Stress Erythropoiesis Research Articles

A bulge uridine in the HIF2α IRE allows IRP1 but not IRP2 to selectively regulate HIF2α expression and ensuing EPO levels

AbstractIron regulatory proteins (IRP1 and IRP2) play a pivotal role in maintaining cellular iron homeostasis by binding to iron-responsive elements (IREs) of target messenger RNAs and regulating the expression of these iron-related genes. Mice and humans who lack functional IRP1 develop erythrocytosis due to erythropoietin (EPO) overproduction, whereas those who lack IRP2 develop microcytic anemia, believed to result from iron deficiency of erythroblasts. Here, we discovered that IRP2 deficiency reduced the expression of hypoxia-inducible factor 2α (HIF2α) and its transcriptional target, EPO, thereby compromising the stress erythropoiesis response to generate red blood cells upon anemia. The distinct consequences of IRP2 and IRP1 on EPO result from the higher binding affinity of the HIF2α IRE for IRP1 than IRP2. This difference in binding affinity arises from a bulge uridine in the upper stem of HIF2α IRE that impairs the ability of IRP2 to bind the IRE. These results reveal that IRP1 and IRP2 play distinct roles in erythropoiesis and unveil an unsuspected IRE binding preference that contributes to the divergent phenotypes observed in IRP1- and IRP2-deficient mammals.

Copper Serum Levels in the Hemodialysis Patient Population.

Copper is an essential element in the diet of mammals, including humans. It plays an important role in the physiological regulation of various enzymes and is consequently involved in several biological processes such as angiogenesis, oxidative stress regulation, neuromodulation, and erythropoiesis. Copper is essential for facilitating the transfer of iron from cells to the bloodstream, which is necessary for proper absorption of dietary iron and the distribution of iron throughout the body. In particular, patients with end-stage renal failure who require renal replacement therapy are at increased risk for disorders of copper metabolism. Many studies on hemodialysis, peritoneal dialysis, and kidney transplant patients have focused on serum copper levels. Some reported mild deficiency, while others reported elevated levels or even toxicity. In some cases, it has been reported that alterations in copper metabolism lead to an increased risk of cardiovascular disease, malnutrition, anemia, or mielopathy. The aim of this review is to evaluate the role of copper in patients undergoing hemodialysis and its potential clinical implications.

Angelica sinensis polysaccharides promote extramedullary stress erythropoiesis via ameliorating splenic glycolysis and EPO/STAT5 signaling-regulated macrophages.

Conventional treatments exhibit various side effects on chronic stress anemia. Extramedullary stress erythropoiesis is a compensatory mechanism, which may effectively counteract anemia. Angelica sinensis polysaccharides (ASP) are the main active ingredient found in Angelica sinensis and exhibit antioxidant and hematopoietic effects. However, the effects of ASP on extramedullary stress erythropoiesis remain to be unclear. Here, we demonstrated the protective effects of ASP on chemotherapeutic drug 5-fluorouracil (5-FU)-induced decline in peripheral blood parameters such as RBC counts, HGB, HCT, and MCH, and the decline of BFU-E colony enumeration in the bone marrow. Meanwhile, ASP promoted extramedullary erythropoiesis, increasing cellular proliferation in the splenic red pulp and cyclin D1 protein expression, abrogating phase G0/G1 arrest of c-kit+ cells in mouse spleen. RT-qPCR and immunohistochemistry further revealed that ASP increased macrophage chemokine Ccl2 genetic expression and the number of F4/80+ macrophages in the spleen. The colony-forming assay showed that ASP significantly increased splenic BFU-E. Furthermore, we found that ASP facilitated glycolytic genes including Hk2, Pgk1, Pkm, Pdk1, and Ldha via PI3K/Akt/HIF2α signaling in the spleen. Subsequently, ASP declined pro-proinflammatory factor IL-1β, whereas upregulating erythroid proliferation-associated genes Gdf15, Bmp4, Wnt2b, and Wnt8a. Moreover, ASP facilitated EPO/STAT5 signaling in splenic macrophages, thus enhancing erythroid lineage Gata2 genetic expression. Our study indicated that ASP may improve glycolysis, promoting the activity of splenic macrophages, subsequently promoting erythroid progenitor cell expansion. Additionally, ASP facilitates erythroid differentiation via macrophage-mediated EpoR/STAT5 signaling; suggesting it might be a promising strategy for stress anemia treatment.

Dysregulation of stress erythropoiesis and enhanced susceptibility to Salmonella Typhimurium infection in AhR-deficient mice.

By acting as an environmental sensor, the ligand-induced transcription factor aryl hydrocarbon receptor (AhR) regulates acute innate and adaptive immune responses against pathogens. Here, we analyzed the function of AhR in a model for chronic systemic infection with attenuated Salmonella Typhimurium (STM). WT and AhR-deficient mice were infected with the attenuated STM strain TAS2010 and analyzed for bacterial burden, host defense functions and inflammatory stress erythropoiesis. AhR-deficient mice were highly susceptible to TAS2010 infection compared with WT mice demonstrated by reduced bacterial clearance and increased mortality. STM infection resulted in macrocytic anemia and enhanced splenomegaly along with destruction of the splenic architecture in AhR-deficient mice. In addition, AhR-deficient mice displayed a major expansion of splenic immature red blood cells, indicative of infection-induced stress erythropoiesis. Elevated serum levels of erythropoietin and interleukin-6 upon infection as well as increased numbers of splenic stress erythroid progenitors already in steady state probably drive this effect and might cause the alterations in splenic immune cell compartments, thereby preventing an effective host defense against STM in AhR-deficient mice. AhR-deficient mice fail to clear chronic TAS2010 infection due to enhanced stress erythropoiesis in the spleen and accompanying destruction of the splenic architecture.

Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells.

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1–3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

Hookworm infection as a model for deepen knowledge of iron metabolism and erythropoiesis in anemia

Over the years, there has been progress in understanding the molecular aspects of iron metabolism and erythropoiesis. However, despite research conducted both in laboratories and living organisms, there are still unanswered questions due to the complex nature of these fields. In this study we investigated the effects of hookworm infection on iron metabolism and how the hosts response to anemia is affected using hamsters infected with Ancylostoma ceylanicum as a model. Our data revealed interesting relationships between infection-induced anemia, erythropoiesis, iron metabolism, and immune modulation, such that the elevated production of erythropoietin (EPO) in renal tissue indicated intensified erythropoiesis in response to anemia. Additionally, the increased expression of the erythroferrone (ERFE) gene in the spleen suggested its involvement in iron regulation and erythropoiesis. Gene expression patterns of genes related to iron metabolism varied in different tissues, indicating tissue-specific adaptations to hypoxia. The modulation of pro-inflammatory and anti-inflammatory cytokines highlighted the delicate balance between immune response and erythropoiesis. Data derived from the investigation of changes induced in iron metabolism and stress erythropoiesis following anemia aid in our understanding of mechanisms related to blood spoliation and anemia, which could potentially be extrapolated or compared to other types or causes of anemia. These findings also contribute to our understanding of the pathophysiology of erythropoiesis in the context of blood loss.

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

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

Lung Epithelium Releases Growth Differentiation Factor 15 in Response to Pathogen-mediated Injury.

GDF15 (growth differentiation factor 15) is a stress cytokine with several proposed roles, including support of stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the cellular sources and downstream effects of GDF15 during pathogen-mediated lung injury are unclear. We quantified GDF15 in lower respiratory tract biospecimens and plasma from patients with acute respiratory failure. Publicly available data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reanalyzed. We used mouse models of hemorrhagic acute lung injury mediated by Pseudomonas aeruginosa exoproducts in wild-type mice and mice genetically deficient for Gdf15 or its putative receptor, Gfral. In critically ill humans, plasma levels of GDF15 correlated with lower respiratory tract levels and were higher in nonsurvivors. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium, and lower respiratory tract GDF15 levels were higher in coronavirus disease (COVID-19) nonsurvivors. In mice, intratracheal P. aeruginosa type II secretion system exoproducts were sufficient to induce airspace and plasma release of GDF15, which was attenuated with epithelial-specific deletion of Gdf15. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and an altered lung transcriptional profile during injury induced by P. aeruginosa type II secretion system exoproducts, which was not recapitulated in mice deficient for Gfral. Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but increased circulating erythrocyte counts. Lung epithelium releases GDF15 during pathogen injury, which is associated with plasma levels in humans and mice and can increase erythrocyte counts in mice, suggesting a novel lung-blood communication pathway.

A bone to pick-cellular and molecular mechanisms of bone pain in sickle cell disease.

The bone is one of the most commonly affected organs in sickle cell disease (SCD). Repeated ischemia, oxidative stress and inflammation within the bone is largely responsible for promoting bone pain. As more individuals with SCD survive into adulthood, they are likely to experience a synergistic impact of both aging and SCD on their bone health. As bone health deteriorates, bone pain will likely exacerbate. Recent mechanistic and observational studies emphasize an intricate relationship between bone remodeling and the peripheral nervous system. Under pathological conditions, abnormal bone remodeling plays a key role in the propagation of bone pain. In this review, we first summarize mechanisms and burden of select bone complications in SCD. We then discuss processes that contribute to pathological bone pain that have been described in both SCD as well as non-sickle cell animal models. We emphasize the role of bone-nervous system interactions and pitfalls when designing new therapies especially for the sickle cell population. Lastly, we also discuss future basic and translational research in addressing questions about the complex role of stress erythropoiesis and inflammation in the development of SCD bone complications, which may lead to promising therapies and reduce morbidity in this vulnerable population.

Unraveling the Role of PIEZO1 in Stressed Erythropoiesis: Implications for Dyserythropoiesis and Potential Therapeutic Targets in Dehydrated Hereditary Stomatocytosis

PIEZO1 is a mechanosensitive cation channel that plays a crucial role in various physiological processes as a mechanical force sensor. In erythrocytes, it is responsible for regulating cellular volume and hydration. Gain-of-function (GoF) mutations in PIEZO1 lead to dehydrated hereditary stomatocytosis (DHS) by slowing down the channel's inactivation kinetics. DHS patients often exhibit a range of clinical presentations, including mild to severe hemolytic anemia, and iron overload. In addition, studies on erythroid progenitor cells from DHS patients have indicated a mutation-dependent delay in erythroid differentiation. Furthermore, studies on constitutive GoF Piezo1 mice have revealed stressed erythropoiesis characterized by increased erythrocyte turnover. The primary aim of this study is to investigate the role of PIEZO1 during stressed erythropoiesis in DHS and identify potential druggable targets. A total of 80 DHS patients were analysed, and their BMRI (reticulocyte count × patient's Hb/normal Hb), EPO, and ERFE levels were measured. The findings revealed mutation-dependent dyserythropoietic features similar to those observed in CDAII patients (characterized by dyserythropoiesis). The engineered erythroid model of DHS, Hudep2-PIEZO1-KI (Hudep2-KI), was subjected to erythroid differentiation for 12 days and compared to Hudep2-WT. A notable reduction in CD235a expression was detected on the last day of differentiation, indicating that PIEZO1-mediated alteration occurs during the late stages of differentiation. Morphological analysis of differentiating cells indicated that KI cells experienced a significant reduction in proliferation rate (1.5-fold increase vs. day 0) compared to Hudep2-WT (3.1-fold increase vs. day 0) starting from day 7. By day 12 of differentiation, KI cells displayed only half the cell count of WT cells, with a substantial decrease in the percentage of orthochromatic normoblasts (13.7% vs. WT 54.5%). This was accompanied by a relative increase in reticulocytes (Hudep2-WT: 45.5% vs. Hudep2-KI: 86.3%). To identify the altered signalling pathways involved in differentiation deregulation, RNAseq was performed at various time points during differentiation. The gene ontology analysis indicated that the most enriched biological processes were related to apoptosis, cellular response to hypoxia, and glycolysis. During the differentiation process, various apoptosis-related processes were found significantly enriched on different days. Upon conducting a single gene analysis, an imbalance between pro-apoptotic and antiapoptotic signals became evident. Specifically, in Hudep2-KI cells compared to the WT, we observed that 70.8% of proapoptotic genes were upregulated. Conversely, the regulation of apoptosis did not seem to impact antiapoptotic signals, with 57.9% of them being upregulated and 42.1% downregulated. This observation led us to suggest that PIEZO1 GoF induces apoptosis by activating pro-apoptotic signals. The findings of this study demonstrate the role of PIEZO1 GOF mutation in stressed erythropoiesis in both DHS patients and the erythroid cellular system. PIEZO1 GOF variants, through their effect on intracellular calcium concentration, influence late-stage differentiation and disrupt several critical pathways, including apoptosis, response to hypoxia, and glycolysis. Ongoing research involves confirming the data in CD34+ cells from DHS patients and PIEZO1 GOF mice. Additionally, the identified deregulated markers hold promise as potential druggable targets for dyserythropoiesis treatment.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

M6A Mediated Ribostasis of RNA Stress Granule Assembly Governs Blood Development and Regeneration

Hematopoietic stem cell (HSC) form diverse cell types and requires transcription and translation regulation. Using CD34+ human hematopoietic stem and progenitor cells (hCD34+) combined with N6-methyladenosine (m6A) sequencing, we identified a novel regulatory mechanism via stress granule regulation during erythropoiesis. Our single-nucleotide resolution m6A analysis of the whole transcriptome identified ~4800 transcripts to be m6A modified and showed demethylation (>2-14 fold) during erythropoiesis. Genome-wide CHIP-seq analysis of hCD34+ cell erythropoiesis with anti-Gata1 showed an enhancer enrichment at the ALKBH5 RNA demethylase gene. Using ALKBH5 CRISPR Cas9 KO strategy combined with hCD34+ cell in-vitro differentiation protocol we observed a block in hCD34+ cell erythropoiesis thus validating Alkbh5 role in erythropoiesis. hCD34+ ALKBH5 KO cells showed a failure of RNA m6A demethylation and discovered stress granule (SGs) transcripts m6A levels to be upregulated (>2-4 fold, p=0.01). Stress granules are membrane-less organelles composed of various RNA binding proteins. SGs form a hub in the cytoplasm that governs RNA metabolism and global proteome by translational regulation. Our analysis showed ATXN2, G3BP1/2, Tia-1,and PABPC1 SGs transcripts are m6a modified at 3'UTR. K562 cell ALKBH5 KO erythropoiesis model led to massive m6A upregulation on ATXN2 3'UTR (>14 fold, p=0.001) and other SGs transcripts. Mass spectrometry analysis of hCD34+ ALKBH5 KO cell proteome showed downregulation of ATXN2(>2 fold, -log P value2), confirming 3' UTR m6A mediated protein synthesis regulation. ALKBH5 KO hCD34+ cells showed aggregation of SGs protein in the cytoplasm due to altered SG protein stoichiometry(>4-10 fold, p=0.001). Zebrafish alkbh5 KO showed defective blood development and anemia. STED microscopy of the whole kidney marrow of alkbh5 KO fish showed the presence of G3BP and ATXN2 SGs with m6A labeled RNAs. alkbh5 KO zebrafish blood and kidney marrow analysis also showed increased myeloid and lymphoid lineages indicating early lineage bias. We used the alkbh5 KO zebrafish model and subjected it to phenyl hydrazine-induced anemia to study its role in stress-induced regeneration. Using FACS analysis of whole kidney marrow, we show red cell recovery failure in alkbh5 KO zebrafish models. Temporal analysis of alkbh5 KO marrow cells during PHZ-induced acute anemia and regeneration showed a systemic failure to resolve SGs compared to controls. We confirmed these results in the mouse ALKBH5 KO model establishing the role of ALKBH5 in stress erythropoiesis. Furthermore, hCD34+ ATXN2 KO combined with Methocult-colony formation assay showed defects in erythroid and myeloid lineages. Super-resolution microscopy of hCD34+ ATXN2 KO cells phenocopied the presence of disease-associated ALS (amyotrophic lateral sclerosis) stress granules with TDP-43, G3BP1/2, PABPC1, and Tia-1 enriched with m6A labeled RNAs. Using the K562 ALKBH5 KO erythropoiesis model and ATXN2 lentiviral overexpression, we effectively resolved stress granules and blocks in erythropoiesis. In summary, m6A marks on ATXN2 mRNA regulate SG biology, affecting HSC differentiation and impacting blood development. SG-mediated regulation of HSC fate may highlight new therapeutic strategies for blood disorders.

Ineffective Erythropoiesis Negatively Impacts the Erythroblastic Island in Sickle Cell Disease

Sickle cell disease (SCD) is a genetic recessive disorder, characterized by painful episodes of vaso-occlusion, chronic hemolytic anemia, and progressive organ failure. It is one of the most common and severe monogenic diseases worldwide. SCD is caused by a point mutation in the β-globin gene, leading to the expression of an abnormal hemoglobin (HbS) that polymerizes under hypoxic conditions, driving red blood cells (RBC) sickling. Peripheral RBCs are extensively studied in SCD, as hemolysis is the major contributor to anemia, but little is known about erythroid differentiation in this pathology. We recently demonstrated the occurrence of ineffective erythropoiesis in the bone marrow (BM) of SCD patients, characterized by the death of a significant proportion of erythroblasts between the polychromatic and the orthochromatic stages. We hypothesize that this increased cell death could negatively impact the central macrophages of the erythroblastic islands (EBIs) as they would engulf high numbers of hemoglobin-rich apoptotic erythroblasts and undergo ferroptosis because of excess iron. To investigate our hypothesis, we used an in vitro system whereby CD14 + monocytes were isolated from peripheral blood and cultured in media supplemented with M-CSF and dexamethasone. This led them to differentiate towards anti-inflammatory tissue macrophages, with an “EBI-like” phenotype, expressing markers such as CD16, CD163, CD169, CD206. Phagocytosis of high numbers of RBCs by “EBI-like” macrophages led to increased cell death, reactive oxygen species, and lipid peroxidation, and to the upregulation of genes involved in ferroptosis pathways such as HO-1 and SLC7A11. This was circumvented by the ferroptosis inhibitors ferrostatin-1 and liproxtatin-1. Furthermore, phagocytosis of apoptotic in vitro-generated SCD erythroblasts also induced ferroptosis in EBI-like macrophages, with increased cell death and lipid peroxidation in a time-dependent manner. Interestingly, while this was the case with hemoglobin-rich, terminally differentiating erythroblasts, phagocytosis of cells without hemoglobin, such as K562 or GPA - progenitor cells did not affect cell viability, indicating that the observed ferroptosis was due to the iron content of the internalized cells. We investigated the effect of increased RBC phagocytosis on the phenotype of surviving macrophages and found an M2 to M1 shift indicating a switch to a pro-inflammatory phenotype under the condition of elevated levels of intracellular iron and oxidative stress. We challenged these findings in vivo using the Townes humanized SCD mouse model. Data on 4-month-old mice showed the expected switch in erythroid production from steady-state erythropoiesis in the BM to a stress erythropoiesis in the spleen. This was associated with a decrease in the number of EBI macrophages (F4/80 HighVCAM+CD169+Ly6G-) in the BM of SS mice when compared to AS and AA mice, and an expansion of red pulp macrophages (RPMs) F4/80 HighCD169+VCAM+. Using a flow cytometry-based assay we found high lipid peroxidation levels in BM EBI macrophages of SS mice as compared to AA, suggesting occurrence of ferroptosis in these cells. Our study suggests that IE in SCD may contribute to anemia, directly by cell death of differentiating erythroblasts, and indirectly by disrupting the erythroblastic island or by inducing its collapse following ferroptosis of the central macrophage. Our work with the Townes mouse model and BM samples from SCD patients will help to elucidate this hypothesis.

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

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.

Rps19 and Rpl5 Play Distinct Roles in hematopoietic Stem Cell maintenance and Erythroid Differentiation

Mutations in ribosomal proteins (RPs) lead to ribosomopathies. Mechanistic basis underlying the failure of erythropoiesis due to mutations in various RPs is yet to be fully defined. Lack of relevant murine models of ribosomapathies has hindered significant progress. To fill this gap, we generated Rps19 and Rpl5 floxed mice using CRISPR/Cas9 technologies and characterized their hematologic phenotype following crossing them to a Vav-iCremice. Vav-iCre;Rps19 fl/+mice are born at expected Mendelian rates with severe macrocytic anemia and reticulocytopenia. These mice die by post-natal day 10 (P10), due to bone marrow failure and absence of splenic stress erythropoiesis. Analysis of fetal erythropoiesis revealed a 40% reduction in the total number of erythroid cells in Vav-iCre;Rps19 fl/+, however erythroblasts at all stages of terminal erythroid differentiation were found. Intriguingly, studies of hematopoietic stem and progenitor cell s demonstrated a progressive exhaustion of the stem cell compartment. scRNAseq studies of Vav-iCre;Rps19 fl/+ fetal livers demonstrated increased expression of RUNX1, a transcription factor implicated in the regulation of ribosome biogenesis. Accordingly, we observed that hematopoietic progenitors of Vav-iCre,Rps19 fl/+had enhanced global protein synthesis along with increased RUNX1 protein, despite ribosomal protein haploinsufficiency demonstrated by polysome profiling. In marked contrast, Vav-iCre;Rpl5 fl/+ mice presented with a more severe phenotype and died perinatally from anemia. Further analysis revealed a failure of erythropoiesis at erythroid progenitor stages in Vav-iCre;Rpl5 fl/+. In striking contrast with the Rps19 model, Vav-iCre;Rpl5 fl/+ mice had an expansion of the hematopoietic stem and progenitor cell compartment that also had an increased protein translation rate . This increased translation occurred in the context of normal expression levels of RUNX1. Interestingly Vav-iCre;Rpl5 fl/+ micehad reduced mTORC1 activity and a dramatic reduction in the expression of other ribosomal proteins. Consistent with previous studies about the pivotal role of mTORC1 in erythropoiesis Vav-iCre,Raptor fl/fl(mTORC1 conditional knockout) had reduced expressions of RPs in fetal liver. In summary, we report different requirements for Rps19 and Rpl5 during fetal hematopoiesis in regulating erythroid progenitor biology. While Rps19 plays an essential role at the HSC level to maintain the generation of erythroid progenitors, Rpl5 is critical once progenitors are committed towards an erythroid fate. Along with the findings that translation is increased in RP haploinsufficient models, our study unravels potential compensatory mechanisms during fetal hematopoiesis.

Stratification of βSβ+ Compound Heterozygotes Based on L-Glutamine Administration and RDW: Focusing on Disease Severity.

Sickle cell disease (SCD) is heterogeneous in terms of manifestation severity, even more so when in compound heterozygosity with beta-thalassemia. The aim of the present study was to stratify βSβ+ patient blood samples in a severity-dependent manner. Blood from thirty-two patients with HbS/β-thalassemia compound heterozygosity was examined for several parameters (e.g., hemostasis, inflammation, redox equilibrium) against healthy controls. Additionally, SCD patients were a posteriori (a) categorized based on the L-glutamine dose and (b) clustered into high-/low-RDW subgroups. The patient cohort was characterized by anemia, inflammation, and elevated coagulation. Higher-dose administration of L-glutamine was associated with decreased markers of inflammation and oxidation (e.g., intracellular reactive oxygen species) and an altered coagulation profile. The higher-RDW group was characterized by increased hemolysis, elevated markers of inflammation and stress erythropoiesis, and oxidative phenomena (e.g., membrane-bound hemoglobin). Moreover, the levels of hemostasis parameters (e.g., D-Dimers) were greater compared to the lower-RDW subgroup. The administration of higher doses of L-glutamine along with hydroxyurea seems to attenuate several features in SCD patients, probably by enhancing antioxidant power. Moreover, anisocytosis may alter erythrocytes' coagulation processes and hemolytic propensity. This results in the disruption of the redox and pro-/anti-inflammatory equilibria, creating a positive feedback loop by inducing stress erythropoiesis and, thus, the occurrence of a mixed erythrocyte population.

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

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