Bone Marrow Erythroid Progenitors Research Articles

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)—The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells

Background aimsEx vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. MethodsWe generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. ResultsMorphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5–7 days. ConclusionsOur results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

A rat model of multicompartmental traumatic injury and hemorrhagic shock induces bone marrow dysfunction and profound anemia.

Severe trauma is associated with systemic inflammation and organ dysfunction. Preclinical rodent trauma models are the mainstay of postinjury research but have been criticized for not fully replicating severe human trauma. The aim of this study was to create a rat model of multicompartmental injury which recreates profound traumatic injury. Male Sprague-Dawley rats were subjected to unilateral lung contusion and hemorrhagic shock (LCHS), multicompartmental polytrauma (PT) (unilateral lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), or naïve controls. Weight, plasma toll-like receptor 4 (TLR4), hemoglobin, spleen to body weight ratio, bone marrow (BM) erythroid progenitor (CFU-GEMM, BFU-E, and CFU-E) growth, plasma granulocyte colony-stimulating factor (G-CSF) and right lung histologic injury were assessed on day 7, with significance defined as p values <0.05 (*). Polytrauma resulted in markedly more profound inhibition of weight gain compared to LCHS (p = 0.0002) along with elevated plasma TLR4 (p < 0.0001), lower hemoglobin (p < 0.0001), and enlarged spleen to body weight ratios (p = 0.004). Both LCHS and PT demonstrated suppression of CFU-E and BFU-E growth compared to naïve (p < 0.03, p < 0.01). Plasma G-CSF was elevated in PT compared to both naïve and LCHS (p < 0.0001, p = 0.02). LCHS and PT demonstrated significant histologic right lung injury with poor alveolar wall integrity and interstitial edema. Multicompartmental injury as described here establishes a reproducible model of multicompartmental injury with worsened anemia, splenic tissue enlargement, weight loss, and increased inflammatory activity compared to a less severe model. This may serve as a more effective model to recreate profound traumatic injury to replicate the human inflammatory response postinjury.

Anemia and Its Connections to Inflammation in Older Adults: A Review.

Anemia is a common hematological disorder that affects 12% of the community-dwelling population, 40% of hospitalized patients, and 47% of nursing home residents. Our understanding of the impact of inflammation on iron metabolism and erythropoiesis is still lacking. In older adults, anemia can be divided into nutritional deficiency anemia, bleeding anemia, and unexplained anemia. The last type of anemia might be caused by reduced erythropoietin (EPO) activity, progressive EPO resistance of bone marrow erythroid progenitors, and the chronic subclinical pro-inflammatory state. Overall, one-third of older patients with anemia demonstrate a nutritional deficiency, one-third have a chronic subclinical pro-inflammatory state and chronic kidney disease, and one-third suffer from anemia of unknown etiology. Understanding anemia's pathophysiology in people aged 65 and over is crucial because it contributes to frailty, falls, cognitive decline, decreased functional ability, and higher mortality risk. Inflammation produces adverse effects on the cells of the hematological system. These effects include iron deficiency (hypoferremia), reduced EPO production, and the elevated phagocytosis of erythrocytes by hepatic and splenic macrophages. Additionally, inflammation causes enhanced eryptosis due to oxidative stress in the circulation. Identifying mechanisms behind age-related inflammation is essential for a better understanding and preventing anemia in older adults.

Expression of microRNA-155 in thalassemic erythropoiesis.

Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in β-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with β-thalassemia/HbE. However, the expression and function of miR-155, especially in β-thalassemia, have not yet been explored. To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from β IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in β-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in β-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in β-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. miR-155 regulates IE in β-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease

AbstractDisordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2–containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

Atg4a Promotes Mitochondrial Quality Control and Hemoglobin Production In Vivo

Mitochondrial quality control is essential for maintaining cellular homeostasis and contributes to the regenerative capacity of hematopoietic stem cells and the differentiation of lineage-committed progeny. During red blood cell (RBC) production, mitochondria contribute to the production of hemoglobin and are the site for heme synthesis. Mitochondrial dysfunction is found in patients with mtDNA mutations, sickle cell disease, and myelodysplastic syndromes; therefore, it is critical to delineate the molecular mediators of mitochondrial quality control during erythropoiesis. While it is established that autophagy contributes to mitochondrial clearance during reticulocyte maturation, less is known about the regulation of mitochondrial quality control in erythroid progenitors. Our previous study identified an erythroid-specific role for the autophagy-related protease ATG4A in human erythropoiesis (Stolla MC et al., Blood Advances, 2022). To better understand the contribution of Atg4a-dependent autophagy to erythropoiesis in vivo, we have developed a new genetically modified mouse model . To evaluate the role of Atg4a in vivo, we disrupted the expression of Atg4a by targeting exons 5/6 using CRISPR/Cas9 in C57/BL6 embryos. Analysis of the peripheral blood of adult Atg4a-/- (KO) and Atg4a+/+ (WT) mice revealed a normal hematocrit (47.5% WT vs. 47% KO), but elevated numbers of red blood cells (10.7 M/μL WT vs. 11.2 M/μL KO, p=0.012) and a compensatory 5.8 % decrease in RBC volume (MCV: 44.3 fl WT vs. 41.7 fl KO, p=0.0004). Consistent with a reduction in RBC size, we observed a modest reduction in total hemoglobin (16.8 g/dl WT vs. 16.0 g/dl KO, p=0.054) and a profound decrease in the mean corpuscular hemoglobin concentration (MCHC: 35.39 g/dl WT vs. 34.17 g/dl KO, p=0.038) in Atg4a -/- (KO) mice. To determine if Atg4a-/- mice are iron deficient, we evaluated iron parameters in peripheral blood. While Atg4a-/- mice had slightly lower values of serum iron (152 μg/dl WT vs. 128.7 μg/dl KO, p=0.21) and total-iron binding capacity (511 μg/dl WT vs. 456 μg/dl KO, p=0.04) they had comparable levels of transferrin saturation (29.9% WT vs. 28.1% KO, p=0.65). Furthermore, we found similar amounts of EPO in the serum of Atg4a-/- and Atg4a+/+ mice (314 pg/ml WT vs. 345 pg/ml KO, p=0.78). Thus, loss of Atg4a leads to a reduction in hemoglobin production and RBC size without overt iron deficiency. To determine if alterations in erythroid maturation contributed to the decrease in MCV and MCHC, we used flow cytometry to quantify bone marrow erythroid progenitors (I), basophilic erythroblasts (II), polychromatic and orthochromatic erythroblasts (III), and reticulocytes (IV) using CD44, Ter119, CD71, and lineage markers (CD11b, B220, Gr-1) in Atg4a-/- mice. Similar numbers of erythroid populations (I-IV) were observed in the bone marrow and spleen of Atg4a-/- and Atg4a+/+ mice suggesting that erythroid maturation was not significantly altered in vivo. Since Atg4a is known to facilitate mitochondrial clearance during human erythropoiesis, we next evaluated mitochondrial dynamics in murine erythroid populations I-IV using dyes for active (TMRM) and total mitochondria (Mitotracker Green, MTG). Compared to Atg4a+/+ mice, Atg4a-/- mice had increased MTG fluorescence in erythroid populations I and II and a 25% decrease in TMRM fluorescence. Therefore, loss of Atg4a significantly reduces the ratio of active-to-total mitochondria in erythroid progenitors and basophilic erythroblasts, indicating that elimination of inactive mitochondria promotes mitochondrial function early in erythropoiesis. Since mitochondria are the site for heme synthesis, we sorted erythroid populations from Atg4a-/- and Atg4a+/+ mice and measured total heme content. While in Atg4a +/+ mice heme content increased by 3-fold between erythroid populations III and IV, heme content did not increase between erythroid populations III and IV from Atg4a-/- mice resulting in significantly reduced heme content in reticulocytes. Our study highlights an important protective role for autophagy in erythropoiesis where it mediates the elimination of dysfunctional mitochondria. Loss of mitochondrial quality control restricts heme production reducing the hemoglobin concentration of circulating RBCs. Further investigation will determine if the absence of Atg4a compromises the response to stress erythropoiesis.

A Genome Scale CRISPR Screen Identifies Novel Genes That Regulate Erythropoietin Production

Erythropoietin (EPO) is a plasma glycoprotein that binds bone marrow erythroid progenitors, stimulating their proliferation and differentiation. EPO is secreted into the circulation by hepatocytes and specialized kidney peritubular fibroblasts, which contribute ~20% and ~80% of the total plasma EPO level, respectively. EPO production is induced during hypoxia, due to enhanced stability of the transcription factor hypoxia-inducible factor (HIF). However, increased HIF activity results in enhanced expression of angiogenesis and oncogenesis promoting genes, raising concerns about clinical strategies that promote EPO production via increasing HIF levels. To define novel regulators of EPO production, we developed a genome-wide functional screen that provides a quantifiable and selectable readout of EPO production. In order to perform such a screen, we generated a reporter HEP3B cell line with homozygous insertion of coding sequences for p2A and eGFP at the endogenous EPO locus. This HEP3B EPO-p2A-eGFP (HEG) cell line expresses equivalent levels of EPO and eGFP proteins, both translated from the same mRNA molecule. Treatment of HEG cells with the HIF stabilizing molecule DMOG results in increased EPO expression, which is abolished by deletion of the hypoxia-responsive-element in the EPO locus. These findings demonstrate the utility of this reporter cell line to study the regulation of EPO production. To identify novel genes that regulate EPO production, we mutagenized the HEG cell line with the GeCKO-v2 CRISPR/Cas9 knock-out library, which delivers a puromycin resistance cassette, SpCas9, and a pooled collection of 123,411 single guide RNAs (sgRNAs) that include 6 independent sgRNAs targeting every human gene. Transduction was performed at a low multiplicity of infection (MOI ~0.3), so that most infected cells receive 1 sgRNA to delete 1 gene only. Puromycin selection was applied from days 1-4 post-transduction and cells were subsequently recovered for 8 days. Cells were then treated with DMOG for 24 hours or left untreated. Cells with increased (top ~10%) or decreased (bottom ~10%) eGFP fluorescence were isolated. This screen was done in biological triplicates. Genes for which sgRNAs are enriched/depleted in eGFP high versus eGFP low DMOG-treated cells likely impact EPO expression independently of the HIF pathway. In contrast, genes for which sgRNAs are enriched/depleted in eGFP high versus eGFP low untreated cells may impact EPO expression in either a HIF dependent or independent manner. As expected, sgRNAs targeting VHL were found to be among the most significantly enriched sgRNAs in eGFP-high versus eGFP-low cells under no treatment conditions. Also as anticipated, sgRNAs targeting genes known to promote EPO production ( HNF4A, WT1, RXRA, and CREBBP) were enriched in eGFP-low versus eGFP-high cells under either DMOG-treated or untreated conditions. To refine our findings, we generated a custom library targeting the top 1,200 candidate regulators of EPO production nominated by the genome-scale screen. Using this library, we performed a secondary CRISPR screen (in biological triplicates) as above. Owning to the 10-fold smaller size of the custom library, the secondary screen was performed with average coverage of &amp;gt;150x throughout the entire screen (including the sorted cell populations). Indeed, the statistical confidence obtained with the secondary screen was superior than that achieved in the genome scale screen. Using a false discovery rate (FDR) cutoff of 1%, gene-level analysis identified 100 and 79 candidate genes whose targeting was associated with increased or decreased EPO production respectively, under either untreated or DMOG-treated conditions. A high degree of concordance was observed for regulators of EPO expression between both screens, performed with and without DMOG treatment. To identify genes that regulate EPO production independent of HIF, we evaluated several of the top genes for which sgRNAs were enriched in eGFP high vs. eGFP low cells treated with DMOG. Using 3 independent sgRNAs targeting each of these candidates, we found 2 genes ( ZNF574 and RBM18), that when deleted, result in increased EPO mRNA, even following DMOG treatment. Additional validation experiments as well as studies aimed to define the role of these 2 genes in the regulation of EPO production are ongoing.

Posttraumatic pneumonia exacerbates bone marrow erythropoietic dysfunction.

Pneumonia is a common complication after severe trauma that is associated with worse outcomes with increased mortality. Critically ill trauma patients also have persistent inflammation and bone marrow dysfunction that manifests as persistent anemia. Terminal erythropoiesis, which occurs in bone marrow structures called erythroblastic islands (EBIs), has been shown to be impacted by trauma. Using a preclinical model of polytrauma (PT) and pneumonia, we sought to determine the effect of infection on bone marrow dysfunction and terminal erythropoiesis. Male and female Sprague-Dawley rats aged 9 to 11 weeks were subjected to either PT (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofracture) or PT with postinjury day 1 Pseudomonas pneumonia (PT-PNA) and compared with a naive cohort. Erythroblastic islands were isolated from bone marrow samples and imaged via confocal microscopy. Hemoglobin, early bone marrow erythroid progenitors, erythroid cells/EBI, and % reticulocytes/EBI were measured on day 7. Significance was defined as p < 0.05. Day 7 hemoglobin was significantly lower in both PT and PT-PNA groups compared with naive (10.8 ± 0.6 and 10.9 ± 0.7 vs. 12.1 ± 0.7 g/dL [ p < 0.05]). Growth of bone marrow early erythroid progenitors (colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte; erythroid burst-forming unit; and erythroid colony-forming unit) on day 7 was significantly reduced in PT-PNA compared with both PT and naive. Despite a peripheral reticulocytosis following PT and PT-PNA, the percentage of reticulocytes/EBI was not different between naive, PT, and PT-PNA. However, the number of erythroblasts/EBI was significantly lower in PT-PNA compared with naive (2.9 ± 1.5 [ p < 0.05] vs. 8.9 ± 1.1 cells/EBI macrophage). In addition to changes in EBI composition, EBIs were also found to have significant structural changes following PT and PT-PNA. Multicompartmental PT altered late-stage erythropoiesis, and these changes were augmented with the addition of pneumonia. To improve outcomes following trauma and pneumonia, we need to better understand how alterations in EBI structure and function impact persistent bone marrow dysfunction and anemia.

Murine erythroid differentiation kinetics in vivo under normal and anemic stress conditions

AbstractOur current understanding of the kinetics and dynamics of erythroid differentiation is based almost entirely on the ex vivo expansion of cultured hematopoietic progenitor cells. In this study, we used an erythroid-specific, inducible transgenic mouse line to investigate for the first time, the in vivo erythroid differentiation kinetics under steady-state conditions. We demonstrated that bipotent premegakaroycyte/erythroid (PreMegE) progenitor cells differentiate into erythroid–committed proerythroblast/basophilic erythroblasts (ProBasoE) after 6.6 days under steady-state conditions. During this process, each differentiation phase (from PreMegE to precolony forming unit-erythroid [PreCFU-E], PreCFU-E to CFU-E, and CFU-E to ProBasoE) took ∼2 days in vivo. Upon challenge with 5-flurouracil (5-FU), which leads to the induction of stress erythropoiesis, erythroid maturation time was reduced from 6.6 to 4.7 days. Furthermore, anemia induced in 5-FU-treated mice was shown to be due not only to depleted bone marrow erythroid progenitor stores but also to a block in reticulocyte exit from the bone marrow into the circulation, which differed from the mechanism induced by acute blood loss.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

Mechanisms of improved erythroid progenitor growth with removal of chronic stress after trauma

BackgroundErythropoietic dysfunction after trauma and critical illness is associated with anemia, persistent inflammation, increased hematopoietic progenitor cell mobilization from the bone marrow, and reduced erythroid progenitor growth. Yet the duration and reversibility of these postinjury bone marrow changes remain unknown. This study sought to determine whether removal of chronic postinjury stress could induce improvements in erythroid progenitor growth. MethodsSprague-Dawley rats (n = 8–11/group) were assigned to the following: naïve, lung contusion and hemorrhagic shock, lung contusion and hemorrhagic shock plus daily chronic stress for 7 days followed by 7 days of routine handling to allow recovery (lung contusion and hemorrhagic shock + chronic stress 7), or lung contusion and hemorrhagic shock plus chronic stress for 14 days (lung contusion and hemorrhagic shock + chronic stress 14). Circulating CD117+CD71+ erythroid progenitors were detected by flow cytometry. Rodents were killed on day 14, and bone marrow erythroid progenitor growth and erythroid transcription factors were assessed. Differences were assessed by analysis of variance (P < .05). ResultsCompared to lung contusion and hemorrhagic shock + chronic stress 14, lung contusion and hemorrhagic shock + chronic stress 7 rodents had improved hemoglobin (8% ± 10% increase vs 6% ± 10% decrease) with fewer mobilized erythroid progenitors (898 × vs 1,524 cells), lower granulocyte-colony stimulating factor levels (3.1 ± 1.1 × pg/mL vs 5.9 ± 1.8 pg/mL), and improved erythroid progenitor growth. Cessation of stress had no impact on erythroid transcription factors GATA-1, GATA-2, LMO2, or KLF1. ConclusionImprovements in erythroid progenitor growth and reduced hematopoietic progenitor cell mobilization were seen 7 days after cessation of chronic stress and were associated with an improvement in hemoglobin. Early bone marrow erythropoietic functional recovery may result from resolution of hematopoietic progenitor mobilization rather than upregulation of pro-erythroid transcription factors. This study suggests that postinjury anemia is reversible and has the potential to improve with the cessation of stress.

The Non-Erythropoietic EPO Analogue (Cibinetide) Preserves Bone Mass in Mice

Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro.Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A).To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups.In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B).Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO.This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MPN-343: The BET Inhibitor Pelabresib Decreases Inflammatory Cytokines, Improves Bone Marrow Fibrosis and Function, and Demonstrates Clinical Response Irrespective of Mutation Status in Myelofibrosis Patients in the Phase 2 MANIFEST Trial

<h3>Context:</h3> Pelabresib (CPI-0610) is a potent, selective BET bromodomain inhibitor under investigation in the ongoing Phase 2 MANIFEST trial, given as monotherapy to ruxolitinib (rux)-intolerant/refractory myelofibrosis (MF) pts in arm 1, as an "add-on" to rux in MF pts who have suboptimal/lost response to rux in arm 2, and as combination therapy with rux in JAKi-naïve MF pts in arm 3. <h3>Objective:</h3> We assessed activity of pelabresib, including changes in inflammatory cytokines (Ck) in blood, improvement of bone marrow (BM) biology, including fibrosis (BMF), erythroid (Ery) progenitor and megakaryocyte (MK) numbers and clustering, and impact of mutational status on clinical response. <h3>Results:</h3> Rapid PD response was demonstrated by median blood <i>IL8</i> mRNA level reduced to 55% of baseline (BL) levels 4 hours post-1st dose of pelabresib in 102 pts, with similar reduction across arms. Elevated blood Ck levels were observed at BL across arms. Reduction of NF-κB and non-NF-κB regulated inflammatory Ck was observed on cycle 1 day 14 (C1D14) across arms and maintained through C9D1 (24 weeks). BMF grading was assessed by local pathologists for BL and post-treatment (most at 24 weeks) biopsies from 116 evaluable pts. Relative BMF improvement of ≥1 grade was observed in 33% (38/116) of all pts, with 21% (6/29) in arm 1, 41% (16/39) in arm 2, and 33% (16/48) in arm 3. BMF grade worsening was observed in only 6% (7/116) pts. Exploratory analyses of Ery and MK lineages by immunohistochemical staining for CD71 and CD61, respectively, on BM biopsy pairs (BL and week 24) revealed an increase in Ery progenitors in 59% (22/37) of pts. Tight clusters of MK, characteristic in MF BM, were observed at BL, and improvement in MK numbers and/or clustering was observed in 65% (24/37) pts. Mutation analysis revealed similar profiles at BL across arms. SVR35 at 24 weeks was similar, regardless of mutational status for each of the 14 genes analyzed. <h3>Conclusions:</h3> Our data demonstrate a robust PD effect of pelabresib in MF pts, indicate broad clinical responses irrespective of mutational status, and suggest the disease-modifying potential of pelabresib by improving BM fibrosis and function.

Altered HSC Programming Drives Aberrant Blood Output Underlying RA Pathogenesis

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder characterized by autoantibody production towards collagen type-II in the synovial joints of the skeletal system, leading to progressive joint damage. RA is accompanied by chronic systemic production of pro-inflammatory cytokines including IL-6, TNF, and IL-1. Importantly, RA patients often suffer from significant hematological complications including production of tissue-damaging macrophages, chronic anemia, impaired lymphopoiesis and suppressed bone marrow function. As blood-forming hematopoietic stem cells (HSC) constitute the root of the immune system, identifying mechanisms by which altered HSC function may contribute to disease could provide key insights into RA pathogenesis and therapeutic intervention.In this study, we used a mouse model of collagen-induced arthritis (CIA), which faithfully recapitulates many features of human autoimmune arthritis, to identify the impact of disease on the hematopoietic compartment, and identify mechanisms underlying alteration in blood lineage output and function. Strikingly, bone marrow hematopoietic populations were significantly altered in CIA mice. This was typified by decreased numbers of peripheral red blood cells (RBC) and bone marrow erythroid progenitors, alongside decreases in bone marrow B cells and common lymphoid progenitors (CLP). On the other hand, we observed increased numbers of platelets, bone marrow granulocytes, and myeloid-biased multipotent progenitors (MPP3). Strikingly, we also observed reductions in the stromal cells comprising the bone marrow niche, with decreases in epithelial, mesenchymal, and osteoblastic cell types, suggesting that the inflammatory effects of RA influence the bone marrow compartment at multiple levels. We functionally validated our phenotypic analyses using colony forming unit (CFU) assays, and observed increased myeloid CFU, with concomitant decreases in erythroid and lymphoid colony forming activity in unfractionated bone marrow cells from CIA mice. Despite these changes, transplantation assays using purified HSCs from CIA and control mice showed that long-term reconstitution potential of HSCs was largely unaltered, suggesting that HSC self-renewal capacity is not significantly impaired. Taken together, these observations suggest that autoimmune arthritis induces substantial remodeling of the bone marrow, resulting in overproduction of myeloid cells at the expense of other lineages.In line with our phenotypic and functional analyses, RNA-seq analysis of HSCs from CIA versus control mice uncovered precocious activation of myeloid-specific gene programs that likely underlie shifts in lineage output. Moreover, in line with our transplantation data, we did not observe significant shifts in the expression of core genes governing HSC self-renewal. Collectively, these data show that the inflammatory environment of RA causes changes to hematopoietic output, leading to an increase in damaging myeloid cell types, further fueling the inflammatory environment and disease progression.We are currently identifying the mechanism underlying myeloid overproduction in the bone marrow, with focus on systemic inflammatory factors and/or shifts in the gastrointestinal microbiota, as potential underwriters of deregulated hematopoiesis and disease development. We are also assessing the functionality of myeloid cells generated by HSCs during RA to determine whether they exhibit a stronger pro-inflammatory phenotype. Ultimately, these studies will increase our understanding of the crosstalk between RA and the hematopoietic system, and potentially opens new avenues for therapeutic intervention to re-establish the hematopoietic system and even rescue RA progression. DisclosuresNo relevant conflicts of interest to declare.

Absence of Interleukin-6 Protects Murine Bone Marrow Erythropoiesis Under Inflammation, in a Process Reversible By Iron Mediated Reactive Oxygen Species Upregulation

Inflammatory states seen in infections and chronic disorders are often characterized by a condition called anemia of Inflammation (AI). Using a mouse model of AI generated by a single injection of the heat killed pathogen Brucella abortus (BA), we have previously shown that mice lacking IL6 (IL6 -KO mice) exhibited protected erythropoiesis in the bone marrow (BM) and a faster recovery from anemia compared to controls. To study the mechanism of IL6 mediated improved erythropoiesis under AI, we investigated erythroid recovery in WT and IL6 -KO mice injected with BA. 72 hours following BA administration, both genotypes showed impaired BM erythropoiesis associated by a surge in inflammatory cytokines such as IFNγ and TNFα and a concurrent increase in mitochondrial ROS (reactive oxygen species), specifically superoxide (SO) in erythroid progenitors. Cytokines levels were normalized after 24 hours. However, during the second phase of erythroid recovery (10-14 days following BA treatment), mice showed a second surge of inflammatory cytokines. During this phase, IL6 -KO mice showed significantly improved BM erythropoiesis and normalization of ROS levels. We analyzed SO levels by Mitosox red, generic ROS production using the chloromethyl derivative of 2′,7′-dichlorofluorescin diacetate and total protein oxidation (by looking at total protein carbonylation) by Western blot. These results were in sharp contrast to WT animals which continued to show upregulated ROS, total protein oxidation and slackened erythroid recovery.Given the extensive association of iron overloading conditions and oxidative stress, we investigated the improved erythroid recovery within IL6 -KO mice in the context of increased iron absorption. We asked if and how BM erythropoiesis would change in IL6 -KO mice lacking Hepcidin (Hamp), a key iron regulatory hormone whose absence induces iron overload. In double knock out mice lacking both IL6 and Hamp (DKO), we observed an initial improved erythroid recovery in the BM, as indicated by increased number of total BM erythroid progenitors and reticulocytes, and improved RBC lifespan. However, all of these factors regressed after 10 days, with a simultaneous increase of cytokine production. The deterioration of erythropoiesis in the BM was accompanied by a concurrent increase in ROS. Of note, compared to non-treated mice, erythroid progenitors in the spleen of DKO-BA did not show increased ROS levels, and this was associated with an increased number of splenic erythroid progenitors and RBC. These results point to a potential role in the microenvironment in influencing erythropoiesis under inflammation. Furthermore, these results suggest that while lack of IL6 protects BM erythropoiesis, iron overload could antagonize it. In fact, iron overloaded IL6 -KO mice (with iron dextran) did show an impairment in BM erythroid recovery and upregulation of ROS, similarly to the DKO. Finally, we hypothesized that while there is a distinct positive correlation between upregulated ROS and weakened BM erythropoiesis, this comes into effect only in the context of inflammation. In other words, we speculated that some level of ROS upregulation is otherwise a physiological byproduct of erythropoietic stress associated with the onset of anemia. In line with this theory, we found that ROS levels were indeed upregulated in BM erythroid progenitors of phlebotomized animals. Our data suggests a model of erythroid recovery under AI wherein inflammation exacerbates ROS production triggered by stress erythropoiesis, and that this phenomenon exquisitely impairs BM erythropoiesis. This negative effect on BM erythropoiesis is mitigated by the absence of IL6, but the lack of IL6 mediated protective function is reversible under conditions of iron overload (Figure 1).Our results, in combination with the upregulation of TNFa, suggest the involvement of non-apoptotic ROS associated programmed cell death such as necroptosis or ferroptosis, which are under investigation. We also have preliminary data pointing to mechanism of increased erythroid iron uptake that could potentially contribute to oxidative stress under AI. With these additional studies, we hope to further characterize the mechanisms surrounding improved BM erythropoiesis in the absence of ROS and IL6. Our studies could potentially characterize cellular responses that can be targeted in conditions of inflammation for therapeutic benefits. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen.

Adult erythropoiesis is a highly controlled sequential differentiation of hematopoietic stem cells (HSCs) to mature red blood cells in the bone marrow (BM). The bones which contain BM are diverse in their structure, embryonic origin, and mode of ossification. This has created substantial heterogeneity in HSCs function in BM of different bones, however, it is not known if this heterogeneity influences erythropoiesis in different bones and different regions of the same bone. In this study, we examined steady state BM erythroid progenitors and precursors from different bones – the femur, tibia, pelvis, sternum, vertebrae, radius, humerus, frontal, parietal bone, and compared all to the femur. Trabecular and cortical regions of the femur were also compared for differences in erythropoiesis. In addition, mouse spleen was studied to determine at which age erythropoietic support by the spleen was lost postnatally. We report that total erythroid cells, and erythroid precursors in the femur are comparable to tibia, pelvis, humerus and sternum, but are significantly reduced in the vertebrae, radius, frontal, and parietal bones. Erythroid progenitors and multipotential progenitor numbers are comparable in all the bones except for reduced number in the parietal bone. In the femur, the epiphysis and metaphysis have significantly reduced number of erythroid precursors and progenitors, multipotential progenitors and myeloid progenitors compared to the diaphysis region. These results show that analysis of erythroid precursors from diaphysis region of the femur is representative of tibia, pelvis, humerus and sternum and have significant implications on the interpretation of the steady-state erythropoiesis finding from adult BM. Postnatal spleen supports erythroid precursors until 6 weeks of age which coincides with reduced number of red pulp macrophages. The residual erythroid progenitor support reaches the adult level by 3 months of age. In conclusion, our findings provide insights to the differences in erythropoiesis between different bones, between trabecular and cortical regions of the femur, and developmental changes in postnatal spleen erythropoiesis.

Ker-050, a Novel Inhibitor of Tgfβ Superfamily Signaling, Induces Red Blood Cell Production By Promoting Multiple Stages of Erythroid Differentiation

Diseases such as myelodysplastic syndrome (MDS) and myelofibrosis (MF) are characterized by ineffective hematopoiesis resulting in one or multiple cytopenias. Disease-causing defects occur across multiple cell lineages and stages of hematopoiesis, making development of an effective treatment for all patients challenging. Current treatment options to address anemia in these diseases target discreet stages in erythropoiesis, whereas defects leading to ineffective hematopoiesis can occur throughout the pathway. Therefore, a treatment that more globally modulates hematopoiesis has the potential to treat broad patient groups. The TGFβ superfamily plays a key role in regulating hematopoiesis; as signaling via SMAD2/3 activation results in cell quiescence, inhibiting precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA ligand trap, promotes hematopoiesis through inhibition of ligands that signal though SMAD2/3, including activins and GDFs. In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to robust, rapid and sustained increases in red blood cells (RBCs), hemoglobin (HGB) and platelets, supporting an effect on the multiple stages of hematopoiesis. Here, we characterize the time course of KER-050-mediated effects on RBC production and changes in erythroid precursor cell populations in mice to characterize the mechanism of action of KER-050 on erythropoiesis. Mice treated with a single dose of a research form of KER-050 (RKER-050, 10mg/kg) had increased RBCs, HGB and hematocrit (HCT) (+8%, +9%, +7%, respectively) 12 hours after administration compared to vehicle-treated mice (VEH), and this effect was further increased on Day 7. There was also a reduction in the number of enucleated erythroid cells in the bone marrow and a parallel increase in the percent of immature reticulocytes (RET) in peripheral blood, suggesting an increased outflux of RET into circulation. This observation is consistent with RKER-050 promoting the maturation of late stage erythroid precursors leading to increases in RBCs, HGB and HCT as early as 12 hours post treatment and remaining 14 days post a single dose. In studies evaluating the effect of RKER-050 on bone marrow erythroid progenitors, a 2-fold increase in late orthochromatic erythroblasts/RETs (EryC) at Days 2 and 7 post-dose was observed. These data are consistent with RKER-050 promoting maturation and release of late-stage erythroid precursors into circulation. RKER-050 also elicited effects on early progenitors. Day 2 post-dose, there was a 2-fold increase in CFU-Es, with a 46% decrease in poly-erythrochromatic/early orthochromatic erythroid precursors (EryB) at Day 4, as compared to VEH. Day 7 post treatment, both CFU-Es and the EryB population returned to VEH levels. The increase in early progenitors appears to replenish the polychromatic erythroblasts (as shown by the return to VEH level of EryB precursors at Day 7), allowing for continued supply of maturing RETs. Consistent with this hypothesis, RKER-050-mediated changes in erythroid precursors continued to Day 14 with significantly increased early progenitor population (BFU-E +24%) and increased late stage erythroid precursors (EryB +40%, EryC 7-fold) while maintaining increased circulating RETs and RBCs. These data demonstrate that the RKER-050 treatment increases early progenitor cells which continue to mature and contribute to the overall upregulation of erythropoietic tone. Surprisingly, RKER-050 treatment resulted in a greater than 2-fold increase in serum levels of erythropoietin (Epo) at Days 4, 7 and 14. These counterintuitive results demonstrate that RKER-050 promotes erythropoiesis while at the same time increasing Epo. This effect may result in a feed-forward effect on the system and result in a sustained upregulation of erythropoietic tone. Overall, these data demonstrate that KER-050 stimulates terminal maturation of late-stage erythroid precursors, expands the early stage precursor population and progresses precursors through erythropoiesis. Additionally, KER-050 increases Epo within the milieu of elevated RBCs. The ability of KER-050 to target multiple stages along the erythropoiesis cascade makes it an appealing therapeutic candidate for diseases that cause anemia due to ineffective erythropoiesis, including MDS and MF, where defects can arise throughout the erythropoietic pathway. Disclosures Feigenson: Keros Therapeutics: Current Employment. Nathan:Keros Therapeutics: Current Employment. Materna:Keros Therapeutics: Current Employment. Gudelsky:Keros Therapeutics: Current Employment. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Nitric oxide-dependent expansion of erythroid progenitors in a murine model of chronic psychological stress.

Anaemia occurs frequently in patients with heart failure and its current treatment lacks clear targets. Emerging evidence suggests that erythroid progenitor cell expansion is an integral part of physiological response to anaemia associated with chronic stress. Understanding the underlying mechanism may provide a novel approach to anaemia management. In this study, we aimed to examine a role for nitric oxide (NO) in the regulation of bone marrow erythroid progenitor response to chronic stress. For this purpose, adult male mice were subjected to 2h daily restraint stress for 7 or 14 consecutive days. The role of NO was assessed by subcutaneous injection with NG-nitro-L-arginine methyl ester, 30min prior to each restraint. Chronic exposure to stress resulted in significantly increased number of bone marrow erythroid progenitors, and blockade of NO biosynthesis prior to daily stress completely prevented stress-induced erythroid progenitor cell expansion. Furthermore, chronic stress exposure led to altered expression of neural, endothelial and inducible nitric oxide synthases (NOS) in the bone marrow, both on mRNA and protein level. Decreased expression of neural and endothelial NOS, as well as reduced expression of NF-kappaB/p65 in bone marrow nuclear cell fraction, was accompanied by elevated bone marrow expression of inducible NOS in chronically stressed animals. This is the first study to demonstrate a role for NO in adaptive response of erythroid progenitors to chronic stress. Targeting NO production may be beneficial to improve bone marrow dysfunction and reduced erythroid progenitor cell expansion in chronic heart failure patients.

ERFE regulation in sickle cell disease: complex but promising.

ERFE regulation in sickle cell disease: complex but promising.

Canonical Signaling By TGF Family Members in Mesenchymal Stromal Cells Is Dispensable for Hematopoietic Niche Maintenance Under Basal and Stress Conditions

The bone marrow contains a complex population of stromal and hematopoietic cells that together generate a unique microenvironment, or niche, to support hematopoiesis. Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche and include CXCL12-abundant reticular (CAR) cells, adipocytes, osteolineage cells, and arteriolar pericytes, all of which have been implicated in hematopoietic stem/progenitor cell (HSPC) maintenance. There also is evidence that adaptive changes in bone marrow stromal cells contributes to recovery from myelosuppresive therapy and the development of certain hematopoietic malignancies. However, the signals that contribute to the development, maintenance, and stress response of bone marrow mesenchymal stromal cells are poorly understood. Here, we test the hypothesis that cytokines of the transforming growth factor superfamily, which include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins/inhibins, provide signals to mesenchymal stromal cells that contribute to basal and stress hematopoiesis responses. To test this hypothesis, we abrogated canonical TGF family signaling in mesenchymal stem/progenitor cells by deleting Smad4 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow. We first performed lineage-tracing studies using Osx-Cre Smad4fl/fl Ai9 mice to show that activation of Osx-Cre at birth (by removal of doxycycline) results in the efficient targeting of bone marrow mesenchymal stromal cells. Moreover, we show that Smad4 mRNA expression is essentially undetectable in sorted mesenchymal stromal cells sorted from the bone marrow of these mice. Basal hematopoiesis and bone marrow stromal cells were analyzed in 6-8 week old Osx-Cre Smad4fl/fl mice. No alterations in the number or spatial organization of CAR cells, osteoblasts, or adipocytes was observed, and expression of key niche factors, including Scf, Cxcl12, and Spp1 was normal. Basal hematopoiesis, including the number of phenotypic HSCs in bone marrow and spleen, also was normal. Recent studies have shown that inhibition of activin signaling by treating with an activin receptor 2 alpha (ACVR2a) ligand trap stimulates erythropoiesis. Although ACVR2a signaling in erythroid progenitors contributes to this effect, two groups showed that inhibition of ACVR2a signaling in bone marrow stromal cells also stimulates erythropoiesis. Thus, we next characterized basal and stress erythropoiesis in Osx-Cre Smad4fl/fl mice. The frequency of phenotypic erythroid progenitors in bone marrow and spleen was similar to control mice. The stress erythropoiesis response was assessed after induction of acute hemolytic anemia by phenylhydrazine treatment. Both the magnitude of anemia and kinetics of erythroid recovery were similar to control mice. Myelosuppressive therapy induces marked alterations in the bone marrow microenvironment that includes an expansion of osteolineage cells and adipocytes, which have been linked to hematopoietic recovery. Thus, we next characterized stress hematopoiesis in Osx-Cre Smad4fl/fl mice in response to 5-fluorouracil (5-FU) treatment. Compared to control mice, the magnitude and duration of neutropenia following 5-FU were similar. Moreover, mouse survival after repeated weekly doses of 5-FU was comparable to control mice. HSPC mobilization by G-CSF is due, in large part, by downregulation of CXCL12 expression in bone marrow mesenchymal stromal cells. A prior study suggested that SMAD signaling negatively regulates CXCL12 expression in stromal cells. Consistent with this finding, we show that treatment of cultured bone marrow derived MSCs with TGF-b1 for 48 hours results in a significant (3.3-fold, P&lt;0.0001) decrease in CXCL12 mRNA expression. Thus, in the final experiments, we characterized G-CSF induced HSPC mobilization in Osx-Cre, Smad4fl/fl or Osx-Cre, Tgfbr2fl/fl mice. HSPC mobilization, as quantified by CFU-C and Kit+ Sca+ lineage- (KSL) cell number in blood or spleen after 5 days of G-CSF treatment was comparable to control mice. Collectively, these data suggest the TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions. Disclosures No relevant conflicts of interest to declare.