Erythroid Progenitor Cells Research Articles

LAG3+ erythroid progenitor cells inhibit HBsAg seroclearance during finite pegylated interferon treatment through LAG3 and TGF-β

HBsAg seroclearance, the ideal aim of anti-hepatitis B virus (HBV) treatment, cannot be achieved easily. Anemia is another common issue for chronic hepatitis B (CHB) patients, which leads to elevation of erythroid progenitor cells (EPCs) and immune suppression in cancer. This study investigated the role of EPCs in HBsAg seroclearance following pegylated interferon-α (PEG-IFN) treatment. CD45+EPC accumulation in CHB patients and an AAV/HBV mice model was found in the circulation and liver by flow cytometry and immunofluorescence tests. Wright-Giemsa staining showed that these pathological CD45+EPCs presented elevated erythroid cells with relative immature morphologies and atypical cells compared with the control cells. CD45+EPCs were associated with immune tolerance and decreased HBsAg seroclearance during finite PEG-IFN treatment. CD45+EPCs suppressed antigen non-specific T cell activation and HBV-specific CD8+T cells, partially through transforming growth factor β (TGF-β). RNA-seq revealed that CD45+EPCs in patients with CHB presented a distinct gene expression profile compared with CD45−EPCs and CD45+EPCs from cord blood. Notably, CD45+EPCs from patients with CHB expressed high level of Lymphocyte-activation gene 3 (LAG3), an immune checkpoint molecule, and were then defined as LAG3+EPCs. LAG3+EPCs diminished the function of antigen presenting cells through LAG3, which was another mechanism by which LAG3+EPCs’ suppressed HBV-specific CD8+T cells. Anti-LAG3 and anti-TGF-β combination treatment decreased serum HBeAg, HBV DNA levels and HBsAg level, as well as HBsAg-expression in hepatocytes during PEG-IFN treatment in the AAV/HBV mice model. Conclusions: LAG3+EPCs inhibited the efficacy of PEG-IFN treatment on HBsAg seroclearance induced by LAG3 and TGF-β. Anti-LAG3, anti-TGF-β and PEG-IFN combination treatment might facilitate HBV clearance.

Anemia of inflammatory: does eiderr knowledge mean better diagnosis and treatment?

Anemia, which is a manifestation of the deterioration of patients' health and performance, is a common concomitant condition in diseases with signs of inflammation activation. This anemia - anemia of inflammation, is caused by disturbances of iron metabolism that lead to iron retention within macrophages, cytokine mediated inhibition of erythropoietin function and erythroid progenitor cell differentiation, and a reduced erytrocyte half-life. Anemia is usually mild to moderate, normocytic and normochromic. It is characterized by low iron circulation, but normal to increased levels of stored ferritin and the hormone hepcidin. The primary therapeutic approach is the treatment of the underlying inflammatory disease. In case of failure, iron supplementation and / or treatment with erythropoietin stimulating agents may be used. Blood transfusions are just an emergency treatment for life-threatening anemia. A new treatment modalities with hepcidin-modifying strategies and stabilizers of hypoxia inducible factors is emerging. However, their therapeutic efficacy needs to be verified and evaluated in clinical trials.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

RNA-regulatory exosome complex suppresses an apoptotic program to confer erythroid progenitor cell survival invivo.

The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors invitro. To determine if this mechanism operates invivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis invivo.

Iron status in chronic inflammatory disease: therapeutic implications.

Anemia of inflammation (AI) is a very frequent clinical condition affecting globally more than a billion people with chronic inflammatory disorders, such as chronic kidney disease, heart failure, and inflammatory bowel disease. It is usually associated with iron deficiency (ID), which imposes a severe additional burden on the recovery from the primary disease. The pathophysiology of iron dysregulation that may ultimately lead to absolute iron deficiency anemia (IDA) during inflammation is multifactorial and includes reduced iron absorption in the bowel, iron retention in macrophages of the reticuloendothelial system, reduction in circulatory half‑life of erythrocytes, inadequate production and activity of erythropoietin, and impaired proliferation and differentiation of erythroid progenitor cells. These result in hypoferremia and iron-restricted erythropoiesis. AI is mostly mild to moderate, normochromic and normocytic with normal or even increased ferritin levels. The current treatment options for AI include iron replacement therapy, treatment with erythropoiesis‑stimulating agents, and red blood cell transfusion. ID management is based on oral or intravenous iron preparations. Given the pathophysiology, treatment with oral iron, although widely used, presents several limitations that impact its effectiveness in patients with chronic inflammatory conditions. Instead, intravenous iron preparations are a valuable option for patients with chronic inflammatory diseases, as they overcome reduced bowel absorption. Novel therapeutic approaches include downregulation of hepcidin synthesis and function, and stabilization of the hypoxia‑inducible factor via inhibition of prolyl hydroxylase domain. Several studies in vitro and in vivo are ongoing; however, the results in humans are still elusive.

Tracking of Human Parvovirus B19 Virus-Like Particles Using Short Peptide Tags Reveals a Membrane-Associated Extracellular Release of These Particles.

B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.

Immunophenotypical profiling of myeloid neoplasms with erythroid predominance using mass cytometry (CyTOF).

Acute erythroid leukemia (AEL) is a disease continuum between Myelodysplastic syndrome (MDS) and Acute myeloid leukemia (AML) with the cellular hallmark of uncontrolled proliferation and impaired differentiation of erythroid progenitor cells. First described by Giovanni di Guglielmo in 1917 AEL accounts for less than 5% of all de novo AML cases. There have been efforts to characterize AEL at a molecular level, describing recurrent alterations in TP53, NPM1 and FLT3 genes. A genomic analysis of AEL cases confirmed its complexity. Despite these advances, the biology underlying erythroid proliferations remains unclear and the prognosis is dismal with a median survival of only 3months for pure erythroid leukemia (PEL). Marker combinations suitable for the identification and characterization of leukemic stem cell (LSC) candidates, monitoring measurable residual disease (MRD) during chemotherapy treatment and the development of innovative targeted therapies are missing. Here, we developed a mass cytometry panel for an in-depth characterization of erythroid and myeloid blast cell populations from human AEL bone marrow samples in comparison to other AML subtypes and healthy counterparts. A total of 8 AEL samples were analyzed and compared to 28 AML samples from different molecular subtypes, healthy bone marrow counterparts (n=5) and umbilical cord blood (n=6) using high-dimensional mass cytometry. Identification of erythroid and myeloid blast populations in high-dimensional mass cytometry data enabled a refined view on erythroblast differentiation stages present in AEL erythroid blasts and revealed immunophenotypical profiles specific to AEL. Profiling of phenotypic LSCs revealed aberrant erythroid marker expression in the CD34+ CD38- stem cell compartment. In addition, the identification of novel candidate surface marker combinations and aberrancies might enhance clinical diagnostics of AEL. We present a high-parameter mass cytometry approach feasible for immunophenotypical analysis of blast and stem cell populations in myeloid neoplasms with erythroid predominance laying the foundation for more precise experimental approaches in the future.

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD=103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

Erythroid-transdifferentiated myeloid cells promote portal vein tumor thrombus in hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is primarily characterized by a high incidence of vascular invasion. However, the specific mechanism underlying portal vein tumor thrombus (PVTT) in HCC remains unclear. As a consequence of myeloid cell developmental arrest, CD71+ erythroid progenitor cells (EPCs) and myeloid-derived suppressor cells play important roles in HCC; however, their roles in PVTT remain unclear. Methods: The role of CD71+ EPCs in the HCC tumor microenvironment (TME) was evaluated via morphological, RNA-sequencing, enzyme-linked immunosorbent assay, and flow cytometric analyses. Co-culture techniques were employed to assess the CD45+ EPCs and their vascular compromising effect. Additionally, the PVTT-promoting function of CD45+ EPCs was explored in vivo in a murine model. Results: The CD45+EPCs in HCC tissues exhibited increased myeloid cell features, including morphology, surface markers, transforming growth factor (TGF)-β generation, and gene expression, compared with those in circulation. Hence, a large proportion of CD45+EPCs, particularly those in TMEs, comprise erythroid-transdifferentiated myeloid cells (EDMCs). Additionally, the expression of C-C chemokine receptor type 2 (CCR2) mRNA was upregulated in CD45+EPCs within the TME. Tumor macrophages from HCC tissues induced substantial migration of CD45+EPCs in a dose-dependent manner. Meanwhile, results from immunofluorescence analyses revealed that these two cell types are positively associated in the TME and circulation. That is, EDMCs are chemoattracted by HCC macrophages mainly via CCR2 from CD45+ EPCs in the circulation. Additionally, the expressions of FX, FVII, FGB, C4b, CFB, and CFH were elevated in CD45+EPCs within the TME compared with those in the spleen. The CD45+EPCs from the HCC TME promoted vessel endothelial cell migration and compromised tube formation through TGF-β and FGB, respectively. Additionally, CD45+EPCs from the TME induced HCC cell migration. HCC macrophage-induced CD45+EPCs to exhibit higher levels of FX, FVII, FGB, and TGF-β. Meanwhile, upregulation of CCAAT/enhancer binding protein beta expression induced FGB and TGF-β generation in CD45+EPCs in the TME. WTAP, a major RNA m6A writer, stabilized FX and FVII mRNA and enhanced their nuclear export in CD45+EPCs from the TME. CD45+EPCs from the TME were positively associated with PVTT and poor prognosis. Splenectomy reduced the level of CD45+EPCs in the circulation and TME, as well as the incidence of microvascular invasion. The incidence of microvascular invasion increased following the transfer of HCC tissue CD45+EPCs to splenectomized HCC-bearing mice. Conclusions: The CD45+EPCs enriched in the HCC microenvironment are EDMCs, which are induced by HCC macrophages to migrate from the circulation to the TME. Subsequently, EDMCs promote PVTT by compromising the blood vessel endothelium, aggravating coagulation, and promoting HCC cell migration.

In vitro production of red blood cells for future transfusion medicine

Large-scale in vitro red blood cell (RBC) production has been attempted in recent years. Potential cell sources for RBC production include hematopoietic stem/progenitor cells, pluripotent stem cells, and immortalized erythroid progenitor cell lines, which can induce enucleated RBCs with characteristics such as oxygen-carrying capacity and deformability. A phase I clinical study of cultured RBCs produced from hematopoietic stem/progenitor cells has revealed a similar in vivo half-life between cultured and native RBCs. Thus, the application of cultured RBCs in blood transfusion is gradually advancing. However, a single transfusion requires a large number of cells, unlike other cell therapies. Therefore, developing a method to mass-produce RBCs from a small culture volume at a low cost is important in the future. This review summarizes the current status and prospects concerning in vitro RBC production using each cell source, which can improve future transfusion medicine.

The role of erythrocytes and erythroid progenitor cells in tumors.

In the current research context of precision treatment of malignant tumors, the advantages of immunotherapy are unmatched by conventional antitumor therapy, which can prolong progression-free survival and overall survival. The search for new targets and novel combination therapies can improve the efficacy of immunotherapy and reduce adverse effects. Since current research targets for immunotherapy mainly focus on lymphocytes, little research has been done on erythrocytes. Nucleated erythroid precursor stem cells have been discovered to play an essential role in tumor progression. Researchers are exploring new targets and therapeutic approaches for immunotherapy from the perspective of erythroid progenitor cells (EPCs). Recent studies have shown that different subtypes of EPCs have specific surface markers and distinct biological roles in tumor immunity. CD45+ EPCs are potent myeloid-derived suppressor cell-like immunosuppressants that reduce the patient's antitumor immune response. CD45- EPCs promote tumor invasion and metastasis by secreting artemin. A specific type of EPC also promotes angiogenesis and provides radiation protection. Therefore, EPCs may be involved in tumor growth, infiltration, and metastasis. It may also be an important cause of anti-angiogenesis and immunotherapy resistance. This review summarizes recent research advances in erythropoiesis, EPC features, and their impacts and processes on tumors.

Blf and the drl cluster synergistically regulate cell fate commitment during zebrafish primitive hematopoiesis.

Hematopoiesis is a highly coordinated process that generates all the body's blood cells, and perturbations in embryonic hematopoiesis may result in illnesses ranging from fetal anemia to various leukemias. Correct establishment of hematopoietic progenitor cell fate is essential for the development of adequate blood cell subpopulations, although regulators of cell fate commitment have not been fully defined. Here, we show that primary erythropoiesis and myelopoiesis in zebrafish embryos are synergistically regulated by blf and the drl cluster, as simultaneous depletion led to severe erythrocyte aplasia and excessive macrophage formation at the expense of neutrophil development. Integrative analysis of transcriptome- and genome-wide binding data revealed that blf and drl cluster genes are responsible for constraining the expression of vasculogenesis-promoting genes in the intermediate cell mass and monocytopoiesis-promoting genes in the rostral blood island. This indicates that blf and drl cluster genes act as determinants of the fate commitment of erythroid and myeloid progenitor cells. Furthermore, a rescue screen demonstrated that Zfp932 is a potential mammalian functional equivalent to zebrafish blf and drl cluster genes. Our data provide insight into conserved cell fate commitment mechanisms of primitive hematopoiesis.

Deficiency of ribosomal protein S26, which is mutated in a subset of patients with Diamond Blackfan anemia, impairs erythroid differentiation.

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis. Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells. Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

A Novel Autosomal Dominant JAK2R715T Mutation Causes Polycythemia Vera in 3 Generations

Polycythemia vera (PV) has high hemoglobin (Hb) concentration caused by clonal proliferation of myeloid cells from a pluripotent stem cell (Prchal, N Engl J Med. 1974). Acquired somatic mutations of JAK2 gene, JAK2V617F and rarely exon 12, are present in virtually all PV patients (Ugo, Nature 2005, Scott N Engl J Med. 2007). The thromboses are the major causes of PV-related morbidity and mortality. A 38-year female suffered an ischemic stroke in 2017 at age 32 with negative thrombophilia work-up. She had elevated hemoglobin (Hb) 18 g/dL with intermittent elevation of leukocytes and normal platelets with borderline low erythropoietin (EPO) levels. JAK2V617F and exon 12 mutation analysis were negative. Her marrow was interpreted as compatible with PV and normal cytogenetics. Her hematologist treated her with phlebotomies. She was referred to us for an additional evaluation and management. Myeloid next generation sequencing (NGS) revealed a previously undescribed mutation JAK2R715T in the pseudokinase domain (JH2) with allelic burden ~50%. Some of her erythroid progenitors (BFU-Es) grew without extrinsic EPO, a hallmark of PV (Prchal, N Engl J Med. 1974). Phlebotomies were stopped; in two months, her Hb had risen to 17.4 g/dL and hematocrit had changed to 48.5% with normal leukocytes and platelets. We obtained JAK2 inhibitor fedratinib on compassionate basis from the manufacturer. On fedratinib, her Hb normalized to 15.2 g/dL with 42.8 % hematocrit. Her iron deficiency was corrected without increase of her Hb. The patient's germline mutation was confirmed by DNA sequencing of her nails. We encouraged her to have her only son and parents tested by NGS. The same JAK2R715T mutation was found in her 10-year-old son's blood sample. He also has elevated Hb at 16.2 g/dL (normal for age 11.5-15.5 g/dL), normal leukocytes and platelets were 400 K/µl. Her asymptomatic 63-year-old mother’ Hb was 17 g/dL, hematocrit 50.5%, and sequencing of her JAK2 also revealed JAK2R715T at ~50%. To assess how the R715T mutation may affect kinase activation, the JH1-JH2 domains of JAK2R715T and JAK2WT were modelled using AlphaFold 2.0 (Jumper, Nature 2021). The overall structure of JAK2R715T JH2 was more rigid than that of JAK2WT, in agreement with DynaMut in silico prediction (PMID: 29718330). -Helix C is shown in dark blue, Y570 and T555 in yellow, and ATP and T715 in red. Detailed comparison of JAK2R715T with JAK2WT JH2 (see Figure) indicated that the rigidification of the structure involved notably residues of the JH2-JH2 trans-dimerization domain centered around -Helix C. This has been suggested to mediate direct JH2-JH2 contacts (Wilmes, Science 2020) and a lengthening of the ß-sheet region surrounding the inhibitory phosphorylation site Tyr570 (Echarri, Mol Cell Biol. 2004). The R715 is in direct vicinity of the ATP binding pocket and promotes a closed lobe conformation through interaction with Thr555 of the nucleotide-binding loop (Bandaranayake, Nat Struct Mol Biol. 2012). We suggest that loss of this interaction may result in a more opened lobe conformation and disturbing of ATP binding to JH2, which regulates JAK2 kinase activity (Hammarén, Proc Natl Acad Sci USA 2015). To evaluate the effect of JAK2R715T in erythroid progenitor cells, the patient's erythroid progenitors derived from peripheral mononuclear cells were grown for 21 days with and without JAK inhibitor (ruxolitinib) following the previously published in vitro expansion protocol (Bruchova, Blood Cells Mol Dis, 2009). Compared to PVs and healthy controls, the patient cells showed increased proliferation. JAK inhibitor ruxolitinib treatment of patient cells induced decreased proliferation and differentiation compared to untreated cells. Assessment of STAT5 phosphorylation on patient's blood cells confirmed increased JAK2 activity to levels even higher than this of patients with confirmed JAK2V617F positive PV. X-chromosome based clonality studies of both females are ongoing. The EPO-independent colonies of her BFU-E is being evaluated for possible acquired conversion to homozygosity by uniparental disomy that is present in PV (Kralovics, Experimental Hematology, 2002). We conclude that this is the first example of dominantly inherited PV phenotype in 3 generations due to a novel germline JAK2R715T gain-of function mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunoregulatory CD71+ Erythroid Cells (CECs) Expand in Multiple Myeloma and Impair Control of L. Monocytogenes Infection

Introduction Although the clinical management of multiple myeloma (MM) patients has significantly improved over recent years, MM remains a largely incurable disease. One of the most common complication of MM, which correlates with the worse clinical outcome, is anemia. Moreover, MM patients are prone to develop life-threatening infections mainly due to the disease-related impairment of the immune system function. Recently, CD71+ erythroid progenitor and precursor cells (CECs) arose as a relevant immunomodulatory population. CECs have been shown to create an immunosuppressive microenvironment via multiple mechanisms including arginase 2 (ARG2), PDL-1, and TGF-beta. It was recently observed in our laboratory that mice bearing late-stage tumors are characterized by the presence of an increased percentage of CECs in their blood and spleens. Moreover, the tumor-associated CECs show elevated levels of arginases. The abovementioned facts may suggest an important role of CECs and their related mechanisms in compromising the immune response in MM. Methods Vk*MYC MM model Recipient C57BL/6 (wild type or ARG2 KO) mice were injected via tail vein with 1 × 106 Vk*MYC cells. M-spike levels were evaluated with serum protein electrophoresis. Five weeks later they were challenged with L. monocytogenes. L.monocytogenes infection and bacterial burden evaluation Mice were injected intraperitoneally with 1 x 103L. monocytogenes cells (strain EGDe). Bacterial burden was analyzed 72h and 7 days post infection. Briefly, spleens and livers of sacrificed mice were isolated, weighed, and homogenized. The homogenates were serially diluted and plated on culture plates. Bacterial burden was calculated using the number of colonies and the dilution factor. Flow cytometry analysis Flow cytometry analyses (immunophenotyping of spleen and bone marrow (BM) cells as well as cytokine beads arrays, Legenplex, BioLegend) were performed using BD Fortessa X-20 flow cytometer operated by BD FACSDiva Software ver. 8.0. The acquired data were re-analyzed using FlowJo™ ver. 10 software. Arginase inhibitor OAT-1746 arginase inhibitor (Molecure SA) was administered intraperitoneally at doses of 5 or 20 mg/kg twice daily, starting from the second or the fifth day before the bacterial challenge, respectively. The treatment was continued until the end of the experiment. Results In Vk*MYC-bearing mice we observed an accumulation of CECs in the blood and spleen in time, correlating with the development of anemia. At the late stage of the disease MM-associated CECs showed less differentiated phenotype and increased expression of arginase-1 (ARG1) and ARG2. MM-associated CECs inhibited T cells activation and proliferation in an ARG and reactive oxygen species-dependent manner. Interestingly, MM development was delayed in ARG2 KO vs WT mice. In the BM aspirates of MM patients we observed increased percentage of CECs expressing high levels of both ARG1 and ARG2 comparing with healthy controls. Moreover, MM impaired control of L. monocytogenes infection as we observed an elevated bacterial burden in the livers and spleens of Vk*MYC-bearing vs control mice. Infected MM-bearing mice had fewer NK cells and activated CD4+ and CD8+ T cells in the spleen than infected control mice. Furthermore, MM altered the dynamics of cytokines involved in the control of intracellular infection. 24h post Listeria challenge we observed their significant decrease followed by an increase at 72h comparing to infected non-MM mice. These results indicate that MM-bearing mice have delayed initial immune response leading to increased bacterial burden followed by excessive cytokines response resulting in sepsis-like condition leading to increased mice mortality. Finally, ARG inhibitor decreased L. monocytogenes burden in MM-bearing mice. Altogether, our results show that accumulation of CECs during MM progression leads to impaired bacterial infection control, at least partially dependent on ARG2 activity. Funding: National Science Centre in Poland [grants No 2017/25/B/NZ6/01139 and 2019/35/B/NZ6/00540]; Polish Ministry of Education and Science [grant No 013/RID/2018/19 (Regional Initiative for Excellence, project budget 12,000,000 PLN)].

Development of the Novel Cytosine Analog Aza-T-Dcyd (AB1) As a Fetal Hemoglobin Inducer to Treat Sickle Cell Disease.

Introduction: Induction of fetal hemoglobin (HbF) expression in sickle cell disease (SCD) patients using small molecules is considered an effective therapeutic strategy to ameliorate severe clinical phenotypes. With this objective in mind, several drug molecules have been tested for HbF induction. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. Studies have reported that DNA methyltransferase inhibitors such as decitabine stimulate γ-globin gene expression through DNA hypomethylation. However, decitabine has cytotoxic side effects and is most effective by intravenous administration. Thus, it is critical to develop alternative safe and orally active hypomethylating agents for inducing HbF. Our lab in collaboration with AkiraBio, conducted preclinical in vitro studies to assess a novel cytosine analog Aza-T-dCyd (AB1), for its ability to induce HbF in human K562 cells and erythroid progenitor cells derived from SCD patients. Methods: K562 cells were cultured in Iscove's Modified Dulbecco's Eagle medium, 10% fetal bovine serum and penicillin/streptomycin. The human erythroid progenitors were generated from peripheral blood mononuclear cells isolated from two SCD patients under IRB exempt protocol as previously established in our lab (Li et al., Haematologica. 2018). The cultured K562 cells were treated either with AB1 alone at gradually increasing concentrations from 3nm to 200nm, decitabine (0.2µM), hydroxyurea (HU100µM) or combined AB1 with HU for 48 hours. The cultured sickle erythroid progenitors were treated with AB1 on day 8 and then harvested on day 10. The harvested cells were analyzed for cell viability and flow cytometry to measure HbF positive cells (F-cells). The γ-globin and β-globin mRNA levels were quantified by qRT-PCR and the protein levels of HbF and DNMT-1 were detected by Western blot technique. Results: Initial studies to evaluate drug toxicity were completed. We observed higher cell viability of K562 cells treated with AB1 10nM and 30nM compared to untreated and HU 100µM (p=0.005) treated cells. Hence, subsequent experiments were conducted with the lower AB1 concentrations. By flow cytometry analysis, AB1 treatment significantly increased F-cells from 13.95% to 20.11% and 22.46% in 10nM and 30nM treated cells (p=0.0002) respectively. Moreover, mean fluorescence intensity analysis showed significant HbF expression in AB1 treated cells compared to untreated cells (p=0.00001). At the transcription level, AB1 increased γ-globin mRNA levels 1.4-fold compared to untreated cells. K562 cells treated with a combination of AB1 and HU at their maximum response concentrations (AB1 10 and 30nM; HU 100 µM) did not show additive effects on F-cells or γ-globin mRNA expression. Other combination concentrations were not assessed. In subsequent studies we investigated the HbF inducing capacity of AB1 in human erythroid progenitors generated from SCD patients (N=2). Normal cell viability was observed in sickle progenitors at 3nm, 6nM and 10nm AB1 treatment. The F-cell level increased from 16.21% to 27.68% at 10nm AB1 (p=0.02). By qRT-PCR assay, a significant 2-fold increase in γ-globin mRNA level was observed in AB1 treated erythroid cells, when normalized with total γ-globin and β-globin mRNA; β globin mRNA levels were unchanged. Finally, when we analyze the effects of AB1 on protein levels of HbF and DNMT-1 by Western blot analysis, 10nM and 30nM AB1 induced HbF and inhibited DNMT-1 expression the sickle erythroid progenitors. Conclusion: Our study findings demonstrate that AB1 induced γ-globin transcription in K562 cells and human sickle erythroid progenitors. Studies to test the effects of AB1 in the Townes SCD mouse model are in progress.

GATA1-Dependent Ceramide Homeostasis Controls Cytokine Signaling and Erythroid Cell Function

Our multi-omic studies in cultured erythroblast cells (G1E-ER-GATA1) revealed that GATA1 regulates the synthesis and/or transport of small molecules, including heme, metal ions, and nucleosides (Liao et al. Cell Rep. 2020, Tanimura et al. Dev. Cell 2018, Zwifelhofer et al. PLoS Genet. 2020). These cells are GATA1-null proerythroblast-like cells stably expressing a β-estradiol-induced allele encoding GATA1 fused to the hormone binding domain of estrogen receptor. Analysis of the GATA1-regulated transcriptome and proteome in G1E-ER-GATA1 cells revealed multiple differentially-expressed components involved in sphingolipid biosynthesis. Sphingolipids are a group of signaling lipids, including ceramides, which regulate cell proliferation and apoptosis, and sphingosine-1-phosphate that controls vascular homeostasis. One of these genes, Degs1, encodes dihydroceramide desaturase (DES) that converts dihydroceramides to ceramides. GATA1 upregulates Degs1 mRNA expression by 12.2-fold and DEGS1 protein by 4.6-fold. GATA1 occupies the promoter and intronic sequences of murine Degs1 locus, suggesting that Degs1 is a GATA1 target gene. Quantitative lipidomics revealed that intracellular ceramide, together with sphingosine and sphingosine-1-phosphate, increased during erythroid maturation, whereas dihydroceramide did not change significantly. To determine the functional significance of GATA1-induced Degs1 expression, we chemically inhibited DES activity with 4-hydroxyphenyl retinamide (4HPR) in β-estradiol-induced G1E-ER-GATA1 cells and quantified sphingolipids using quantitative lipidomics. 4HPR increased dihydroceramide 7.8-fold at 24 h and 14-fold at 48 h, while ceramide was reduced 1.7 and 1.9-fold, respectively, which was associated with reduced cell growth, hemoglobinization, and GATA1-dependent gene activation. Treating primary fetal liver HSPCs with 4HPR significantly reduced the number of CFU-E by 4.8-fold (p<0.0001) and BFU-E by 4.8-fold (p<0.0001), but not CFU-GM. Furthermore, treating the cells with C6-dihydroceramide (C6-dhCer) or C6-ceramide (C6-Cer), which elevates intracellular ceramides, strongly impaired erythroid, but not myeloid, colony formation. Thus, disrupting dihydroceramide or ceramide homeostasis is deleterious to mouse erythroid progenitor cells. To elucidate the mechanistic underpinnings of the erythroid cell hypersensitivity to (dh)ceramides, we asked if disrupting ceramide homeostasis impacts stem cell factor (SCF) and erythropoietin (Epo) signaling, which are essential for erythroid cell proliferation, survival, and differentiation. Inhibiting DES with 4HPR or treating the cells with C6-dhCer or C6-Cer significantly reduced SCF-dependent induction of p-AKT and p-ERK and Epo-dependent induction of p-AKT, p-ERK, and p-STAT5, suggesting that both SCF and Epo signaling are inhibited by (dh)ceramides. Short-term C6-Cer treatment impaired Epo-dependent activation of p-AKT and p-ERK, but not p-STAT5, suggesting a differential sensitivity of downstream signaling pathways. In addition, C6-Cer reduced SCF-induced p-AKT without affecting activation of its receptor, c-Kit, indicating that ceramides inhibit cytokine signaling post-receptor activation. Since ceramide activates protein phosphatase PP2A in vitro, we asked if PP2A mediates ceramide-dependent inhibition of cytokine signaling. PP2A inhibition with okadaic acid rescued ceramide-dependent reduction of p-AKT and p-ERK. Furthermore, two distinct PP2A activators, FTY-720 and DT-061, impacted SCF and Epo-dependent signaling similar to C6-Cer, suggesting that ceramide inhibits cytokine signaling via activation of PP2A. Finally, we demonstrated that Epo-dependent GATA1 phosphorylation is impaired by 4HPR or C6-Cer. These findings establish a paradigm in which GATA1 controls sphingolipid biosynthetic enzymes and the generation of regulatory lipids that restrict cytokine signaling, thereby impacting erythrocyte development and function. As inflammatory mediators implicated in anemia of inflammation elevate dihydroceramides and ceramides, we propose that the integrated transcriptional, metabolic and signaling mechanism has broad importance in erythroid biology and pathology.

Anemia-Activated Cis Regulatory Requirements for Erythroid Regeneration

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. Whether anemia-activated transcription and underlying usage of cis-regulatory elements to control gene expression are unique to anemia or overlap with physiological erythropoiesis is an unresolved question regarding anemia response mechanisms. We previously discovered that a cis-regulatory transcriptional enhancer at the Samd14 locus (S14E), defined by a CANNTG-spacer-AGATAA (named E-box-GATA) composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. Our working model proposes that the S14E is part of an ensemble of similar anemia-responsive cis-elements promoting erythroid precursor activities. To determine whether loci containing a cohort of S14E-like cis-elements are transcriptionally upregulated in anemia, we conducted single cell RNA-sequencing of Kit+ cells isolated from mouse spleen during the recovery phase of acute anemia. Single-cell regulatory network inference and clustering (SCENIC) analysis - used to predict transcription factors involved in regulatory mechanisms - revealed that anemia-activated transcripts co-expressed with Gata1 and Tal1 were localized in specific populations of erythroid cells. We identified 65 anemia-activated genes associated with GATA1/TAL1-occupied S14E-like cis-elements including Samd14, suggesting common transcriptional mechanisms are involved in restoring homeostasis. Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), we found the chromatin accessibility of S14E-like cis-elements associated with synovial sarcoma X-2 interacting protein (Ssx2ip) and tubulin tyrosine ligase-like12 (Ttll12) was significantly increased in anemia. To test S14E-like function in transcriptional activation, we targeted these sequences using sgRNA-guided Cas9 and dCas9-KRAB in mouse and human erythroid cell lines (G1E, K562 and HUDEP-2). Both SSX2IP and TTLL12 S14E-like enhancers were required for maximal gene transcription. These data demonstrate that a cohort of S14E-like cis-elements promote anemia-activated gene expression. Ssx2ip is a poorly studied gene that is upregulated by anemia in erythroid precursors (19.2-fold, p<0.0001). To connect cis-regulatory activation in anemia with erythroid regeneration, we next tested Ssx2ip function in erythroid precursor cells isolated from a mouse model of acute anemia. shRNA mediated knockdown of Ssx2ip decreased the percentage of early stage (CD71lowTer119-) erythroid precursors 4.4-fold (p=0.001). Ssx2ip knockdown decreased burst-forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) colony forming potential of erythroid precursors by 1.3-fold (p=0.008) and 1.6-fold (p<0.0001), respectively. To investigate a possible role for Ssx2ip in cell proliferation, we conducted BrdU incorporation and cell proliferation assays. These experiments revealed that Ssx2ip is an important determinant of erythroid progenitor cell cycle progression, and proliferation in anemia. Together, we provide rigorous evidence that S14E-like cis-elements associate with anemia-activated genes, activate gene expression, and that the anemia-activated Ssx2ip gene plays important roles in erythroid precursor cells. Our results highlight a common mechanism of gene activation that regulates erythroid regeneration via S14E-like enhancers. Ongoing efforts will establish mechanisms of Ssx2ip activities in erythropoiesis and functional requirements of additional cohorts of S14E-like enhancers involved in gene regulation during anemia recovery. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, and anemia recovery. Given the role of Ssx2ip in erythroid regeneration, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia.

Exploring the Functional Role of an Alternative mRNA Transcript of the EPO Gene

Erythropoietin (EPO) is the key regulator of erythropoiesis and stimulates the proliferation and differentiation of erythroid progenitor cells into mature erythrocytes. In a previous study, we identified a frame-shift mutation in the EPO gene that causes familial erythrocytosis with elevated erythropoietin (EPO) levels by initiating excess production of EPO protein from an alternative EPO mRNA [1]. This alternative non-coding mRNA is transcribed from a promoter (P2) located in intron 1, whereas the full length mRNA that encodes the EPO protein is transcribed from the P1 promoter located upstream of exon 1. Large parts of intron 1 sequences, including the P2 transcript are conserved among different species (Figure 1A), suggesting that the P2 transcript could have an as yet unknown physiological function. To study the putative function of the P2 transcript, we performed experiments in a human cell line, Hep3B, which can produce EPO. We used the CRISPR/Cas9 system to generate mutant Hep3B clones with targeted deletions in intron 1 of the EPO gene. Close to the P2 transcription start site in intron 1, we identified three GATA-consensus motifs that might act as promoter elements for the P2 transcript. In the first round of CRISPR/Cas9 mutagenesis, we targeted the first two GATA elements and expected to knock down the expression of the P2 transcript. Targeted Hep3B cells were sorted and deposited as single cells to derive clones that were genotyped by next generation sequencing. We obtained 2 clones with homozygous deletions in EPO intron 1 and one clone that was compound heterozygous (Figure 1A). In these Hep3B clones we assessed EPO mRNA expression by qPCR and EPO protein levels in the supernatant by ELISA (Figure 1B). Unexpectedly, the P2 transcript was still detectable in all three clones carrying deletions in intron 1 that eliminated GATA boxes 1 and 2 (data not shown). EPO P1 mRNA expression and EPO protein levels in the supernatant were slightly reduced in most of the clones. These data indicate that the GATA box 1 and 2 motifs are not required for generating the P2 transcript. We have now performed a second round of CRISPR/Cas9 mutagenesis. In one series we targeted all three GATA-motifs through a 186 bp deletion, and in a second series we targeted a highly conserved region upstream of the GATA boxes by a 93 bp deletion (Figure 1A). We obtained 4 clones homozygous for the 93 bp deletion and the clones for the 186 bp deletion are currently being expanded. The analysis of EPO mRNA and protein in these additions CRISPR/Cas9 clones will be presented. Our study shows that the GATA motifs 1 and 2 are not required for function of the P2 promoter in intron 1 of the EPO gene. However, deleting these two motifs in two different cell clones decreased the P1 full-length EPO mRNA expression and EPO protein production in Hep3B cells by 70.2% and 37.2%, respectively. Additional clones with deletion of all three GATA motifs and a highly conserved region upstream of the GATA boxes will likely provide more insights into the physiological role of the P2 transcript.