 (IL-7) promotes survival of murine pro- and pre-B cells against stress levels of corticosterone (Cs). In short-term, 16-h, bone marrow cultures IL-7 abrogated Cs-induced apoptosis and cell cycle arrest in pro-B cells by decreasing apoptosis 60% and completely restoring the cell cycle. IL-7 also reduced Cs-induced apoptosis by 36% in pre-B cells and 24% in IgM(+) B cells, but did not restore deficits in the cell cycle. Among pro- and pre- B cells, substantial protection against high, pharmacological, levels of Cs was also provided by IL-7. Interestingly, stem cell factor, while reducing spontaneous apoptosis in pro-B cells, did not protect against Cs-induced death, either alone or with IL-7. In conclusion, IL-7 has potential immunotherapeutic value since it provides substantial protection to pro- and pre-B cells against the adverse effects of Cs.     

Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.

Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.     

Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.