Macrophages In Erythroblastic Islands Research Articles

Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Deciphering the regulatory landscape of murine splenic response to anemic stress at single-cell resolution

AbstractStress erythropoiesis can be influenced by multiple mediators through both intrinsic and extrinsic mechanisms in early erythroid precursors. Single-cell RNA sequencing was conducted on spleen tissue isolated from mice subjected to phenylhydrazine and serial bleeding to explore novel molecular mechanisms of stress erythropoiesis. Our results showed prominent emergence of early erythroblast populations under both modes of anemic stress. Analysis of gene expression revealed distinct phases during the development of emerging erythroid cells. Interestingly, we observed the presence of a "hiatus" subpopulation characterized by relatively low level of transcriptional activities that transitions between early stages of emerging erythroid cells, with moderate protein synthesis activities. Moreover, single-cell analysis conducted on macrophage populations revealed distinct transcriptional programs in Vcam1+ macrophages under stress. Notably, a novel marker, CD81, was identified for labeling central macrophages in erythroblastic islands (EBIs), which is functionally required for EBIs to combat anemic stress. These findings offer fresh insights into the intrinsic and extrinsic pathways of early erythroblasts' response to stress, potentially informing the development of innovative therapeutic approaches for addressing anemic-related conditions.

Spatial Mapping Reveals Distinct Erythroid Niches in Mice and Humans during Development and Stress

Like hematopoiesis, erythropoiesis requires erythroid niche nurturing and support. While extensive studies have revealed various components and functions of the HSC niches, the erythroid niche is less characterized. The most studied erythroid niche is the bone marrow erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. However, most of the reported studies of EBI involve in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. Here, we utilized multiple spatial mapping technologies, including spatial transcriptomics and whole-mount imaging studies, to characterize erythropoiesis in mouse and human hematopoietic tissues during development and under stress. We first utilized the Visium platform from 10x Genomics to study the spatial transcriptomic profiles of the E14.5 mouse fetal liver, which is the major organ for murine fetal definitive erythropoiesis. The Visium platform contains barcoded spots with a diameter of 55 µm, which is suboptimal for single-cell analyses but ideal for the investigation of cell-cell interactions within their microenvironment. We found that each hepatic lobe exhibited different cluster distributions, indicating distinct cell compositions among different anatomic loci. Further deconvolution of the clusters and co-analysis with corresponding single-cell RNA sequencing data revealed that there was a higher positive correlation between erythroid cells and C1q+ macrophages compared to other macrophages, suggesting that C1q+ macrophages are more likely to be EBI macrophages in mouse. C1q+ macrophages exhibited high expression of known genes involved in EBI macrophage function and erythropoiesis. Cyclic immunofluorescence (CyCIF) and whole-mount immunofluorescence analyses further demonstrated the co-localization of C1q+ macrophages with erythroid cells in the E14.5 fetal liver. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed across different stages of fetal liver development, newborn bone marrow, and adult spleen under physiologic and stress conditions. These findings demonstrate that C1q+ macrophages as EBI macrophages are conserved across different hematopoietic organs in mice. We next applied the same technologies to human hematopoietic tissues during development and stress. In contrast to the mouse, we did not observe a strong positive correlation between erythroid cells and C1q+ macrophages or other macrophages in the human fetal liver. Instead, there is a strong association between erythroid progenitor and maturing erythroid cells. This strong association is also found in different developmental stages of the human fetal liver as well as the bone marrow, which becomes even stronger in regenerating human bone marrow after chemotherapy. We also recapitulated these findings in a human bone marrow organoid model. These results demonstrate the erythroid cell self-sustaining capacities in human erythropoiesis. Taken together, our findings reveal the distinct erythroid niches in mice and humans during development and stress, which provides a comprehensive understanding of erythropoiesis during evolution.

Erythrophagocytosis Mediated By Circulating Monocytes Is Induced By Distinct Mechanisms Depending on the Disease

Stress erythropoiesis (SE) occurs when the steady state erythropoiesis is no longer sufficient to sustain the number of healthy red blood cells (RBC) needed in the circulation. There is an extension of this activity to extramedullary sites, contributing to the increase in cell production in the erythroblastic island (EBI) niche, and altered or damaged RBCs are then cleared by macrophages. Some diseases have a state of chronic SE. Sickle cell anemia (SCA) affects multiple aspects of RBCs, leading to recurrent vaso-occlusive crisis, hemolytic anemia, and inflammation. Polycythemia Vera (PV), an acquired neoplastic disease with mutations in JAK2 kinase, is characterized by the overproduction of RBCs, increasing blood viscosity, and resulting in a higher risk of thrombotic events. We previously showed that circulating monocytes (MC) of both diseases have a phenotype similar to the EBIs macrophages, with higher expression of CD169, CD163, CD206 and VCAM-1, and also participate in the phagocytosis of RBCs, function typically carried out by macrophages. Therefore, the aim of this study was to determine whether the increased phagocytic activity found in these patients was due to the alterations on the MCs phenotype or if it was an influence of the RBCs, as well as investigate the consequences to the iron metabolism after phagocytosis. MCs from peripheral blood were isolated and incubated for 2 hours with previously stained RBCs, to allow the phagocytosis to happen. After this period, the remaining RBC were lysed and the antibodies anti-intracellular heme-oxigenase-1 (HO-1) and anti-membrane ferroportin (FPN) were used for flow cytometry analysis. The statistical analysis was done by paired t-student test (P<0.05). When exposed to SCA RBCs, MCs from SCA patients and healthy controls (HC) show higher phagocytic capability, than when exposed to HC RBCs (MC HC 22.9±8 vs 9.4±1.1, **P=0,0099; MC SCA 33.6±11.4 vs 16.4±11, **P=0,0054; respectively). The same happens to PV and HC MCs: when exposed to PV RBCs: they show higher phagocytosis compared to the presence of HC RBCs (MC HC 9.3±4.4 vs 2.8±4.7, **P=0.0065; MC PV 21.3±6.1 vs 11±6.8, *P=0.0146; respectively). Investigating the role of MCs in this activity, we compared SCA, PV and HC MCs in the presence of patients RBCs. SCA MCs internalized RBCs similarly to HC MCs (P=0.0699); on the other hand, PV MCs presented higher phagocytic capability when compared to HC MCs (**P=0.0022). In all groups, the expressions of both HO-1 and FPN were higher in those MCs that participated in the phagocytosis than in those that didn't contribute to this function (HO-1: MC SCA 91.9±4 vs 65.9±16.8, **P=0.0098, MC PV 92.1±5.5 vs 61.7±15.5, ***P=0.0007; FPN: MC SCA 81.8±13.8 vs 66.5±15.7, ***P=0.0003, MC PV 70.9±17 vs 53.2±16.5, **P=0.0013 respectively). This indicates an increase in the ability to degrade heme and export iron post-phagocytosis. The results suggest that RBCs from patients with SCA are the ones responsible for the increase in the phagocytic activity by MCs, indicating that RBCs clearance is not an innate function of SCA MCs, even though they are active participants in the circulation. For PV, both RBCs and MCs seem to contribute to the increase of this process in the circulation of patients. The higher expression of molecules related to the iron metabolism suggests a participation of circulating MCs to the iron disponibility, helping with the maintenance of stock for the erythrocytic production. Even though SCA and PV have different pathophysiology, they both present SE and the circulating MCs from both groups show an important participation in the clearance of RBCs, either by an intrinsic function of MCs or a direct influence of changes in the RBCs, suggesting the important contribution of MCs to avoid oxidative stress and help with iron disponibility for erythropoiesis during the course of SE.

Dysfunction of CD169+ macrophages and blockage of erythrocyte maturation as a mechanism of anemia in Plasmodium yoelii infection

Plasmodium parasites cause malaria with disease outcomes ranging from mild illness to deadly complications such as severe malarial anemia (SMA), pulmonary edema, acute renal failure, and cerebral malaria. In young children, SMA often requires blood transfusion and is a major cause of hospitalization. Malaria parasite infection leads to the destruction of infected and noninfected erythrocytes as well as dyserythropoiesis; however, the mechanism of dyserythropoiesis accompanied by splenomegaly is not completely understood. Using Plasmodium yoelii yoelii 17XNL as a model, we show that both a defect in erythroblastic island (EBI) macrophages in supporting red blood cell (RBC) maturation and the destruction of reticulocytes/RBCs by the parasites contribute to SMA and splenomegaly. After malaria parasite infection, the destruction of both infected and noninfected RBCs stimulates extramedullary erythropoiesis in mice. The continuous decline of RBCs stimulates active erythropoiesis and drives the expansion of EBIs in the spleen, contributing to splenomegaly. Phagocytosis of malaria parasites by macrophages in the bone marrow and spleen may alter their functional properties and abilities to support erythropoiesis, including reduced expression of the adherence molecule CD169 and inability to support erythroblast differentiation, particularly RBC maturation in vitro and in vivo. Therefore, macrophage dysfunction is a key mechanism contributing to SMA. Mitigating and/or alleviating the inhibition of RBC maturation may provide a treatment strategy for SMA.

Regulatory Effect and Mechanism of Erythroblastic Island Macrophages on Anemia in Patients with Newly Diagnosed Multiple Myeloma.

To examine the clinical characteristics and anemia-related factors in patients with newly diagnosed multiple myeloma (NDMM), as well as the effect and mechanism of erythroblastic islands (EBIs) and EBI macrophages in NDMM patients with anemia. We collected and analyzed clinical data to find anemia-related factors. Using flow cytometry, the numbers and ratios of erythroblasts and EBI macrophages were determined. RNA sequencing (RNA-seq) was used to determine the differences of EBI macrophages in NDMM patients with or without anemia. Based on the clinical characteristics of NDMM patients with anemia, MCV, abnormal levels of albumin, osteolytic lesions, and Durie-Salmon (DS) stage are risk factors for anemia. Patients with anemia have fewer erythroblasts, erythroblastic islands (EBIs), and EBI macrophages in their bone marrow than patients without anemia. RNA-seq analysis of EBI macrophages from the bone marrow of patients with and without anemia revealed that macrophages from patients with anemia are impaired and tend to promote the production of interleukin-6, which has been demonstrated to be an essential survival factor of myeloma cells and protects them from apoptosis. In NDMM patients with anemia, EBI macrophages are impaired, which causes anemia in those patients. Our finding highlights the significance of EBI macrophages in anemia in NDMM patients and provides a new strategy for recovery from anemia in these patients.

Macrophage deficiency in CSF1R-knockout rat embryos does not compromise placental or embryo development.

Macrophages are an abundant cell population in the placenta and developing embryo and appear to be involved in processes of vascularization, morphogenesis, organogenesis, and hematopoiesis. The proliferation, differentiation, and survival are dependent on signals from the macrophage colony-stimulating factor receptor, CSF1R. Aside from the role in macrophages, Csf1r mRNA is highly expressed in placental trophoblasts. To explore the function of macrophages and Csf1r in placental and embryonic development, we analyzed the impact of homozygous Csf1r null mutation (Csf1rko) in the rat. In late gestation, IBA1+ macrophages were abundant in control embryos in all tissues, including the placenta, and greatly reduced in the Csf1rko. CSF1R was also detected in stellate macrophage-like cells and in neurons using anti-CSF1R antibody but was undetectable in trophoblasts. However, the neuronal signal was not abolished in the Csf1rko. CD163 was most abundant in cells forming the center of erythroblastic islands in the liver and was also CSF1R dependent. Despite the substantial reduction in macrophage numbers, we detected no effect of the Csf1rko on development of the placenta or any organs, the relative abundance of vascular elements (CD31 staining), or cell proliferation (Ki67 staining). The loss of CD163+ erythroblastic island macrophages in the liver was not associated with anemia or any reduction in the proliferative activity in the liver, but there was a premature expansion of CD206+ cells, presumptive precursors of liver sinusoidal endothelial cells. We suggest that many functions of macrophages in development of the placenta and embryo can be provided by other cell types in their absence.

Heme oxygenase 1 in erythropoiesis: an important regulator beyond catalyzing heme catabolism.

Heme oxygenase 1 (HO-1), encoded by the HMOX-1 gene, is the main heme oxygenase that catalyzes the degradation of heme into iron, carbon monoxide, and biliverdin. HMOX-1 gene expression is stimulated by oxidative stress and regulated at transcriptional and post-transcriptional levels. After translation, subcellular location and protein stability of HO-1 are also altered by different extracellular and intracellular stimuli. HO-1 plays a key role in regulating iron homeostasis and cell protection and has become a new target for disease treatment. Erythropoiesis is a tightly controlled, iron-dependent process that begins with hematopoietic stem cells and maturates to red blood cells. HO-1 is expressed in hematopoietic stem/progenitor cells, hematopoietic niche cells, erythroblasts, and especially erythroblastic island and phagocytic macrophages. HO-1 functions importantly in the entire erythroid development process by influencing hematopoietic stem cell proliferation, erythroid lineage engagement, terminal erythroid differentiation, and even senescent RBC erythrophagocytosis. HO-1 is also related to stress erythropoiesis and certain red blood cell diseases. Elucidation of HO-1 regulation and function in erythropoiesis will be of great significance for the treatment of related diseases.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

A Putative Erythroid Synapse Linking Developing Erythroblasts and Macrophages in Erythroblastic Islands

An estimated 25% of the global population suffer from anemia, a major cause being deficient erythropoiesis. Mammalian erythropoiesis in the adult occurs primarily in the bone marrow (BM), with the spleen being an active site of extramedullary erythropoiesis during anemic stress. Here, we examine regulators of the anemic stress response using an unbiased transcriptomic approach to compare FACS-sorted splenic Ter-119+ erythroid precursors, from untreated mice or phenylhydrazine (PHZ)-treated mice exhibiting chemically-induced anemia. Transcriptomic analyses revealed unexpected expression of a broad range of immune markers in untreated mouse splenic erythroid cells, most which were conspicuously diminished in expression during PHZ-induced anemic stress. These surface antigens, previously unreported on erythroid cells, include markers of B lymphocytes (CD22, CD74), T lymphocytes (CD2, CD3ε) and leukocytes including CD11a/LFA-1, CD45, CD48, CD53, as well as the homing receptors L-selectin and PSGL-1. Immunophenotyping confirmed expression of these immune markers on a subpopulation of steady state BM and splenic erythroid precursors, with a significant reduction in immune marker-expressing erythroid cells seen in the spleen during anemic stress. Erythropoiesis is governed by a close relationship between erythroid precursors and central macrophages in multicellular clusters termed erythroblastic islands (EBIs). Confocal microscopy demonstrated localization of the immune markers CD2, CD3ε, CD11a and PSGL-1 on Ter-119+ mouse erythroid precursors with frequent clustering of markers seen at intercellular contact points where erythroid cells interact with the central EBI macrophage. PHZ-treated mice exhibited a significantly reduced frequency of EBIs in both the BM and spleen compared to control mice. PHZ-treated mice also demonstrated downregulated Interferon Regulatory Factor-8 (Irf8) transcripts on transcriptomic analysis of splenic erythroid precursors. Irf8-deficient mice have been reported to have a profoundly reduced erythroid compartment. There are also reports that Irf8 may be a regulator of macrophage morphology. We therefore studied Irf8-deficient mice to evaluate whether Irf8 has a role in EBI-regulation. BM analysis of Irf8-deficient mice showed a reduced frequency of EBI-like structures compared to wildtype (WT) mice. Scanning electron micrographs revealed that Irf8-/- macrophages are smaller with fewer erythroid precursors attached compared to WT macrophages. Irf8-/- EBIs harbored no identifiable reticulocytes in contrast to WT EBIs. EBI rosette reconstitution assays demonstrated that Irf8-/- erythroid precursors were unable to bind to WT or Irf8-/- macrophages, whereas Irf8-/- macrophages could interact with WT erythroid cells. Immunophenotyping of Irf8-/- erythroid precursors revealed that immune marker expression (CD2, CD38, CD43, CD45, PSGL-1) was significantly lower compared to WT counterparts. These data suggest that Irf8 may regulate both macrophage morphology and erythroid cell surface phenotype influencing erythropoiesis. The newly uncovered immune markers expressed by erythroid cells are also involved in formation of immunological synapses between lymphocytes and macrophages. This led us to question whether an analogous synapse-like structure may exist in EBIs. Serial block face scanning electron microscopy (SBF-SEM) was used to assess EBI morphology at the nanometer scale. SBF-SEM micrographs show erythroblasts extending pseudopods towards the macrophage for attachment. Three-dimensional volumetric reconstruction of SBF-SEM micrographs revealed en face spatial topologies present on the surface of EBI central macrophages similar to those seen in immunological synapses. Our findings suggest that immune markers, possibly mediated by Irf8, may function to form synaptic-like interactions between erythroid cells and macrophages that result in EBIs - an "erythroid synapse". Failure in forming these interactions may result in alterations in EBI formation with potential downstream impacts on erythropoiesis. Improved understanding of how EBIs form and function may aid in the development of therapeutic agents to combat anemia.

Acute Anemic Stress Is Modulated By Intercellular Mitochondria Transport from Erythroblastic Island Macrophages to Early Erythroblasts Via CD47-Sirpα Interactions

Stress erythropoiesis can be modulated by multiple cellular and molecular mediators from early stages of erythroblasts and precursors via cell intrinsic and extrinsic mechanisms. To investigate novel cell autonomous and non-autonomous mechanisms to overcome erythroid stress, we adopted phenylhydrazine (PHZ) induced acute anemic stress in mice and analyzed bulk RNA-seq on splenic early erythroblasts (Ter119+CD44high), and found significant enrichment of the early erythroid progenitor populations, as well as up-regulation of cell-cycle, DNA replication, proliferation and mitochondrial metabolism related genes (Gata1, Tal1, Sox6, E2Fs, Lin54, Foxo3 etc), suggesting that the most prominent erythroblastic cell-autonomous responses to stress include proliferation and energy metabolism. Next, we aim to explore the cell extrinsic pathways to mediate stress erythropoiesis from the perspective of the erythroid niche, i.e., erythroblastic islands (EBIs) macrophages. Intercellular mitochondria transfer is an emerging topic with implications in multiple biological processes, while it remains elusive whether mitochondria can be transported intercellularly within the EBIs, and if such a phenomenon is functionally relevant. In our study, we infused EBI macrophages into PHZ or serial bleeding induced anemic mice, and demonstrated that exogenous EBI macrophage infusion accelerated the erythroid regeneration in both modes of acute anemic stressors in a dynamic manner. More interestingly, we infused EBI macrophage isolated from mito-Dendra2 mice that express trackable mitochondria, and observed that mitochondria are transferred from EBI macrophages to early stages of erythroblasts. We conducted in vitro co-culture of EBI macrophages isolated from mito-Dendra2 mice and early erythroblasts isolated from PHZ treated tdTomato-Vav-Cre mice, and observed that mitochondria transfer mainly occurs via cell-cell contact mediated direct uptake during erythroid stress. Single cell RNA-sequencing (scRNA-seq) revealed that early erythroblasts receiving mitochondria exhibit up-regulation of a myriad of of proliferation signatures that are enriched in RNA binding (Rps2, Rpl28, Rpl10a, etc.), translation/protein synthesis (Eif5a, Eef1g, Ybx1, etc.), ATP metabolism (Atp5o, Atp5d, Atp6v0e, etc.) related molecular functions. The result pointed to the functional importance of intercellular mitochondria transfer to mediate the bioenergetic profiles of recipient erythroblasts by enhancing their protein/ATP synthesis and proliferation. Furthermore, scRNA-seq analysis revealed a subset of metabolically active erythroid populations marked by CD47 that receive higher abundance of mitochondria, as well as exhibiting enrichment in proliferation signatures, suggesting a potential role of the CD47 labelled erythroblasts in mitochondria transfer mediated erythroid regeneration. Notably, CD47 or Sirpα blockade in vivo leads to remarkable decline in both mitochondria transfer events as well as their mediated erythroid recovery. Thus, we postulate that there is a clear association between CD47 and mitochondria transfer mediated stress erythropoiesis. Collectively, our study explored the cell intrinsic and extrinsic pathways of early erythroblasts' responses to overcome stress, and shed light on the regulatory mechanisms of mitochondria transfer through CD47-SIRPα mediated cell-cell contacts and/or their downstream signaling in the context of stress erythropoiesis.

Mitochondria transfer mediates stress erythropoiesis by altering the bioenergetic profiles of early erythroblasts through CD47.

Intercellular mitochondria transfer is a biological phenomenon implicated in diverse biological processes. However, the physiological role of this phenomenon remains understudied between erythroblasts and their erythroblastic island (EBI) macrophage niche. To gain further insights into the mitochondria transfer functions, we infused EBI macrophages in vivo into mice subjected to different modes of anemic stresses. Interestingly, we observed the occurrence of mitochondria transfer events from the infused EBI macrophages to early stages of erythroblasts coupled with enhanced erythroid recovery. Single-cell RNA-sequencing analysis on erythroblasts receiving exogenous mitochondria revealed a subset of highly proliferative and metabolically active erythroid populations marked by high expression of CD47. Furthermore, CD47 or Sirpα blockade leads to a decline in both the occurrence of mitochondria transfer events and their mediated erythroid recovery. Hence, these data indicate a significant role of mitochondria transfer in the enhancement of erythroid recovery from stress through the alteration of the bioenergetic profiles via CD47-Sirpα interaction in the early stages of erythroblasts.

Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis

AbstractThe erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

GM-CSF impairs erythropoiesis by disrupting erythroblastic island formation via macrophages

Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.

Transcriptional Control of Gene Expression and the Heterogeneous Cellular Identity of Erythroblastic Island Macrophages.

During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.

Monocytes from Patients with Polycythemia Vera Express Molecules Related to Stress Erythropoiesis and Have Increased Erythrocyte Phagocytosis

Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease.We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC).Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17).In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p<0.0001; 312.3±15.1 vs 368.8±12.1, p=0.0174; 13,923±2256 vs 22,792±3211, p=0.0161; 1,234±96 vs 1,498±58.9 p=0.004, respectively). Fpn expression was not significantly different. A lower expression of CD172 in the C-MC suggests less inhibitory signaling for erythrophagocytosis in those cells. Although C-MC and I-MC have been previously linked to erythropoiesis, we saw a larger number of the investigated molecules being more expressed in NC-MC, supporting their possible involvement in regulating erythropoiesis. Our results suggest that MCs in PV could be more likely to attach erythroid cells and could therefore contribute to form EBIs if differentiated to macrophages. Higher molecule expression was associated to a higher percentage of MCs containing intracellular CD235a (0.35±0.07 vs 4.48±1.08, p<0.0001). This is evidence of circulating PV MCs performing erythrophagocytosis and supports a role for them in RBC clearance.Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients. DisclosuresFertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

AbstractThe erythropoietin receptor (EpoR) has traditionally been thought of as an erythroid-specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In this study, we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice, in which a very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes, and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further shown by the EpoR-tdTomato-Cre–mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles for EPO/EpoR in hematopoiesis that warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

PPARγ is essential for the development of bone marrow erythroblastic island macrophages and splenic red pulp macrophages.

Tissue-resident macrophages play a crucial role in maintaining homeostasis. Macrophage progenitors migrate to tissues perinatally, where environmental cues shape their identity and unique functions. Here, we show that the absence of PPARγ affects neonatal development and VCAM-1 expression of splenic iron-recycling red pulp macrophages (RPMs) and bone marrow erythroblastic island macrophages (EIMs). Transcriptome analysis of the few remaining Pparg-deficient RPM-like and EIM-like cells suggests that PPARγ is required for RPM and EIM identity, cell cycling, migration, and localization, but not function in mature RPMs. Notably, Spi-C, another transcription factor implicated in RPM development, was not essential for neonatal expansion of RPMs, even though the transcriptome of Spic-deficient RPMs was strongly affected and indicated a loss of identity. Similarities shared by Pparg- and Spic-deficient RPM-like cells allowed us to identify pathways that rely on both factors. PPARγ and Spi-C collaborate in inducing transcriptional changes, including VCAM-1 and integrin αD expression, which could be required for progenitor retention in the tissue, allowing access to niche-related signals that finalize differentiation.

Erythroblastic Island Macrophages Shape Normal Erythropoiesis and Drive Associated Disorders in Erythroid Hematopoietic Diseases

Erythroblastic islands (EBIs), discovered more than 60 years ago, are specialized microenvironments for erythropoiesis. This island consists of a central macrophage with surrounding developing erythroid cells. EBI macrophages have received intense interest in the verifications of the supporting erythropoiesis hypothesis. Most of these investigations have focused on the identification and functional analyses of EBI macrophages, yielding significant progresses in identifying and isolating EBI macrophages, as well as verifying the potential roles of EBI macrophages in erythropoiesis. EBI macrophages express erythropoietin receptor (Epor) both in mouse and human, and Epo acts on both erythroid cells and EBI macrophages simultaneously in the niche, thereby promoting erythropoiesis. Impaired Epor signaling in splenic niche macrophages significantly inhibit the differentiation of stress erythroid progenitors. Moreover, accumulating evidence suggests that EBI macrophage dysfunction may lead to certain erythroid hematological disorders. In this review, the heterogeneity, identification, and functions of EBI macrophages during erythropoiesis under both steady-state and stress conditions are outlined. By reviewing the historical data, we discuss the influence of EBI macrophages on erythroid hematopoietic disorders and propose a new hypothesis that erythroid hematopoietic disorders are driven by EBI macrophages.

Impact of Erythropoietin Production by Erythroblastic Island Macrophages on Homeostatic Murine Erythropoiesis.

Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1–10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated.