Island Macrophages 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.

Generation, characterization, and use of EKLF(Klf1)/CRE knock-in mice for cell-restricted analyses.

EKLF/Klf1 is a tissue-restricted transcription factor that plays a critical role in all aspects of erythropoiesis. Of particular note is its tissue-restricted pattern of expression, a property that could prove useful for expression control of a linked marker or enzymatic gene. With this in mind, we fused the CRE recombinase to the genomic EKLF coding region and established mouse lines. We find by FACS analyses that CRE expression driven by the EKLF transcription unit recapitulates erythroid-restricted expression with high penetrance in developing embryos. We then used this line to test its properties in the adult, where we found EKLF/CRE is an active and is a robust mimic of normal EKLF expression in the adult bone marrow. EKLF/CRE is also expressed in erythroblastic island macrophage in the fetal liver, and we demonstrate for the first time that, as seen during embryonic development, EKLF is also expressed in adult BM-derived erythroblastic island macrophage. Our data also support lineage studies showing EKLF expression at early stages of hematopoiesis. The EKLF/CRE mouse lines are novel reagents whose availability will be of great utility for future experiments by investigators in the red cell field.

Hematopoietic and stromal DMP1-Cre labeled cells form a unique niche in the bone marrow

Skeletogenesis and hematopoiesis are interdependent. Niches form between cells of both lineages where microenvironmental cues support specific lineage commitment. Because of the complex topography of bone marrow (BM), the identity and function of cells within specialized niches has not been fully elucidated. Dentin Matrix Protein 1 (DMP1)-Cre mice have been utilized in bone studies as mature osteoblasts and osteocytes express DMP1. DMP1 has been identified in CXCL12+ cells and an undefined CD45+ population. We crossed DMP1-Cre with Ai9 reporter mice and analyzed the tdTomato+ (tdT+) population in BM and secondary hematopoietic organs. CD45+tdT+ express myeloid markers including CD11b and are established early in ontogeny. CD45+tdT+ cells phagocytose, respond to LPS and are radioresistant. Depletion of macrophages caused a significant decrease in tdT+CD11b+ myeloid populations. A subset of CD45+tdT+ cells may be erythroid island macrophages (EIM) which are depleted after G-CSF treatment. tdT+CXCL12+ cells are in direct contact with F4/80 macrophages, express RANKL and form a niche with B220+ B cells. A population of resident cells within the thymus are tdT+ and express myeloid markers and RANKL. In conclusion, in addition to targeting osteoblast/osteocytes, DMP1-Cre labels unique cell populations of macrophage and stromal cells within BM and thymus niches and expresses key microenvironmental factors.

Leveraging Transcriptomic Data to Study Erythromyeloblastic Island (EMBI) Macrophages: Using Spic-GFP As a Marker for Embi Macrophages

We recently demonstrated that the majority of the erythroblastic islands in the mouse bone marrow are supporting not only terminal erythropoiesis but also granulopoiesis, being common niches for terminal hematopoiesis (Romano, Seu, et al. Blood 2022). Using single cell RNA-sequencing (scRNA-seq) and Cellular Indexing of Transcriptome and Epitopes by Sequencing (CITE-seq) we demonstrated several macrophage populations captured from EMBI-enriched bone marrow. Notably, the population of the classically defined EBI macrophages characterized by the genes Mertk, Dnase2a, and Hmox1, also showed sensitive and specific expression of the heme-induced Spic transcription factor (Figure 1). In agreement with the CITE-seq data, imaging flow cytometry (IFC) of EMBIs from SpiC igfp/igfp mouse bone marrow demonstrated that at least 80% of the central macrophages were GFP +, confirming their expression of Spic (Figure 2). After stimulation with erythropoietin (Epo), which leads to an increase of both the number and size of EMBIs, both Spic-GFP-positive and negative central macrophages doubled in number, as determined by IFC studies. Moreover, Spic-GFP + EMBI macrophages isolated from mouse bone marrow reconstituted efficiently into EMBIs after incubation with CD71 + and CD11b + cells isolated from another ( Spic-GFP neg) bone marrow. Characterizing EMBIs by IFC allows for identification of phenotypic characteristics of macrophages participating in the EMBI structure. The strong and intimate interactions between the erythroblasts and granulocytic precursors to the central macrophage facilitates the preservation of EMBIs and their analysis by IFC; however, these same strong interactions add a layer of complexity to methods that require single cells, such as scRNA-seq. Previous attempts to capture EMBI macrophages have had lower than expected yields. Utilizing the Spic-GFP reporter mouse model permitted the use of harsher dissociation by trypsinization and sorting based on GFP positivity, optimizing EMBI macrophage recovery. The resulting captures yielded thousands of EMBI macrophages based on cells expressing Adgre1, Vcam1, Hmox1, DNase2a, and Mertk that show a marked degree of transcriptional heterogeneity in other macrophage surface markers. Comparison of the captures from control (saline-) and Epo-stimulated EMBI macrophages revealed an emergent macrophage population, increasing up to 25-fold after Epo in comparison to control. This Epo-induced macrophage population has approximately 900 globally distinguishing genes including markers characteristic of monocytes such as Ace (encoding the angiotensin-converting enzyme), implicating monocyte recruitment to serve as stress erythropoiesis-supporting macrophages within the murine bone marrow. Additional clusters of macrophages with the characteristics of classically defined EMBI macrophages are also increasing with Epo- stimulation, indicating heterogeneity within the Spic-GFP + population. To verify these transcriptomic results on the protein level, we performed IFC with CD169, Mertk, and other EMBI macrophage markers providing further insights to the heterogeneity of their expression on the central macrophages within intact EMBIs. We anticipate that the Spic-GFP + reporter mouse will be a useful tool in EMBI studies, providing a specific and sensitive marker to trace and isolate the majority of EMBI macrophages.

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.

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.

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.

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.

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis.

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.

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.

48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p< 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.

A Shift in Myeloid Cell Phenotype via Down Regulation of Siglec-1 in Island Macrophages of Bone Marrow Is Associated With Decreased Late Erythroblasts Seen in Anemia of Critical Illness

Burn injury has been shown to significantly dampen erythropoiesis in both burn patients and in murine models. Our previous findings elucidated the erythropoietin independent defects in red cell development stages involving erythroid progenitor production and late stage erythroblast enucleation processes. We hypothesized that macrophages (MØ) in erythroblast islands (EBI) could be yet another roadblock impeding erythropoiesis following burn injury. Here we highlight that the methodology to study EBI can be achieved with single cell suspensions using a simple technique such as flow cytometry, as obtaining the central erythroblast island macrophages (EBIMØs) of interest is a delicate process. We elucidated the requisite of EBIMØ from the erythroblast as well as the MØ perspective. In addition to the primary erythropoiesis organ, the bone marrow (BM), spleens were also examined for extra-medullary erythropoiesis. Femurs and spleens were harvested from adult mice (B6D2F1) subjected to 15% total body surface area (TBSA) scald burn (B) or sham burn (S). Total bone marrow cells (TBM) and splenocytes were probed for total erythrons, early and late erythroblasts and EBIMØ by flow cytometry. There was only a marginal increase in the number of EBIMØ after burn, but, between the signatures of EBIMØ, Siglec-1 expression (MFI) was reduced by 40% in B with and a parallel 44% decrease in TBM erythrons in the BM. There were more (2.5-fold) EEBs and less LEBs (2.4-fold) per million TBM cells in B; with a corresponding decrease in Siglec-1 and Ly6G expressions in EBIMØ associated with EEB. Conversely, extra-medullary erythropoiesis was robust in spleens from B. Not only were the numbers of EBIMØs increased in B (p < 0.002), both EEBs and LEBs associated with EBIMØ were higher by 30 and 75%, respectively. Importantly, an increase in Siglec-1 and Vcam1 expressing F480+ splenic macrophages was observed after burn injury. Therefore, stagnant EEBs in the BM after burn injury could be due to low Siglec1 expressing EBIMØ, which perhaps impede their maturation into LEBs and reticulocytes. Repercussion of myeloid cell phenotype specific to BM after burn injury could plausibly account for a defective late stage RBC maturation resulting in anemia of critical illness.Summary Sentence: Characterization of erythroblast island macrophages (EBIMØ) in the bone marrow and spleen at different stages of erythropoiesis after burn injury.

Nrf2 Null Mice Are Deficient in CD169+ Macrophages, Associated with Impaired Erythroid Response and Delayed Recovery from Acute Blood Loss

Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.