Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-19
Author(s):  
Miao Lin ◽  
Vijay Menon ◽  
Raymond Liang ◽  
Tasleem Arif ◽  
Laura Breda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Double Mutant ◽  
Complete Blood Count ◽  
Globin Gene ◽  
Mutant Mice ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Wild Type ◽  
The Status

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p<0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking >=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

Metabolic Pathways Control Normal and Beta-Thalassemic Erythroid Cell Maturation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 369-369 ◽  
Author(s):  
Geníis Camprecióos ◽  
Xin Zhang ◽  
Valentina D'Escamard ◽  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Metabolic Pathways ◽  
Research Funding ◽  
Mtor Signaling ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Abstract 369 Erythroid cell maturation requires the integration of erythropoietin receptor (EpoR)-mediated signaling pathways and transcriptional programs of erythroid cell proliferation and differentiation largely orchestrated by GATA-1 and its transcriptional partners. Although red blood cells (RBC) purely rely on glycolysis for energy production, and metabolic processes specifically autophagy have been implicated in erythroid maturation, the potential involvement of metabolic pathways in the control of erythroid cell production is not known. Foxo3 transcription factor is a direct target of GATA-1, functionally regulated by EpoR signaling and essential for the redox regulation of erythropoiesis. Mammalian target of rapamycin (mTOR) kinase is a critical regulator of metabolic processes. We have found that generation and cycling of early erythroid precursors is controlled by a redox-mediated Foxo3-mTOR signaling. Terminal erythroid maturation is specifically compromised in Foxo3-deficient mice. Terminal maturation involves nuclear condensation, enucleation, mitochondrial clearance and complete adoption (or conversion) of glycolytic pathway by erythroid cells. We found RBC to be significantly decreased in Foxo3 mutant bone marrow and peripheral blood. This was in contrast to the increase of polychromatophilic erythroblasts associated with an increase in the total TER119+ population in Foxo3 mutant bone marrow likely reflecting a compensatory mechanism. Notably, using DRAQ5, a DNA-binding fluorescent dye, we found the rate of erythroblasts' enucleation in Foxo3 mutant mice to be significantly compromised. During erythroid maturation Riok3 and Mxi1 transcripts encoding for two important regulators of fetal liver erythroid enucleation are highly upregulated in the bone marrow and robustly expressed in the adult normoblasts and reticulocytes. In agreement with defective enucleation expression of both Riok3 and Mxi1 is highly reduced in Foxo3 mutant erythroblasts and reticulocytes. Interestingly, a two-week in vivo treatment of wild type (WT) and Foxo3−/− mice with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, increased significantly the rate of enucleation in a Foxo3-dependent manner, suggesting that mTOR requires Foxo3 activity in supporting erythroid cell maturation. Importantly, targeting mTOR ameliorates beta-thalassemia as inhibition of mTOR signaling by rapamycin treatment improved erythroid cell maturation in the bone marrow, resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase) as well as significant reduction in reticulocyte production of beta-thalassemic intermedia (th3/+) mice. Combination of thiazole orange (an RNA and DNA probe) with DRAQ5 determined that in addition to enucleation, the relative production of reticulocytes is also decreased significantly in Foxo3 mutant bone marrow. Strikingly, 6,1% of RBC (CD71 negative) in the peripheral blood of Foxo3−/− animals contained mitochondria (CD71−Mito+) as compared to 0.7 % of wild type RBC. Autophagy is strongly implicated in late stage erythroid cell maturation and mitochondrial removal from reticulocytes. In agreement with a function for Foxo3 in control of mitochondrial removal, expression of Ulk1 (Atg1) and Nix (Bnip3l) both regulators of mitochondrial clearance via autophagy was highly downregulated in Foxo3 mutant normoblasts and reticulocytes. The expression of Nix was notable since Nix was upregulated over 40 fold in wild type but not in Foxo3 mutant reticulocytes as evaluated by the Fluidigm™ microfluidics array technology. These results are consistent with the expression pattern of Foxo3 that is highly upregulated with erythroid maturation and is the most highly expressed in normoblasts. Collectively our results indicate that Foxo3 has a key function in the regulation of terminal erythroid cell maturation. They also suggest that rapamycin may be considered for the treatment of beta-thalassemia. These results are consistent with the model of FOXO3a induction during late human erythroid cell maturation. Based on these studies we propose that Foxo3 coordinates metabolic pathways with the transcriptional program of terminal erythroid cell maturation. Understanding this metabolic program is likely to impact efficient RBC production in culture. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Loss of Foxo3 Reduces Erythroblast Apoptosis and Enhances RBC Production in Beta-Thalassemic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 756-756 ◽  
Author(s):  
Raymond Liang ◽  
Genís Campreciós ◽  
Carolina L. Bigarella ◽  
Saghi Ghaffari
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Mutant Mice ◽  
Mrna Levels ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Qrt Pcr

β-thalassemia arises as a result of mutations in the β-globin gene. As a consequence erythropoiesis, the process that insures the daily generation of billions of red blood cells (RBCs), becomes disrupted. Ineffective erythropoiesis is a major contributor to the β-thalassemic anemia and is partially due to aberrant apoptosis during late stages of erythroid maturation. Despite the importance of apoptosis, the underlying molecular mechanisms regulating this process in β-thalassemia erythroblasts are not fully elucidated. One potential mechanism involves the transcription factor Foxo3, which under specific contexts can act as a positive regulator of apoptosis, but is also an essential transcriptional regulator of terminal erythroblast maturation. Foxo3 has a range of outputs that it can execute from sustaining cellular integrity by mitigating oxidative stress to inducing apoptosis under conditions of overwhelming stress. Given these functions, we sought to determine if Foxo3 played a role in maintaining RBC maturation in β-thalassemic mice. To address this, we used Hbbth3/+ (th3/+) mice that display a phenotype similar to β-thalassemia intermedia, and produced double mutant Foxo3-/-/Th3/+ mice. The th3/+ mice display a mild erythroblast apoptotic phenotype. We hypothesized that loss of Foxo3 may exacerbate the β-thalassemic phenotype. On the contrary, we found that loss of Foxo3 in a β-thalassemic background improved RBC numbers and hemoglobin concentration (by 1g/dl, n=10 mice) in double mutant mice compared to th3/+ mice. Furthermore, double mutant mice had a statistically significant lower frequency of apoptosis (2 fold less) during bone marrow erythroblast maturation as measured by flow cytometry analysis of annexin V-binding and 7AAD staining in distinct erythroblast stages resolved by TER119, CD44 and cell size (n=3 mice per genotype). We predicted that high levels of oxidative stress may prematurely activate FOXO3 during erythroblast maturation in β-thalassemic mice. In turn, activated FOXO3 may potentially promote apoptosis in these cells. To evaluate this, we examined FOXO3 levels by qRT-PCR and immunofluorescence in FACS sorted populations of erythroblasts (TER119+,CD44,FSC) or erythroid progenitors (TER119-,c-KIT+,CD71HI) acquired from bone marrow of at least 3 mice per genotype. Our data show increased mRNA levels of Foxo3 in early erythroblasts, corresponding to increased FOXO3 protein expression in erythroid progenitors from β-thalassemic mice relative to wild-type mice. We also examined the activation status of p53, as it is also a major regulator of apoptosis that can be triggered by oxidative stress. Nuclear p53 levels were greater in β-thalassemic as compared to wild-type erythroid progenitors based on immunofluorescence analysis of sorted cells from bone marrow of 3 mice per genotype. These results suggest a higher level of active p53 in β-thalassemic erythroid progenitors. Our results provide evidence that FOXO3, a factor normally critical for erythroblast maturation, may cooperate with aberrantly active p53 to induce apoptosis in β-thalassemic erythroblasts. In support of this, downstream p53 targets including Gadd45a and p21 that are also Foxo3 targets were significantly upregulated in β-thalassemic erythroblasts relative to wild-type erythroblasts as determined by qRT-PCR of cDNA produced from 3 mice per genotype. To more closely examine the mechanism of decreased apoptosis in double mutant Foxo3-/-/Th3/+ erythroblasts, we compared the expression of multiple genes involved in apoptosis by qRT-PCR of sorted erythroblast populations from at least 3 mice per genotype. We found multiple pro-apoptotic genes including, Cycs, Tnfsf10, Puma, and Bim expressed at significantly lower levels at various erythroblast stages in double mutant compared to β-thalassemic erythroblasts. Together, our data suggests Foxo3 becomes inappropriately and prematurely activated in erythroid progenitors and early erythroblasts in the context of β-thalassemia and cooperates with p53 to promote apoptosis. These findings raise the possibility that cooperation of Foxo3 and p53 in β-thalassemic erythroblasts might contribute to the ineffective erythropoiesis of β-thalassemic mice. They also suggest the possibility that as a homeostatic maintaining factor, Foxo3 behaves differently in the context of disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Exploring the Mechanisms of Thalassemic Erythropoiesis Improvement Caused By Bone Marrow Tfr2 Deletion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3624-3624
Author(s):  
Antonella Nai ◽  
Irene Artuso ◽  
Maria Rosa Lidonnici ◽  
Sandro Altamura ◽  
Giacomo Mandelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Double Mutant ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Globin Gene ◽  
Proteasome Activity ◽  
Mutant Mice ◽  
Erythroid Cells ◽  
Ineffective Erythropoiesis ◽  
Rbc Count

Abstract Transferrin receptor 2 (TFR2), the type 3 hemochromatosis gene, is an activator of the iron hormone hepcidin in the liver and a partner of erythropoietin (EPO) receptor in erythroid cells. The loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity inducing erythrocytosis in mice (Nai et al, Blood 2015). We explored whether deletion of BM Tfr2 improves anemia and ineffective erythropoiesis in β-thalassemias, iron-loading anemias due to recessive β-globin gene mutations. We generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through BM transplantation (BMT). Deletion of BM Tfr2 ameliorates RBC morphology with consistent and persistent increase of RBC count and Hb levels in thalassemic mice, accompanied by reduced iron accumulation. Around 22 weeks after BMT the improvement fades in double mutantanimals: Hb levels return comparable to those of Hbbth3/+mice, while RBC count persists higher. Anemia improvement in double mutant mice reduces serum EPO levels and improves erythropoiesis, in particular 22 weeks after BMT. Overall these data prove that the loss of the beneficial effect of deleting Tfr2 is not accounted for by erythropoiesis failure, but likely by exhaustion of splenic iron consumed by the enhanced erythropoiesis. In order to elucidate the molecular mechanisms of the phenotype improvement, we investigated whether the EPO-EPOR signaling pathway is overactive, as occurs in Tfr2 null erythroid cells (Nai et al, Blood 2015). Taking into account that Tfr2BMKO/Hbbth3/+ mice have lower serum EPO than Hbbth3/+, the expression levels of target genes of the EPOR-JAK2-STAT5 (Erfe and Bcl-xl) and of EPOR-PI3K-AKT pathway (Fasl, Epor and Ccng2) is consistent with the signaling being inappropriately active in double mutant mice. To start unraveling the global molecular/cellular processes underlying the remarkable phenotype amelioration, we performed RNAseq analysis on spleen samples from double mutant and Hbbth3/+ control mice at the time point of maximal erythropoiesis improvement (22 weeks post BMT). Spleens are enlarged in both genotypes with about 80% Ter119+ (erythroid) cells in both. In total we identified 2796 genes (1997 protein coding) differentially regulated between the two genotypes. The analysis of iron-related genes reveals a strong reduction of the expression of the iron exporter Fpn, Hmox1 and Alas2, suggestive of decreased hemolysis and/or of free heme accumulation in double mutants. Gene ontology analysis reveals enrichment of genes involved in cell cycle and proliferation, mitochondrial function, as well as proteasome activity and of most of the antioxidant targets (Sod1, Sod2, Fth1, Txn1, Txn2, Gstpi) of the canonical NF-kB pathway. Underrepresented genes are those involved in lipid handling, leukocyte/lymphocyte differentiation and coagulation. In summary, the RNAseq patterns indicate an increased spleen erythroid commitment and a mitochondrial metabolic shift, similar to the shift occurring during hematopoietic development to sustain erythroid proliferation and differentiation. We speculate that this effect is mediated by the enhanced EPO sensitivity. Interestingly EPO directly stimulates mitochondrial genes expression in adipocytes. Also the increased proteasome activity may significantly contribute to the improved erythropoiesis, since proteasomal degradation is required in the process of erythroblast enucleation. Finally, the activation of the NF-kB antioxidant response may contrast ROS increase and limit ineffective erythropoiesis. In conclusion, targeting erythroid TFR2 might become a novel erythropoiesis stimulating approach, worth to be tested in other forms of anemia. Disclosures Muckenthaler: Novartis: Research Funding. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Myeloid Dendritic Cells Contribute to Hematopoietic Homeostasis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 30-30
Author(s):  
Jingzhu Zhang ◽  
Daniel C. Link ◽  
Teerawit Supakorndej ◽  
Mahil Rao
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Preliminary Analysis ◽  
Double Mutant ◽  
Immune Surveillance ◽  
Mutant Mice ◽  
Independent Effect ◽  
Wild Type ◽  
Myeloid Dendritic Cells ◽  
Osteoblast Function

Abstract Dendritic cells (DCs) are antigen-presenting cells that are distributed throughout the body, and their main function is thought to be immune-surveillance. There is considerable phenotypic and functional heterogeneity of dendritic cells that tracks, in part, with their tissue localization. Myeloid dendritic cells (mDC), also known as conventional dendritic cells, are DCs with a myeloid origin. A previous study showed that perivascular mDCs in the bone marrow provide signals that regulate the survival of mature B cells (Sapoznikov et al., Nat Immunol, 2008). Using Cx3cr1gfp/+mice, we show that mDCs, defined as CX3CR1-GFP-bright, MHCII-bright cells, represent 0.2 ± 0.08% of bone marrow cells. They are localized to both venous sinusoids and arterioles in the bone marrow, placing them in the perivascular stem niche, along with CXCL12-abundant reticular (CAR) cells and Nestin-GFP+ cells. To assess the contribution of mDCs to the regulation of hematopoiesis, we used two independent mouse models to ablate mDCs: CD11cDTR and Zbtb46DTR mice. We previously reported that ablation of mDCs induces modest hematopoietic stem/progenitor (HSPC) mobilization. We show that mDC ablation also suppresses osteoblast function, with expression of osteocalcin mRNA (a marker of mature osteoblasts) decreasing 3.5-fold after mDC ablation (from 18.7 ± 9.9 to 5.3 ± 3.0; P < 0.05). To our surprise, mDC ablation (in both models) was associated with a significant loss of bone marrow macrophages. Prior studies have shown that macrophage ablation results in a loss of mature osteoblasts and modest HPSC mobilization. Thus, it is not clear whether mDCs have an independent effect on HSPC trafficking and osteoblast function. To address this issue, we first asked whether the macrophage loss after mDC ablation was mediated in a non-cell autonomous fashion. Mixed bone marrow chimeras were established containing both Zbtb46DTR and wild-type hematopoietic cells. Upon treatment with diphtheria toxin, we observed depletion of Zbtb46DTR but not wild-type mDCs (as expected). In contrast, a similar decrease in both Zbtb46DTR and wild-type macrophages was observed. These data show that the decrease in macrophages is an indirect consequence of mDC ablation and suggest that mDCs generate signals that contribute to macrophage retention and/or survival in the bone marrow. To further address the role of macrophages in this phenotype, we generated mice expressing Zbtb46-DTR alone, CD169-DTR alone (previously shown to ablate macrophages), or mice carrying both Zbtb46-DTR and CD169-DTR. As reported previously, ablation of macrophages induces a modest mobilization of Kit+ Sca1+ lineage- (KSL) cells to the spleen (7.1 ± 3.2 x104 versus 3.0 ± 1.2 x104 for PBS treated mice; P =0.06). Ablation of mDCs also induces modest mobilization (9.1 ± 2.5 x104 versus 5.8 ± 3.5 x104 for PBS treated mice; P < 0.05). Preliminary analysis of double mutant mice (n = 4) suggest an additive effect of combined mDC and macrophage ablation on HSPC mobilization with 25.9 ± 18.4 x104 KSL cells per spleen (P < 0.05 compared with macrophage alone ablation). Likewise, preliminary analysis suggests that the magnitude of osteoblast suppression (as measured by osteocalcin expression) is greater in double mutant mice. Collectively, these data suggest that bone marrow mDCs, in addition to a possible role in immune surveillance, contribute to blood homeostasis through multiple mechanisms. Specifically, mDCs appear to generate signals that are required for macrophage retention and/or survival in the bone marrow. mDCs also regulate HSPC trafficking and osteoblast function through a macrophage independent mechanism. Studies are underway to identify signals generated by mDCs that mediate these biological responses. Disclosures No relevant conflicts of interest to declare.

RNA Sequencing Of Adult Bone Marrow Erythroid Populations Reveals Significant Transcriptional Deregulation In Foxo3-/- Mutant Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3416-3416
Author(s):  
Genís Campreciós ◽  
Xin Zhang ◽  
Yan Kou ◽  
Avi Ma'ayan ◽  
Saghi Ghaffari
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Expression Profiles ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Wild Type ◽  
Adult Bone Marrow ◽  
Erythroid Maturation ◽  
Adult Bone

Abstract Transcriptional control of last stages of erythropoiesis is a complex and well orchestrated process controlled by lineage-specific transcription factors. The precise contribution of the different transcription factors to this multistep process has not been fully elucidated. Foxo3 is a transcription factor that is required for terminal erythroid maturation and Foxo3 mutant mice exhibit ineffective erythropoiesis. In order to gain further insight into the contribution of Foxo3 to the control of adult terminal erythroid maturation we analyzed the transcriptome of three adult bone marrow erythroid precursor populations: pro-, basophilic and polychromatophilic erythroblasts from wild type and Foxo3-/- mice. Populations were FACS sorted according to their TER119 and CD44 cell surface expression and FSC properties. RNA was then isolated and sequenced using the Illumina GaII platform. Genes were grouped into 3 categories according to their expression during erythroid cell maturation using the Short Time Series Expression Miner (STEM) program: no change (4577 genes), down-regulated (2868 genes) or up-regulated (2637) (Figure 1). Enrichment analysis of groups of genes using the ChEA database identified Myb, Meis1, Runx1, Fli1 and PU.1 as the main transcription factors regulating gene repression over erythroid maturation. In contrast, ChEA identified known erythroid transcription factors like Gata1, Eklf and Tal1 to drive the up-regulation of many of the erythroid-specific genes. This analysis also enabled the identification of putative novel transcription factors implicated in erythroid cell maturation. Interestingly, the difference between WT and Foxo3-/- cells increased gradually from pro- to polychromatophilic erythroblasts in correlation with increased Foxo3 expression during these steps of maturation. Strikingly, pathway enrichment analysis detected several immune-related pathways such as Toll-like receptors, TGF-β and IL-1 signaling as expressed in maturing wild type erythroid cells and significantly deregulated in Foxo3-/- cells. The expression of a number of these immune genes in erythroid cells has been validated by qRT-PCR. In addition, among others, a cluster of genes from the autophagy pathway was noted to be significantly down-regulated in Foxo3 mutant erythroid cells. In order to better dissect Foxo3 transcriptional control during erythroid maturation, STEM analysis of Foxo3-/- samples revealed an unexpected number of differences compared to WT. Most remarkably the STEM analysis identified that 90% of the 1198 genes that are continuously up-regulated during erythroid maturation from pro- to polychromatophilic are highly compromised in their level of expression during erythroid maturation in the absence of Foxo3. Interestingly, this group was also enriched for Foxo3 direct target genes as determined by ChIP-seq studies. We also identified a subset of genes whose expression increased from pro- to basophilic erythroblasts but decreased thereafter in the absence of Foxo3 in contrast to wild type cells. Interestingly, ChEA analysis on this group identified a subset of genes that are targets of Gata1, Eklf and Tal1 that may require Foxo3 for their full expression at the last stages of erythroid cell maturation. In conclusion, we present an unbiased genome-wide approach using RNA sequencing of adult bone marrow erythroid cells to study the contribution of Foxo3 to the regulation of gene expression at the last stages of erythroid cell maturation. This analysis enabled us to identify novel genes and pathways whose function in the control of red cell generation requires further investigations.Fig. 1Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples.Fig. 1. Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Current Perspective of Beta Thalassemia and Its Treatment Strategies

2019 ◽  
Author(s):  
Shaukat Ali ◽  
Shumaila Mumtaz ◽  
Hafiz Abdullah Shakir ◽  
Hafiz Muhammad Tahir ◽  
Tafail Akbar Mughal
Keyword(s):  
Genetic Testing ◽  
Blood Transfusion ◽  
Dna Analysis ◽  
Complete Blood Count ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
Thalassemia Minor

Thalassemia is genetic blood disease cause by absence or decrease of one or more of the globin chain synthesis. Beta thalassemia is characterized by one or more mutations in beta globin gene. Absence or reduced amount the of beta globin chains cause ineffective erythropoiesis which leads to anemia. Beta thalassemia has been further divided into three main forms: Thalassemia minor/silent carrier, major and intermedia. More severe form is thalassemia major in which patients depend upon blood transfusion for survival and high level of iron occur as a consequence of consistent blood transfusion. Over loaded iron invokes the synthesis of reactive oxygen species that are toxic in redundancy and triggering the impairment to vascular, endocrine and hepatic system. Thalassemia can be diagnosed and detected through various laboratory tests such as blood smear, prenatal testing (genetic testing of amniotic fluid), DNA analysis (genetic testing) and complete blood count. Treatment of thalassemia intermedia is symptomatic but it can also be managed by splenectomy and folic supplementation. While thalassemia major can be treated by transplantation of bone marrow, regular transfusion of blood and iron chelation treatment, stimulation of fetal hemoglobin production, hematopoietic stem cell transplantation and gene therapy.

The affinity of hemoglobin for oxygen affects ventilatory responses in mutant mice with Presbyterian hemoglobinopathy

2003 ◽  
Vol 285 (4) ◽  
pp. R747-R753 ◽  
Author(s):  
Masahiko Izumizaki ◽  
Masakatsu Tamaki ◽  
Yo-ichi Suzuki ◽  
Michiko Iwase ◽  
Takuji Shirasawa ◽  
...  
Keyword(s):  
Minute Ventilation ◽  
Globin Gene ◽  
Open Circuit ◽  
Mutant Mice ◽  
Whole Body ◽  
Wild Type ◽  
Peripheral Tissues ◽  
Ventilatory Responses ◽  
Significant Difference ◽  
In The Wild

The purpose of this study was to test whether chronically enhanced O2 delivery to tissues, without arterial hyperoxia, can change acute ventilatory responses to hypercapnia and hypoxia. The effects of decreased hemoglobin (Hb)-O2 affinity on ventilatory responses during hypercapnia (0, 5, 7, and 9% CO2 in O2) and hypoxia (10 and 15% O2 in N2) were assessed in mutant mice expressing Hb Presbyterian (mutation in the β-globin gene, β108 Asn → Lys). O2 consumption during normoxia, measured via open-circuit methods, was significantly higher in the mutant mice than in wild-type mice. Respiratory measurements were conducted with a whole body, unrestrained, single-chamber plethysmograph under conscious conditions. During hypercapnia, there was no difference between the slopes of the hypercapnic ventilatory responses, whereas minute ventilation at the same levels of arterial PCO2 was lower in the Presbyterian mice than in the wild-type mice. During both hypoxic exposures, ventilatory responses were blunted in the mutant mice compared with responses in the wild-type mice. The effects of brief hyperoxia exposure (100% O2) after 10% hypoxia on ventilation were examined in anesthetized, spontaneously breathing mice with a double-chamber plethysmograph. No significant difference was found in ventilatory responses to brief hypoxia between both groups of mice, indicating possible involvement of central mechanisms in blunted ventilatory responses to hypoxia in Presbyterian mice. We conclude that chronically enhanced O2 delivery to peripheral tissues can reduce ventilation during acute hypercapnic and hypoxic exposures.

New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4195-4195
Author(s):  
Elena Levantini ◽  
Francesca Bertolotti ◽  
Francesco Cerisoli ◽  
Anna L. Ferri ◽  
Elisa Brescia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blood Cell ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Regulatory Gene ◽  
Embryonic Stem ◽  
Mutant Mice ◽  
Wild Type ◽  
Cell Production

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

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