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Stress erythropoiesis: definitions and models for its study

  • Robert F Paulson
    Correspondence
    Offprint requests to: Robert F. Paulson, Center for Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Sciences, 108 Research Building A, Penn State University, University Park, PA 16802.
    Affiliations
    Center for Molecular Immunology and Infectious Disease and the Department of Veterinary and Biomedical Sciences, Penn State University, University Park, PA

    Intercollege Graduate Program in Genetics, Penn State University, University Park, PA
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  • Sneha Hariharan
    Affiliations
    Intercollege Graduate Program in Genetics, Penn State University, University Park, PA
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  • Jane A Little
    Affiliations
    Department of Medicine, University of North Carolina Comprehensive Sickle Cell Disease Program, Chapel Hill, NC
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Open AccessPublished:August 01, 2020DOI:https://doi.org/10.1016/j.exphem.2020.07.011

      Highlights

      • Stress erythropoiesis is a complex response to anemic stress.
      • Increased steady-state erythropoiesis is driven by Epo.
      • Inflammation impairs steady-state erythropoiesis and induces BMP4-dependent stress erythropoiesis.
      • BMP4-dependent stress erythropoiesis is highly conserved in mouse and human.
      Steady-state erythropoiesis generates new erythrocytes at a constant rate, and it has enormous productive capacity. This production is balanced by the removal of senescent erythrocytes by macrophages in the spleen and liver. Erythroid homeostasis is highly regulated to maintain sufficient erythrocytes for efficient oxygen delivery to the tissues, while avoiding viscosity problems associated with overproduction. However, there are times when this constant production of erythrocytes is inhibited or is inadequate; at these times, erythroid output is increased to compensate for the loss of production. In some cases, increased steady-state erythropoiesis can offset the loss of erythrocytes but, in response to inflammation caused by infection or tissue damage, steady-state erythropoiesis is inhibited. To maintain homeostasis under these conditions, an alternative stress erythropoiesis pathway is activated. Emerging data suggest that the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway is integrated into the inflammatory response and generates a bolus of new erythrocytes that maintain homeostasis until steady-state erythropoiesis can resume. In this perspective, we define the mechanisms that generate new erythrocytes when steady-state erythropoiesis is impaired and discuss experimental models to study human stress erythropoiesis.
      Steady-state erythropoiesis has an enormous capacity to generate new erythrocytes. It is estimated that adult humans produce 2.5 × 106 erythrocytes per second [
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      ]. Production and turnover are finely balanced to maintain oxygen delivery to the tissues while avoiding problems with blood viscosity associated with overproduction. Steady-state erythroid progenitors are derived from immature megakaryocyte erythroid progenitors and multipotential myeloid progenitors [
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      ]. They develop in close proximity with macrophages in a specialized niche referred to as an erythroblastic island (EBI) [
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      ]. These structures are conserved between rodents and humans [
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      ]. Despite the capacity of steady-state erythropoiesis to produce erythrocytes, there are times when it is unable maintain erythroid homeostasis. Simple blood loss can lead to increased steady-state erythropoiesis, but the situation is more complex when inflammation caused by infection or tissue damage inhibits steady-state erythropoiesis. At these times, an alternative erythropoiesis pathway is required and the bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis pathway predominates (for review, see [
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      ]). BMP4-dependent stress erythropoiesis has a different strategy than steady-state erythropoiesis. Instead of constantly producing new erythrocytes, the stress erythron produces a bolus of new erythrocytes to maintain homeostasis until steady-state erythropoiesis can resume normal erythroid output [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
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      • Harandi OF
      • Hedge S
      • Wu DC
      • McKeone D
      • Paulson RF
      Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors.
      ,
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ,
      • Perry JM
      • Harandi OF
      • Porayette P
      • et al.
      Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.
      ]. In this perspective, we outline the characteristics of the BMP4-dependent stress erythropoiesis pathway and discuss the utility of different experimental systems to model human stress erythropoiesis.

      Stress erythropoiesis is a stem cell-based tissue regeneration response

      Stress erythropoiesis is a catchall phrase that describes the increase in erythroid output in response to anemic stress. However, this response is more than just increasing steady-state erythropoiesis. Early studies in mice analyzed the recovery from phenylhydrazine (PHZ)-induced acute hemolytic anemia. These data suggested that bone marrow steady-state erythroid progenitors migrated to the spleen, where they differentiated in response to the increased serum erythropoietin (Epo) levels induced by tissue hypoxia [
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      ]. Subsequent analysis of mouse strains with mutations that impair stress erythropoiesis revealed that this model was incorrect [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ,
      • Perry JM
      • Harandi OF
      • Porayette P
      • et al.
      Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.
      ]. The increase in erythropoiesis during recovery came from progenitor cells that were distinct from steady-state erythroid progenitors and whose development was regulated by many signals that are not involved in the development of steady-state erythroid cells. Analysis of flexed-tail (f) mutant mice established a role for BMP4 signaling in the recovery from anemia, and for the purposes of this review we refer to this response as BMP4-dependent stress erythropoiesis [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ]. Before we discuss the mechanisms that regulate this pathway, we need to define the differences between BMP4-dependent stress erythropoiesis and increased steady-state erythropoiesis. The BMP4-dependent pathway is best understood in mice, and our discussion of the mechanisms that regulate this process will focus on those data. Pro-inflammatory cytokines and alarmins inhibit steady-state erythropoiesis and promote myelopoiesis, to drive the development of myeloid effector cells [
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      ,
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      ]. To compensate for the loss of steady-state erythropoiesis, BMP4-dependent stress erythropoiesis is induced. Unlike steady-state erythropoiesis, inflammatory signals act as inducers of this pathway [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ]. In mice, the BMP4-dependent stress erythropoiesis pathway is extramedullary, occurring in the adult spleen and liver [
      • Lenox LE
      • Shi L
      • Hegde S
      • Paulson RF
      Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling.
      ]. In response to PHZ-induced anemia, a population of stress burst-forming units erythroid (BFU-E) is expanded in the spleen, while at the same time the production of steady-state BFU-E in the bone marrow decreases [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ]. This switch in erythroid production from steady-state erythropoiesis to BMP4-dependent stress erythropoiesis is a common feature of experimental anemias induced by diverse treatments ranging from PHZ injection to models of sterile inflammation [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ,
      • Harandi OF
      • Hedge S
      • Wu DC
      • McKeone D
      • Paulson RF
      Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors.
      ,
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Bennett LF
      • Liao C
      • Paulson RF
      Stress erythropoiesis model systems.
      ]. In contrast to these treatments, many researchers use treatment with erythropoietin (Epo) to induce stress erythropoiesis, often referred to as Epo stress. Treatment with Epo does not induce the BMP4-dependent stress erythropoiesis pathway. Although some articles have reported increased erythropoiesis in the spleens of mice treated with Epo, this observation is due to the differentiation of committed late-stage erythroid progenitors (colony-forming units erythroid [CFU-E] and erythroblasts) in the spleen and not BMP4-dependent stress erythropoiesis [
      • Suzuki M
      • Ohneda K
      • Hosoya-Ohmura S
      • et al.
      Real-time monitoring of stress erythropoiesis in vivo using Gata1 and beta-globin LCR luciferase transgenic mice.
      ]. In addition, Epo treatment skews steady-state hematopoiesis to favor erythropoiesis by increasing the commitment of immature progenitors to the erythroid lineage. In many ways, the action of Epo is the opposite of the action of pro-inflammatory cytokines (Figure 1), as Epo increases steady-state erythropoiesis while decreasing steady-state myelopoiesis [
      • Tusi BK
      • Wolock SL
      • Weinreb C
      • et al.
      Population snapshots predict early haematopoietic and erythroid hierarchies.
      ,
      • Grover A
      • Mancini E
      • Moore S
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      Erythropoietin guides multipotent hematopoietic progenitor cells toward an erythroid fate.
      ,
      • Singh RP
      • Grinenko T
      • Ramasz B
      • et al.
      Hematopoietic stem cells but not multipotent progenitors drive erythropoiesis during chronic erythroid stress in EPO transgenic mice.
      ]. The role for Epo in increasing steady-state erythropoiesis is also observed in phlebotomy-induced anemia. Careful phlebotomy does not induce substantial tissue damage and inflammation and is a weak inducer of the BMP4-dependent pathway.
      Figure 1
      Figure 1Schematic of alternate responses to anemia. Left: Steady-state erythropoiesis is increased by increased levels of Epo. Right: Inflammation compromises steady-state erythropoiesis, which leads to activation of the BMP4-dependent stress erythropoiesis pathway. The megakaryocyte pathway is not shown to simplify the diagram.
      The primary differences between these processes are in the progenitor cells, the signals that regulate their proliferation and commitment to differentiation, and the niche where BMP4-dependent stress erythropoiesis occurs. As described above, in mice, BMP4-dependent stress erythropoiesis is extramedullary. In general, stress erythropoiesis is often referred to as splenic erythropoiesis. This characterization is misleading. Fully grown adult mice (>8–10 weeks old) exhibit little steady-state erythropoiesis in the spleen, but in response to inflammatory stress, the spleen is the primary site of BMP4-dependent stress erythropoiesis. However, splenectomized mice are equally capable of responding to anemic stress. In this case, the liver becomes the site of BMP4-dependent stress erythropoiesis. Despite the change in site, liver stress erythropoiesis utilizes the same signals and progenitor cells that are observed in the spleen [
      • Lenox LE
      • Shi L
      • Hegde S
      • Paulson RF
      Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling.
      ]. Because of these observations, BMP4-dependent stress erythropoiesis can be thought of as extra-medullary. Although this pathway is conserved in humans, it has not been established that human BMP4-dependent stress erythropoiesis occurs in the spleen. Extramedullary erythropoiesis is observed in many pathological conditions such as anemia, malignancy, and infection, but the role of the BMP4-dependent pathway in these situations has not been investigated [
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      ].
      The origin and development of immature progenitors are major differences between steady-state and BMP4-dependent stress erythropoiesis. Steady-state erythroid progenitors are derived from multipotential progenitors that adopt the erythroid fate. The direct precursor of erythroid progenitor cells is a megakaryocyte–erythroid progenitor (MEP) [
      • Karamitros D
      • Stoilova B
      • Aboukhalil Z
      • et al.
      Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.
      ,
      • Pronk CJ
      • Rossi DJ
      • Mansson R
      • et al.
      Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.
      ]. In contrast, stress erythroid progenitors are derived from short-term hematopoietic stem cells (ST-HSCs) that are characterized as CD34+Kit+Sca1+Lineageneg18 [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. In the bone marrow, these cells have the potential to generate all cell lineages; however, upon homing to the spleen, signals in the splenic micro-environment commit these cells to the erythroid lineage. The key signals in this commitment step are hedgehog and BMP4 [
      • Perry JM
      • Harandi OF
      • Porayette P
      • et al.
      Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.
      ]. It is most likely Indian hedgehog (Ihh) that regulates this commitment, as Sonic hedgehog (Shh) is not expressed in the red pulp of the spleen and Desert hedgehog (Dhh) appears to have a negative effect on stress erythropoiesis [
      • Lau CI
      • Outram SV
      • Saldana JI
      • et al.
      Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog.
      ]. Hedgehog signaling induces ST-HSCs to express BMP4, and it is the two signals acting together that is required for the specification of the stress erythroid lineage. Mutations in the hedgehog signaling pathway do not affect steady-state erythropoiesis. In contrast, loss of hedgehog signaling prevents maintenance of the BMP4-dependent pathway. In response to PHZ-induced anemia, the BMP4-dependent pathway generates new erythrocytes over the 7-day recovery period. After this initial red blood cell (RBC) generation, it takes 21 days before the mouse can respond again to a second anemic challenge. Mutations in hedgehog signaling completely inhibit the regeneration of the pathway, preventing subsequent responses to anemia. Furthermore, activation of hedgehog signaling in the bone marrow leads to the development of stress progenitors, suggesting that the compartmentalization of hedgehog signaling restricts stress erythropoiesis to extramedullary sites [
      • Perry JM
      • Harandi OF
      • Porayette P
      • et al.
      Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.
      ].
      Although BMP4 and Hedgehog signals restrict the ST-HSCs to the stress erythroid lineage, the immature stress erythroid progenitors (SEPs) maintain stem cell properties. Immature SEPs can be broken down into three populations based on their expression of Kit, Sca1, CD34, and CD133 (Figure 2A) [
      • Harandi OF
      • Hedge S
      • Wu DC
      • McKeone D
      • Paulson RF
      Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors.
      ,
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. All three of these populations retain their ability to self-renew and can be serially transplanted [
      • Harandi OF
      • Hedge S
      • Wu DC
      • McKeone D
      • Paulson RF
      Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors.
      ,
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. Following lineage restriction, the SEPs proliferate, but do not differentiate, which generates a transient amplifying population of progenitor cells. This stage in development is characterized by the expression of stem cell markers and a lack of expression of the erythroid program. This amplification step is necessary to generate enough progenitors so that when they differentiate, sufficient erythrocytes will be made to maintain homeostasis until steady-state erythropoiesis can resume. The signals that drive this expansion include growth and differentiation factor 15 (GDF15) and canonical Wnt signaling [
      • Chen Y
      • Xiang J
      • Qian F
      • et al.
      Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation.
      ,
      • Hao S
      • Xiang J
      • Wu DC
      • et al.
      Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche.
      ]. Mutations in either of these pathways impair the expansion of immature SEPs but have little effect on steady-state erythropoiesis [
      • Hao S
      • Xiang J
      • Wu DC
      • et al.
      Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche.
      ,
      • Cobas M
      • Wilson A
      • Ernst B
      • et al.
      Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.
      ]. In addition, Kit receptor and its ligand stem cell factor (SCF) are required for the proliferation of immature SEPs. Mutations in the Kit receptor impair the proliferation of immature SEPs to the point that mice with severe loss-of-function alleles of Kit lack SEPs in the spleen [
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ]. However, unlike mutations GDF15 and the Wnt signaling pathway, mutation of Kit or SCF exhibit a macrocytic anemia, indicating a need for this signaling pathway in both steady-state and BMP4-dependent stress erythropoiesis [
      • Russell ES
      Hereditary anemias of the mouse: a review for geneticists.
      ]. The expansion of immature SEPs is followed by a transition to differentiation. At this stage during their development, the proliferating immature populations of SEPs acquire the ability to differentiate and initiate the erythroid gene expression program. The progenitors lose their ability to self-renew and can no longer be serially transplanted. The signal that drives this transition is Epo, but in this instance Epo is not acting on erythroid progenitor cells (as it would during terminal differentiation), but rather on macrophages in the micro-environment. Epo signaling alters the signals generated by the macrophages, inhibiting the expression of Wnt factors, which promote proliferation and increasing the production of prostaglandins (PGJ2 and PGE2), which promote differentiation [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ,
      • Chen Y
      • Xiang J
      • Qian F
      • et al.
      Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation.
      ]. During this transition, SEPs lose the expression of stem cell markers and start expressing the Epo receptor, which drives terminal differentiation. Other signals that contribute to this transition include corticosteroids that act through the glucocorticoid receptor, which promote the expansion of the population of committed erythroid progenitors. Mutation of the glucocorticoid receptor impairs stress erythropoiesis at this stage. Corticosteroids work in concert with secreted SCF to drive the proliferation of committed progenitor cells [
      • Bauer A
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      The glucocorticoid receptor is required for stress erythropoiesis.
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      ,
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      ,
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      The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro.
      ,
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      ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.
      ].
      Figure 2
      Figure 2Model of BMP4-dependent stress erythropoiesis. (A) Schematic of the four steps from migration of ST-HSCs into the spleen to terminal differentiation. After specification, the status of Epo receptor expression in the SEPs is indicated. Cell surface markers that identify the populations of SEPs are shown. KSL=Kit+Sca1+Lineage. The signals known to participate at each stage are indicated below. (B) Schematic of the development of the stress erythropoiesis niche in the spleen.
      In both steady-state and stress erythropoiesis, BFU-E represent the most immature committed erythroid progenitor as defined by colony assays. Stress BFU-E differ from steady-state BFU-E in that they can form BFU-E colonies in medium containing only Epo. Maximal stress BFU-E production is observed when cells are plated with Epo, BMP4, and SCF at 2% O2 [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ,
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. When compared with bone marrow steady-state BFU-E, stress BFU-E generate larger colonies, which is consistent with stress BFU-E having a greater capacity to generate new erythrocytes [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ]. Despite these differences, the generation of erythrocytes from CFU-E and erythroblasts in both steady state and the BMP4-dependent pathway require the same genes regulated by key erythroid transcription factors like Gata1, Scl, and Klf1 [
      • Crispino JD
      • Weiss MJ
      Erythro-megakaryocytic transcription factors associated with hereditary anemia.
      ]. The only difference is that Kit receptor expression is maintained in stress erythroblasts [
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ].
      Similar to all types of hematopoiesis, BMP4-dependent stress erythropoiesis relies on interactions with the micro-environment. During each of the stages of stress erythropoiesis, SEPs interact with monocytes and macrophages in the micro-environment. Eliminating macrophages in vivo or in vitro blocks the development of SEPs [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ,
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ,
      • Chow A
      • Huggins M
      • Ahmed J
      • et al.
      CD169(+) macrophages provide a niche promoting erythropoiesis under homeostasis and stress.
      ,
      • Ramos P
      • Casu C
      • Gardenghi S
      • et al.
      Macrophages support pathological erythropoiesis in polycythemia vera and beta-thalassemia.
      ]. The interaction between macrophages and developing erythroid progenitors is a common theme observed in both steady-state and BMP4-dependent stress erythropoiesis. These interactions are mediated by adhesion molecules that are expressed by macrophages in both the bone marrow and the spleen. However, mutations revealed that certain adhesion molecules have a greater role in stress erythropoiesis while others function in steady-state erythropoiesis [
      • Ulyanova T
      • Jiang Y
      • Padilla S
      • Nakamoto B
      • Papayannopoulou T
      Combinatorial and distinct roles of alpha(5) and alpha(4) integrins in stress erythropoiesis in mice.
      ,
      • Ulyanova T
      • Padilla SM
      • Papayannopoulou T
      Stage-specific functional roles of integrins in murine erythropoiesis.
      ]. For example, the α4 integrin mutation exhibits little effect in steady-state erythropoiesis but exhibits a delayed recovery from PHZ-induced acute anemia [
      • Ulyanova T
      • Jiang Y
      • Padilla S
      • Nakamoto B
      • Papayannopoulou T
      Combinatorial and distinct roles of alpha(5) and alpha(4) integrins in stress erythropoiesis in mice.
      ,
      • Ulyanova T
      • Padilla SM
      • Papayannopoulou T
      Stage-specific functional roles of integrins in murine erythropoiesis.
      ]. In contrast, mutation of Maea leads to a defect in steady-state erythropoiesis that is compensated by stress erythropoiesis [
      • Wei Q
      • Boulais PE
      • Zhang D
      • et al.
      Maea expressed by macrophages, but not erythroblasts, maintains postnatal murine bone marrow erythroblastic islands.
      ]. The roles of adhesion molecules in regulating BMP4-dependent stress erythropoiesis are complicated by the nature of the adhesion molecules that can function as heterodimers, such as α4β1 and α5β1 integrins or monomers such as Maea and their interactions, which can be homotypic like Maea–Maea or heterotypic like α4β1–Vcam1 (for discussion of this complexity, see [
      • Ulyanova T
      • Georgolopoulos G
      • Papayannopoulou T
      Reappraising the role of alpha5 integrin and the microenvironmental support in stress erythropoiesis.
      ]). In addition to these adhesive interactions, signaling in macrophages plays a key role in regulating SEP development. As described above, Epo signaling in macrophages induces a change in the signals from those that promote proliferation to those that promote differentiation. Macrophages within steady-state erythroblastic islands also express the Epo receptor, but the effects of Epo-dependent signaling in steady-state EBI macrophages are not known and do not appear to increase prostaglandin production as observed in the spleen [
      • Li W
      • Wang Y
      • Zhao H
      • et al.
      Identification and transcriptome analysis of erythroblastic island macrophages.
      ].
      Not only are the signals from the micro-environment and the interaction between progenitors and macrophages in EBIs different in stress erythropoiesis, the development of the niche is different. Steady-state erythropoiesis maintains EBIs in the bone marrow for constant production of erythrocytes [
      • Seu KG
      • Papoin J
      • Fessler R
      • et al.
      Unraveling macrophage heterogeneity in erythroblastic islands.
      ,
      • Jacobsen RN
      • Forristal CE
      • Raggatt LJ
      • et al.
      Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80(+)VCAM1(+)CD169(+)ER-HR3(+)Ly6G(+) erythroid island macrophages in the mouse.
      ,
      • Jacobsen RN
      • Nowlan B
      • Brunck ME
      • et al.
      Fms-like tyrosine kinase 3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse.
      ,
      • Jacobsen RN
      • Perkins AC
      • Levesque JP
      Macrophages and regulation of erythropoiesis.
      ,
      • Kaur S
      • Raggatt LJ
      • Millard SM
      • et al.
      Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment.
      ]; however, the splenic stress erythropoiesis niche is induced by inflammation and develops in concert with SEPs [
      • Liao C
      • Prabhu KS
      • Paulson RF
      Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia.
      ]. The pro-inflammatory signals that inhibit bone marrow erythropoiesis play two roles in stress erythropoiesis. They promote SEP proliferation and the recruitment of monocytes into the spleen to form the niche [
      • Hao S
      • Xiang J
      • Wu DC
      • et al.
      Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche.
      ,
      • Liao C
      • Prabhu KS
      • Paulson RF
      Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia.
      ]. Monocytes mature into macrophages as SEPs proliferate and transition to stress BFU-E, which illustrates the coordinate development of progenitors and the splenic stress erythropoiesis niche (Figure 2B). In addition to monocytes and macrophages, the development of the stress erythropoiesis niche also includes other elements. Type 1 conventional dendritic cells and monocytes are derived from a common progenitor [
      • Merad M
      • Sathe P
      • Helft J
      • Miller J
      • Mortha A
      The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.
      ]. These dendritic cells are recruited into the niche and play a key role in stress erythropoiesis [
      • Liao C
      • Prabhu KS
      • Paulson RF
      Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia.
      ]. They express SCF, and loss of the niche population of dendritic cells impairs recovery from anemia [
      • Kim TS
      • Hanak M
      • Trampont PC
      • Braciale TJ
      Stress-associated erythropoiesis initiation is regulated by type 1 conventional dendritic cells.
      ]. Although infection or tissue damage increases pro-inflammatory cytokine production, which skews hematopoiesis toward the production of myeloid effector cells and inhibits steady-state erythropoiesis, these signals lead to increased production and mobilization of monocytes and dendritic cells, which subsequently home to the spleen, leading to the development of the stress erythroid niche [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ,
      • Pietras EM
      Inflammation: a key regulator of hematopoietic stem cell fate in health and disease.
      ,
      • Pietras EM
      • Mirantes-Barbeito C
      • Fong S
      • et al.
      Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal.
      ,
      • Liao C
      • Prabhu KS
      • Paulson RF
      Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia.
      ,
      • Boldin MP
      • Taganov KD
      • Rao DS
      • et al.
      miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice.
      ,
      • Oduro Jr, KA
      • F Liu
      • Tan Q
      • et al.
      Myeloid skewing in murine autoimmune arthritis occurs in hematopoietic stem and primitive progenitor cells.
      ,
      • Zhao JL
      • Ma C
      • O'Connell RM
      • et al.
      Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis.
      ]. In addition, inflammation induces the production of corticosteroids by the adrenal gland, which act on this niche to promote the maturation monocytes into EBI macrophages [
      • Falchi M
      • Varricchio L
      • Martelli F
      • et al.
      Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.
      ,
      • Heideveld E
      • Hampton-O'Neil LA
      • Cross SJ
      • et al.
      Glucocorticoids induce differentiation of monocytes towards macrophages that share functional and phenotypical aspects with erythroblastic island macrophages.
      ,
      • Heideveld E
      • Masiello F
      • Marra M
      • et al.
      CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield.
      ]. This tight coordination of signals coupled with tissue-specific responses to inflammatory signals ensures that the mobilization of the immune response is robust without compromising erythroid homeostasis.
      The role of inflammation in regulating stress erythropoiesis is similar to other tissue regeneration responses. For example, transient inflammation initiates regeneration following injury in the intestinal epithelium and in skeletal muscle. In these systems, the recruitment of monocytes and macrophages into the sites of injury provides key signals to promote the expansion and differentiation of tissue-resident stem cells to repair these tissues [
      • Tidball JG
      Regulation of muscle growth and regeneration by the immune system.
      ]. Similar to BMP4-dependent stress erythropoiesis, pro-inflammatory micro-environments are associated with the expansion of immature progenitors, while anti-inflammatory or pro-resolving signals are associated with differentiation. This reliance on resolution of inflammatory signals for terminal differentiation provides a basis for the observation that chronic inflammation impairs regeneration [
      • Tidball JG
      Regulation of muscle growth and regeneration by the immune system.
      ,
      • Asfaha S
      Intestinal stem cells and inflammation.
      ,
      • Giannakis N
      • Sansbury BE
      • Patsalos A
      • et al.
      Dynamic changes to lipid mediators support transitions among macrophage subtypes during muscle regeneration.
      ,
      • Rathinam VAK
      • Chan FK
      Inflammasome, inflammation, and tissue homeostasis.
      ]. In erythropoiesis, chronic inflammation leads to anemia. Many of these chronic anemias, including sickle cell disease, hemolytic anemia, and the anemia of chronic disease (ACD), have underlying inflammatory components that may contribute to the pathology of the anemia [
      • Weiss G
      • Ganz T
      • Goodnough LT
      Anemia of inflammation.
      ]. Hemolysis releases hemoglobin and heme that become pro-inflammatory mediators, while, in the case of ACD, infections and tissue damage induce inflammatory responses that inhibit steady-state erythropoiesis [
      • Weiss G
      • Ganz T
      • Goodnough LT
      Anemia of inflammation.
      ,
      • Canesin G
      • Hejazi SM
      • Swanson KD
      • Wegiel B
      Heme-derived metabolic signals dictate immune responses.
      ,
      • Nader E
      • Romana M
      • Connes P
      The red blood cell–inflammation vicious circle in sickle cell disease.
      ]. Inflammatory signals also increase hepcidin production leading to the sequestration of iron and act on phagocytes, increasing their turnover of erythrocytes [
      • Cassat JE
      • Skaar EP
      Iron in infection and immunity.
      ,
      • Libregts SF
      • Gutierrez L
      • de Bruin AM
      • et al.
      Chronic IFN-gamma production in mice induces anemia by reducing erythrocyte life span and inhibiting erythropoiesis through an IRF-1/PU.1 axis.
      ,
      • Soares MP
      • Weiss G
      The Iron Age of host–microbe interactions.
      ]. Resolution of the underlying cause of inflammation is the best way to treat ACD. In mouse models of sterile inflammatory disease, the mice develop anemia. The BMP4-dependent stress erythropoiesis pathway is active in these models and promotes the initial recovery from the anemia; however, the mice develop generalized inflammatory disease, which leads to a relapsing anemia [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ]. These data highlight a weakness of this regenerative pathway. Unlike the constant production of steady-state erythropoiesis, BMP4-dependent stress erythropoiesis makes a bolus of erythrocytes and then must start over to generate a second wave of erythrocytes. Because of this strategy, chronic inflammatory stress presents a problem for the BMP4-dependent stress erythropoiesis pathway. Constant pro-inflammatory signals prevent the transition to differentiation by maintaining a pro-proliferation micro-environment, which limits erythrocyte production and erodes the ability of this pathway to increase erythroid production to maintain homeostasis.

      Model systems to study stress erythropoiesis

      The goal of studying stress erythropoiesis in model organisms is to exploit the experimental advantages of these systems to understand the process in humans in sufficient detail that we can then develop new therapies for human anemia. The vulnerability of model systems will always be in how well the mechanisms that regulate stress erythropoiesis in a model organism are conserved in human stress erythropoiesis. It is difficult to answer this question because human stress erythropoiesis is not easily studied. As mentioned above, most of what we know about BMP4-dependent stress erythropoiesis has come from the study of mice. When comparative studies were done, the data from these studies revealed that BMP4-dependent stress erythropoiesis was highly conserved between mouse and human. Later in this section, we discuss the data on murine and rat systems and how they compare with data on human stress erythropoiesis.
      Because of experimental imitations, the study of human erythropoiesis has been limited to studying anemic patients, which are observational data, while the culture of primary erythroid progenitors isolated from patients or generated from CD34+ cells isolated from cord blood, bone marrow, or peripheral blood has yielded more mechanistic data. Although cultures of purified CD34+ cells have been useful in studying human steady-state erythropoiesis, stress erythropoiesis is more complex and includes interactions between progenitor cells and a complex micro-environment and niche. From the study of murine stress erythropoiesis, we developed a model for BMP4-dependent stress erythropoiesis (Figure 2) [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ,
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ]. This model is recapitulated in an in vitro culture system that uses unfractionated bone marrow cells cultured in medium containing BMP4, Shh, GDF15, SCF, and Epo [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. This culture generates a monocyte-derived macrophage micro-environment. By manipulating the factors in the medium, we can separate the expansion phase from the differentiation phase. During the expansion phase, the medium lacks Epo, and the immature SEP populations are generated. These in vitro-generated SEPs are transplantable, providing erythroid short-term radioprotection, exhibit self-renewal in vivo, but are erythroid restricted. Addition of Epo to the medium results in changes in the micro-environment that promote a transition of SEPs from self-renewing stem cell-like progenitors to committed erythroid progenitors. This culture system has been invaluable for the study of stress erythropoiesis and the results obtained in vitro correlate with in vivo models. A major strength of this culture system is that it can be applied to human bone marrow [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. Analysis of human bone marrow cultures revealed that they required the same growth factors and generated a similar series of SEP populations with the exception that Sca1 was not a marker for the human SEPs. Manipulating these cultures has provided an experimental platform to dissect the BMP4-dependent stress erythropoiesis pathway in humans. Human cultures form the same monocyte-derived macrophage stromal layer that supports the proliferation and differentiation of SEPs. The response of macrophages in the niche is conserved between humans and mice. The comparison of in vitro–generated human SEPs with previously identified human stress erythroid progenitors isolated from the peripheral blood of anemic patients revealed that the in vitro-derived SEPs exhibited the characteristics of patient-derived progenitors. Human stress erythropoiesis is associated with the expression of fetal hemoglobin (HbF). Gamma (γ) globin, which replaces β-globin in HbF is silenced in adults through the action of the BCL11A repressor complex [
      • Liu N
      • Hargreaves VV
      • Zhu Q
      • et al.
      Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch.
      ,
      • Menzel S
      • Garner C
      • Gut I
      • et al.
      A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15.
      ,
      • Sankaran VG
      • Menne TF
      • Xu J
      • et al.
      Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
      ]. Healthy adults usually exhibit 1%–5% HbF+ cells in circulation [
      • Diepstraten ST
      • Hart AH
      Modelling human haemoglobin switching.
      ,
      • Vinjamur DS
      • Bauer DE
      • Orkin SH
      Recent progress in understanding and manipulating haemoglobin switching for the haemoglobinopathies.
      ]. However, in response to anemia and bone marrow transplant, the percentage of HbF+ cells increases, which suggests that stress erythropoiesis may reactivate the fetal erythroid program [
      • Alter BP
      • Rappeport JM
      • Huisman TH
      • Schroeder WA
      • Nathan DG
      Fetal erythropoiesis following bone marrow transplantation.
      ,
      • Alter BP
      • Rosenberg PS
      • Day T
      • et al.
      Genetic regulation of fetal haemoglobin in inherited bone marrow failure syndromes.
      ]. The increase in HbF+ cells is also observed when anemia is induced in nonhuman primates [
      • DeSimone J
      • Biel SI
      • Heller P
      Stimulation of fetal hemoglobin synthesis in baboons by hemolysis and hypoxia.
      ]. The source of these HbF+ erythrocytes is unclear, but analysis of erythroid progenitors in thalassemia and sickle cell disease patients identified CD34+KIT+ progenitors that also expressed CD235a. These cells, when cultured in vitro, gave rise to HbF+ erythrocytes [
      • Luck L
      • Zeng L
      • Hiti AL
      • Weinberg KI
      • Malik P
      Human CD34(+) and CD34(+)CD38(–) hematopoietic progenitors in sickle cell disease differ phenotypically and functionally from normal and suggest distinct subpopulations that generate F cells.
      ,
      • Mathias LA
      • Fisher TC
      • Zeng L
      • et al.
      Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage.
      ]. CD34, KIT, and CD235a (mouse Ter119) are markers observed on murine and human BMP4-dependent SEPs. In vitro-derived human SEPs express low levels of BCL11a, which leads to high levels of γ-globin and HbF. Similarly, murine SEPs do not express Bcl11a and exhibit higher levels of εy- and βh1-globin [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. Comparison of the properties of human and murine SEPs generated in vitro with those of SEPs isolated from anemic patients underscores the conservation of BMP4-dependent stress erythropoiesis in mouse and human.
      In vivo analysis of BMP4-dependent stress erythropoiesis in mice has relied primarily on two experimental systems, erythroid short-term radioprotection (STR) after bone marrow transplant and PHZ-induced acute hemolytic anemia [
      • Bennett LF
      • Liao C
      • Paulson RF
      Stress erythropoiesis model systems.
      ]. After transplant, erythroid STR is maintained by stress erythropoiesis, which generates erythrocytes in the first 2 weeks after transplant, maintaining erythroid homeostasis until donor stem cells can engraft and begin steady-state erythropoiesis [
      • Harandi OF
      • Hedge S
      • Wu DC
      • McKeone D
      • Paulson RF
      Murine erythroid short-term radioprotection requires a BMP4-dependent, self-renewing population of stress erythroid progenitors.
      ,
      • Hao S
      • Xiang J
      • Wu DC
      • et al.
      Gdf15 regulates murine stress erythroid progenitor proliferation and the development of the stress erythropoiesis niche.
      ]. Erythroid STR is a powerful system to analyze the development of SEPs and the stress erythropoiesis niche in the spleen. The role of the BMP4-dependent stress erythropoiesis pathway in generating new erythrocytes after stem cell transplant in humans has not been directly addressed. However, analysis of erythropoiesis after transplant revealed that patients exhibit a transient increase in HbF cells [
      • Meletis J
      • Papavasiliou S
      • Yataganas X
      • et al.
      'Fetal' erythropoiesis following bone marrow transplantation as estimated by the number of F cells in the peripheral blood.
      ,
      • Weinberg RS
      • Schofield JM
      • Lenes AL
      • Brochstein J
      • Alter BP
      Adult 'fetal-like' erythropoiesis characterizes recovery from bone marrow transplantation.
      ]. This increase is also observed in nonhuman primates [
      • Humbert O
      • Peterson CW
      • Norgaard ZK
      • Radtke S
      • Kiem HP
      A nonhuman primate transplantation model to evaluate hematopoietic stem cell gene editing strategies for beta-hemoglobinopathies.
      ]. These observations are consistent with our data indicating that human SEPs generated in vitro express high levels of γ-globin and HbF and suggest that human BMP4-dependent stress erythropoiesis contributes to erythroid homeostasis after transplant.
      Historically, the use of PHZ to induce acute hemolytic anemia has been used to test murine mutations for defects in stress erythropoiesis. This protocol allows for the study of proliferation and differentiation of progenitor cells in the spleen and the concurrent development of the niche. Changes in bone marrow erythropoiesis can easily be assessed at the same time. Using this system, we have shown that unlike steady-state erythropoiesis, which constantly produces new erythrocytes, BMP4-dependent stress erythropoiesis is cyclical. The time from induction of anemia until the pathway can fully respond to a secondary challenge is 28 days [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ,
      • Perry JM
      • Harandi OF
      • Porayette P
      • et al.
      Maintenance of the BMP4-dependent stress erythropoiesis pathway in the murine spleen requires hedgehog signaling.
      ]. The mobilization of ST-HSCs from the bone marrow and their homing to the spleen constitute a regulated process. Normal adult mice do not have circulating ST-HSCs in the peripheral blood that can be cultured in vitro to form SEPs. However, following PHZ-induced anemia, we observe an increase in peripheral blood mononuclear cells (PBMCs) that can generate SEPs when cultured (Supplementary Figure E1, online only, available at www.exphem.org). We see a peak at 60 hours after PHZ, which is a time when the mouse is nearing recovery from anemia. Similarly, normal human donors do not have PBMCs that give rise to SEPs when cultured. In contrast, when we cultured PBMCs from sickle cell disease patients, 7 of 10 patients generated stress BFU-E. In each case in which we observed stress BFU-E, culturing PBMCs led to the generation of CD34+CD133+Kit+ SEPs (Supplementary Figure E2, online only, available at www.exphem.org). These data further underscore the conservation of BMP4-dependent stress erythropoiesis between humans and mice and indicate that patients with sickle cell disease mobilize this conserved stress erythropoiesis pathway.
      Given its role in the inflammatory response, two other in vivo models have been used to study BMP4-dependent stress erythropoiesis in the context of inflammatory anemia. Injection of heat-kill Brucella abortus (HKBA) induces an inflammatory anemia in approximately 7 days, and the mice recover over the next 21 days [
      • Gardenghi S
      • Renaud TM
      • Meloni A
      • et al.
      Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus.
      ,
      • Kim A
      • Fung E
      • Parikh SG
      • et al.
      A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin.
      ]. HKBA injection mimics human anemia of inflammation. A second kinetically similar model uses lipopolysaccharide and the β-glucan zymosan A to induce a generalized chronic inflammatory disease [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ,
      • Millot S
      • Andrieu V
      • Letteron P
      • et al.
      Erythropoietin stimulates spleen BMP4-dependent stress erythropoiesis and partially corrects anemia in a mouse model of generalized inflammation.
      ]. Anemia develops in approximately 7 days and resolves over the next 21 days. This model rapidly induces stress erythropoiesis before the mice exhibit overt anemia. Although they initially recover, the mice progress to relapsing chronic anemia [
      • Bennett LF
      • Liao C
      • Quickel MD
      • et al.
      Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C.
      ]. There are other infection-based models in which stress erythropoiesis and anemia have been studied. The cecal ligation and puncture (CLP) model results in a polymicrobial infection leading to anemia and is a model for sepsis [
      • Mishra SK
      • Choudhury S
      Experimental protocol for cecal ligation and puncture model of polymicrobial sepsis and assessment of vascular functions in mice.
      ]. Similarly, infection with Salmonella leads to splenic stress erythropoiesis [
      • Jackson A
      • Nanton MR
      • O'Donnell H
      • Akue AD
      • McSorley SJ
      Innate immune activation during Salmonella infection initiates extramedullary erythropoiesis and splenomegaly.
      ]. These models are more complex models of inflammatory anemia induced by infection in which the role of the adaptive immune system must be considered.
      Analysis of the BMP4-dependent stress erythropoiesis pathway in mice has laid the foundation for the characterization of a conserved pathway in humans, which is supported by the data from human in vitro cultures and the analysis of SEPs in the peripheral blood of patients. Despite these findings, the role of BMP4-dependent stress erythropoiesis in responding to anemic stress in humans is questioned. Most of the uncertainty comes from the extramedullary nature of murine stress erythropoiesis. In C57BL/6 mice, the strain in which the BMP4-dependent pathway has been most characterized, overt splenomegaly is observed and there is a significant expansion of SEPs in the spleen during the recovery period [
      • Lenox LE
      • Perry JM
      • Paulson RF
      BMP4 and Madh5 regulate the erythroid response to acute anemia.
      ]. However, depending on the treatment used to induce anemia and the strain of mice used in the experiment, the splenomegaly and expansion of SEP populations in the spleen are variable, and some strains exhibit little splenomegaly [
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ]. In humans, the location of stress erythropoiesis is confounded by the lack of experimental data. Extramedullary hematopoiesis and erythropoiesis are observed in humans and are associated with pathological conditions such as malignancy and anemia. Whether these cases reflect BMP4-dependent stress erythropoiesis is not known. In some cases, responses in the bone marrow can further complicate the interpretation. For example, in hemolytic anemia, bone marrow hypercellularity with an expansion of erythroid progenitors is observed, but splenomegaly is also seen [
      • Tabbara IA
      Hemolytic anemias: diagnosis and management.
      ]. It is not known where compensatory erythropoiesis occurs.
      In addition to mice, rats have historically been used to study stress erythropoiesis. In an Experimental Hematology article, Zhang et al. [
      • Zhang J
      • Liu Y
      • Han X
      • et al.
      Rats provide a superior model of human stress erythropoiesis.
      ] suggested that rats are a superior model for human stress erythropoiesis. The authors based that idea on the observation that rats, like humans, have abundant bone marrow space, which could allow increased bone marrow erythropoiesis in response to anemic stress. In contrast, the marrow space in mice is more restricted. The authors found that ACI inbred rats respond to PHZ-induced anemia by increasing the percentage of Kit+ and late-stage erythroblasts in the bone marrow to a greater extent than in the spleen. The authors did not observe an increase in BMP4 expression in the spleen at the time points assayed. The use of inbred strains illustrates both the strengths and the weakness of the rodent system. Inbred strains reduce experimental variability, but different inbred strains exhibit distinct responses to anemic stress [
      • Perry JM
      • Harandi OF
      • Paulson RF
      BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors.
      ]. Analysis of the literature reveals that the rat response to anemic stress varies between inbred strains. As in C57BL/6 mice, the responses of Wistar and Long Evans rats to PHZ-induced anemia and Wistar rats to a model of inflammatory anemia induce compensatory erythropoiesis in spleen rather than bone marrow [
      • Mazur A
      Metabolism of the stimulated rat spleen: I. Ferrochelatase activity as an index of tissue erythropoiesis.
      ,
      • Petakov M
      • Biljanovic-Paunovic L
      • Jovcic G
      • et al.
      The influence of acute sterile inflammation on erythropoiesis in rats.
      ,
      • Carmichael RD
      • Orlic D
      • Lutton JD
      • Gordon AS
      Effects of anemia and hypertransfusion on neonatal marrow and splenic erythrocytic colony-forming units in vitro.
      ,
      • Orlic D
      • Wu JM
      • Carmichael RD
      • et al.
      Increased erythropoiesis and 2′5′-A polymerase activity in the marrow and spleen of phenylhydrazine-injected rats.
      ]. Furthermore, Sprague-Dawley rats not only increase splenic erythropoiesis in response to PHZ and pregnancy; they also induce BMP4-dependent stress erythropoiesis in a model of lung injury and chronic stress [
      • Alamo IG
      • Kannan KB
      • Loftus TJ
      • et al.
      Severe trauma and chronic stress activates extramedullary erythropoiesis.
      ,
      • Mattsson R
      • Mattsson A
      • Lindahl-Kiessling K
      Anemia causes erythropoiesis and increased antibody synthesis in the spleen of the pregnant mouse.
      ]. We have observed similar strain-specific differences in inbred mice [
      • Lenox LE
      • Shi L
      • Hegde S
      • Paulson RF
      Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling.
      ]. Despite its use in early studies of erythropoiesis and stress erythropoiesis, the rat has lagged behind the mouse as an experimental system. The reagents for the analysis of rat hematopoiesis are not well developed. There are fewer mutant strains of rats, although new mutants could be efficiently developed using Crispr/Cas9 genome editing techniques [
      • Smalley E
      CRISPR mouse model boom, rat model renaissance.
      ]. Antibodies to cell surface markers that are well correlated with human cell surface markers have not been developed. Despite these weaknesses, the experimental techniques described above for the murine system have been used or could be easily adapted to the rat system. A more informative use of the rat system to study stress erythropoiesis would only add to our knowledge of stress erythropoiesis.
      In addition to rodents, other vertebrate systems have contributed to the study of stress erythropoiesis. Non-human primates have been used to study the regulation of fetal hemoglobin in response to anemia. These studies indicated that responses in baboons mimic human responses to anemia [
      • Lavelle D
      • DeSimone J
      • Heller P
      Fetal hemoglobin reactivation in baboon and man: a short perspective.
      ]. Although these studies have played an important preclinical role in the development of compounds to reactivate the expression of γ-globin in adults, the cost of these models and the experimental limitations make their routine use unlikely.
      Zebrafish as a vertebrate model organism provides a powerful genetic system in which to study erythropoiesis and stress erythropoiesis. The use of standard genetic screens and chemical genetic screens have identified a number of mutations that affect hematopoiesis and erythropoiesis [
      • Avagyan S
      • Zon LI
      Fish to learn: insights into blood development and blood disorders from zebrafish hematopoiesis.
      ,
      • Konantz M
      • Schurch C
      • Hanns P
      • et al.
      Modeling hematopoietic disorders in zebrafish.
      ]. Analysis of these mutants has identified new developmental processes that are highly conserved in vertebrates. Although only a few studies have looked at erythroid regeneration, treatment with PHZ leads to increased erythropoietic activity in the caudal hematopoietic tissue [
      • Lenard A
      • Alghisi E
      • Daff H
      • et al.
      Using zebrafish to model erythroid lineage toxicity and regeneration.
      ]. Whereas other studies have suggested a role for BMP signaling in erythroid regeneration [
      • McReynolds LJ
      • Tucker J
      • Mullins MC
      • Evans T
      Regulation of hematopoiesis by the BMP signaling pathway in adult zebrafish.
      ]. The use of zebrafish to study stress erythropoiesis is promising albeit not yet fully developed.

      Conclusions

      The study of stress erythropoiesis provides important insight into the mechanisms by which the hematopoietic system compensates for the loss of erythrocytes and erythroid production. New studies indicate that stress erythropoiesis is integrated into the inflammatory response. A better understanding of these mechanisms will enable us to exploit these pathways and develop new therapeutics to treat anemia. Experimentally, the murine system is the most advanced and exhibits high conservation with human stress erythropoiesis. However, no single experimental system is perfectly informative, and only the integration of data from all these experimental systems will promote our understanding of human stress erythropoiesis.

      Acknowledgments

      Work in the Paulson lab was funded by National Institutes of Health (NIH) Grants DK080040, DK119865, and HL146528 and by NIFA-USDA Hatch Funds Project No. PEN04605, Accession No. 1010021. Work in the Little lab was funded by CWRU and Cleveland University Hospitals Medical Center. We thank Margherita Cantorna for comments on the article.

      Supplemental Data and Methods

      Figure E1
      Figure E1Generation of stress BFU-E from cultured peripheral blood mononuclear cells isolated from phenylhydrazine (PHZ) treated mice. Mice were treated with a single dose of PHZ (100 mg/Kg mouse). At the indicated timepoints mice were euthanized and analyzed. Hematocrit measurements are indicated in red (N=2-5 per time point). Peripheral blood mononuclear cells were isolated cultured in Stress erythropoiesis differentiation media (SEDM) for 5 days. Stress BFU-E in the cultures were measured by colony assay in methylcellulose media containing Epo cultured for 7 days. The total number of stress BFU-E in the expanded progenitor cells was calculated and graphed in blue. N=3-5 per time point.
      Figure E2
      Figure E2Generation of stress BFU-E from peripheral blood mononuclear cells isolated from Sickle Cell Anemia patients. Peripheral blood mononuclear cells were isolated from patients (UPN9 and UPN 15) with sickle cell disease or normal controls (HC2) and cultured in stress erythropoiesis differentiation media (SEDM) for 5 days. (Top panels and bottom left) Representative flow cytometry analysis of PBMCs from patients with sickle cell disease or normal controls (HC2) before and after culture in SEDM media for 5 days. (Bottom right) Total number of stress BFU-E produced in each culture was calculated and graphed. UPN denotes a patient with sickle cell disease and HC1 and HC2 are normal controls. ND no BFU-E colonies detected.

      Supplemental methods

      Induction of phenylhydrazine induced acute hemolytic anemia [
      • Bennett LF
      • Liao C
      • Paulson RF
      Stress erythropoiesis model systems.
      ] and isolation PBMCs. 6 to 8-week-old C57BL/6 mice (Taconic) were injected with a single dose of phenylhydrazine (100mg/kg mouse in sterile PBS). Peripheral blood was isolated by cardiac puncture at the indicated time points and diluted into sterile PBS. The diluted peripheral blood cells were layered onto Histopaque 1077 (Sigma-Aldritch) and centrifuged. Peripheral blood mononuclear cells (PBMCs) were isolated from the interface and washed twice in PBS + 2% fetal bovine serum.
      Stress erythropoiesis cultures. Stress erythropoiesis cultures were performed as previously done [
      • Bennett LF
      • Liao C
      • Paulson RF
      Stress erythropoiesis model systems.
      ,
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ]. In short, PBMCs were cultured in stress erythropoiesis differentiation media (SEDM) containing IMDM media supplemented with 20% fetal bovine serum + Shh (25ng/mL) + BMP4 (15ng/mL) + GDF15 (30ng/mL) + SCF (15ng/mL) + Epo (3ng/mL) and cultured at 2%O2 for 5 days. Nonadherent progenitor cells were assayed for stress BFU-E formation by plating 1 x 105 expanded cells/ mL of methylcellulose media (M3334 StemCell Technologies, Vancouver, BC, Canada), which contains 3 U/mL Epo. BFU-E colonies were stained with acid benzidine stain and counted after 5-7 days of culture. Total number of BFU-E in the expanded cells was calculated.
      Isolation of PBMCs from Sickle cell anemia patients. De-identified peripheral blood samples from patients suffering from sickle cell disease was obtained from Case Western Reserve University Hospital. PBMCs were then isolated from peripheral blood using Histopaque 1077 as described above for murine PBMCs. Control PBMCs were purchased from ReachBio. PBMCs obtained were plated at 1 x 106 cells/ml stress erythropoiesis differentiation media containing human growth factors. The cells were cultured for 5 days at 2% O2. After 5 days of culture, the expanded nonadherent progenitor cells were plated at 1x105 cells per ml in methylcellulose media (H4330 StemCell Technologies, Vancouver, BC, Canada) for assay of stress BFU-E colony formation. After 5 days of culture, colonies were stained with acid benzidine and counted. Total number of BFU-E in the expanded cells was calculated.
      Flow cytometric analysis of human PBMCs. Prior to culture in SEDM media and after 5 days of culture in SEDM, human PBMC were analyzed for the expression of KIT, CD34 and CD133 as previously described [
      • Xiang J
      • Wu DC
      • Chen Y
      • Paulson RF
      In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.
      ].

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