Infused wild-type macrophages reside and self-renew in the liver to rescue the hemolysis and anemia of Hmox1-deficient mice

AbstractHeme oxygenase 1 (HMOX1), the inducible enzyme that catabolizes the degradation of heme into biliverdin, iron, and carbon monoxide, plays an essential role in the clearance of senescent and damaged red blood cells, systemic iron homeostasis, erythropoiesis, vascular hemostasis, and oxidative and inflammatory stress responses. In humans, HMOX1 deficiency causes a rare and lethal disease, characterized by severe anemia, intravascular hemolysis, as well as vascular and tissue damage. Hmox1 knockout (KO) mice recapitulated the phenotypes of HMOX1-deficiency patients and could be rescued by bone marrow (BM) transplantation that engrafted donor’s hematopoietic stem cells into the recipient animals after myeloablation. To find better therapy and elucidate the contribution of macrophages to the pathogenesis of HMOX1-deficiency disease, we infused wild-type (WT) macrophages into Hmox1 KO mice. Results showed that WT macrophages engrafted and proliferated in the livers of Hmox1 KO mice, which corrected the microcytic anemia, rescued the intravascular hemolysis, restored iron homeostasis, eliminated kidney iron overload and tissue damage, and provided long-term protection. These results showed that a single macrophage infusion delivered a long-term curative effect in Hmox1 KO mice, obviating the need for BM transplantation, and suggested that the HMOX1 disease stems mainly from the loss of viable reticuloendothelial macrophages. Our work provides new insights into the etiology of HMOX1 deficiency and demonstrates the potential of infusion of WT macrophages to prevent disease in patients with HMOX1 deficiency and potentially other macrophage-related diseases.

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Myeloid-biased HSC require Semaphorin 4A from the bone marrow niche for self-renewal under stress and life-long persistence

10.1101/2022.01.12.476097 ◽

2022 ◽

Author(s):

Dorsa Toghani ◽

Sharon Zeng ◽

Elmir Mahammadov ◽

Edie I. Crosse ◽

Negar Seyedhassantehrani ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Knock Out ◽

Inflammatory Stress ◽

Surface Receptor ◽

Key Drivers

Tissue stem cells are hierarchically organized. Those that are most primitive serve as key drivers of regenerative response but the signals that selectively preserve their functional integrity are largely unknown. Here, we identify a secreted factor, Semaphorin 4A (Sema4A), as a specific regulator of myeloid-biased hematopoietic stem cells (myHSC), which are positioned at the top of the HSC hierarchy. Lack of Sema4A leads to exaggerated myHSC (but not downstream balanced HSC) proliferation after acute inflammatory stress, indicating that Sema4A enforces myHSC quiescence. Strikingly, aged Sema4A knock-out myHSC expand but almost completely lose reconstitution capacity. The effect of Sema4A is non cell-autonomous, since upon transplantation into Sema4A-deficient environment, wild-type myHSC excessively proliferate but fail to engraft long-term. Sema4A constrains inflammatory signaling in myHSC and acts via a surface receptor Plexin-D1. Our data support a model whereby the most primitive tissue stem cells critically rely on a dedicated signal from the niche for self-renewal and life-long persistence.

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Heme Oxygenase 1 Deficiency Compromises Stress Responses of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.1353.1353 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1353-1353

Author(s):

Yu-An Cao ◽

Amy J. Wagers ◽

Holger Karsunky ◽

Hui Zhao ◽

Robert Reeves ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Heme Oxygenase ◽

Stress Responses ◽

Mitogen Activated Protein Kinase ◽

Heme Oxygenase 1 ◽

Repeated Treatment ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) must be able to balance their self-renewal and differentiation activities in order to preserve their compartment in response to hematopoietic insults for efficient and life-long hematopoiesis while ensuring sufficient blood production to meet the increased hematopoietic demand. Mechanism(s) that regulate this balance during stress hematopoiesis remain to be fully understood. Heme oxygenase 1 (HO-1) is an important stress-inducible protein and a key enzyme of heme degradation that produces iron, bilirubin, and carbon monoxide (CO). CO is a gaseous regulator of cellular responses to a variety of insults. HO-1 deficiency results in reduced stress defense due to inadequate levels of its products. We report here that mice lacking one allele of HO-1 (HO-1−/+ mice) recovered more rapidly from myelotoxic injury and that sufficient HO-1−/+ bone marrow (BM) transplants engrafted lethally irradiated hosts with accelerated kinetics. These effects of HO-1 deficiency on regenerative hematopoiesis were associated with accelerated cell division of lineage-depleted (lin-) Sca-1+ cells. However, in other experimental settings, the converse can be seen in HO-1 deficiency. Repeated treatment of HO-1−/+ mice with 5-FU significantly reduced the number of HSCs in the marrow, relative to wild-type animals, and following adoptive transfer, the HO-1−/+ HSC compartment was smaller compared to that of HO-1+/+ in recipient animals. As a consequence, limited numbers of HO-1−/+ BM cells failed to provide adequate radio-protection of lethally irradiated recipients, and HO-1−/+ HSCs showed significantly compromised capacity to serially repopulate myeloablated hosts. We also noted that expression levels of the cyclin-dependent kinase inhibitor p21Cip/WAF1 were lower in lin- HO-1−/+ BM cells in contrast to that in HO-1+/+ cells under steady-state conditions, and that p38 mitogen-activated protein kinase (p38MAPK) was insufficiently activated in HO-1−/+ multipotent progenitors (MPPs) after heme challenge, compared to that in wild-type MPPs. We propose a model wherein HO-1 plays an intracellular regulatory and cyto-protective role in maintaining HSC compartment, especially during stress hematopoiesis. It follows that reduced levels of p38MAPK activation, presumably due to insufficient production of CO when HO-1 is deficient, result in accelerated proliferation of MPPs. This would potentially skew the balance between self-renewal and differentiation of HSC and deplete the primitive cell compartment, leading to ultimate hematopoietic exhaustion.

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Ex Vivo Expansion and Long-Term Hematopoietic Reconstitution Ability of Sorted CD34+CD59+ Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽

10.1182/blood.v112.11.2324.2324 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2324-2324

Author(s):

Juan Xiao ◽

Bing Han ◽

Wanling Sun ◽

Yuping Zhong ◽

Yongji Wu

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Peripheral Blood Cell ◽

Intravascular Hemolysis ◽

Blood Cell Count ◽

Normal Cells ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT). Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo. Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. The long-term hematopoietic supporting ability of expanded CD34+CD59+ cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency(SCID) mice in vivo. Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P<0.01 ), they remained strong colony-forming capacity even after expansion ( no difference was noticed in CFCs or LTC-IC of PNH CD34+CD59+ cells before and after expansion, P>0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells. Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

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Lig4 Is Essential for Maintaining HSC Homeostasis

Blood ◽

10.1182/blood.v124.21.606.606 ◽

2014 ◽

Vol 124 (21) ◽

pp. 606-606

Author(s):

Ji Hye Park ◽

Robert S Welner ◽

Daniel G. Tenen ◽

Catherine T. Yan

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Steady State ◽

Microarray Analysis ◽

Wild Type ◽

Stress Injury ◽

Repair Capacity ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Blood cells of all lineages are generated from small pools of long-term hematopoietic stem cells (LT-HSCs) that continually replenish throughout life. LT-HSCs regulate the balanced turnover of all mature blood lineages by switching between self-renewal, differentiation and quiescence, thereby maintaining hematopoietic homeostasis in steady state and in response to injury. In any given cell, some DNA damage may remain despite the action of DNA repair processes, including in LT-HSCs. Over time, HSCs lose their long-term capability to self-renew due to misrepair of DNA breaks and increased accumulation of DNA damage, resulting in loss of regenerative plasticity and immune fitness. The accrual of DNA damage is the principal factor that contributes to functional decline in HSC renewal and in the immune system during ageing. In recent studies, the capacity of aging HSCs to self-renew is shown to be dependent on DNA repair pathways, with non-homologous end-joining (NHEJ) as the principle pathway implicated in DNA repair in quiescent HSCs from ex-vivo cell-based assays. Although NHEJ in particular has been implicated in this process in LT-HSCs, there has so far been very little evidence of this activity in vivo. DNA Ligase IV (Lig4), which catalyzes the end-ligation of broken DNA ends mediated by NHEJ, has no known functions outside of NHEJ. Because a deficiency in Lig4 in mice is embryonic lethal, here we assessed the role of Lig4 in HSC homeostasis by assaying HSC functions in a knockin mouse model of a hypomorphic homozygous R278H mutation in Lig4 that had been identified in the first DNA Lig4 Syndrome patient. The R278H mutation significantly impairs the end-ligation function of the Lig4 protein, and mice homozygous for the R278H mutation (Lig4R/R) showed diminished DSB repair capacity and age-dependent lymphopenia that implicated potential HSC defects. Consistent with a defect in NHEJ, we show the Lig4 R278H mutation severely limited HSC self-renewal. Lig4R/R HSC reconstitutions were skewed towards the myeloid lineage and resulted in severely reduced chimerism, confirming the capacity of HSCs to self-renew requires functional DNA repair. Next, we examined if there is increased DNA damage with/without ionizing irradiation (IR). Lig4R/R LT-HSCs showed an increase in reactive oxygen species (ROS), abnormal cycling and increased apoptosis from accumulated DNA damage in steady state and slow DNA double strand breaks (DSBs) repair kinetics in response to low dose IR because of improper Lig4 function. This led us to check the LT-HSC pool more carefully. It has been shown that the HSC pool is intact and phenotypically increased with age. Strikingly, we found that the HSCs in both young and old Lig4R/R mice are markedly reduced to 20% of wild-type levels. The severe LT-HSC reduction and lethality of disease in Lig4R/Rmice was completely rescued by transplantation with wild type bone marrow. These evidences support the notion of a critical role for Lig4 in maintenance of the LT-HSC pool. In a recent study, it was reported that the steady state pool of murine adult LT-HSCs can be further distinguished into quiescent (~20%) and variably cycling (~80%) populations. Since LT-HSCs in young Lig4R/R mice are maintained in steady state at 20% of WT HSCs, we hypothesized that the reduced pool of LT-HSCs in the Lig4R/R mice is caused by the loss of cycling LT-HSCs that continually replenish blood lineages during aging. To compare these populations, microarray analysis was done on the pool of WT and Lig4R/R LT-HSCs, versus sorted populations of quiescent and variably cycling LT-HSCs. Microarray analysis clearly showed that the Lig4R/RLT-HSCs correlated with the quiescent LT-HSCs, indicating NHEJ regulates the homeostasis of the faster cycling LT-HSC pool. Our study suggest that the slowest cycling LT-HSCs serve to replenish the overall LT-HSC pool and HSC homeostasis is maintained by capacity of faster cycling LT-HSC pool to revert to quiescence in response to stress/injury. Additionally, defective NHEJ depletes the faster cycling LT-HSC pool and underlies early HSC exhaustion in Lig4R/R mice. Our findings demonstrate for the first time a physiological role for Lig4 in the maintenance of HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

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Antidiabetic Potential of the Heme Oxygenase-1 Inducer Curcumin Analogues

BioMed Research International ◽

10.1155/2013/918039 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Yong Son ◽

Ju Hwan Lee ◽

Yong-Kwan Cheong ◽

Hun-Taeg Chung ◽

Hyun-Ock Pae

Keyword(s):

Oxidative Stress ◽

Er Stress ◽

Heme Oxygenase ◽

Stress Responses ◽

Therapeutic Potential ◽

Heme Oxygenase 1 ◽

Low Grade ◽

Cellular Protection ◽

Inflammatory Stress ◽

Curcumin Analogues

Although there is a therapeutic treatment to combat diabetes, the identification of agents that may deal with its more serious aspects is an important medical field for research. Diabetes, which contributes to the risk of cardiovascular disease, is associated with a low-grade chronic inflammation (inflammatory stress), oxidative stress, and endoplasmic reticulum (ER) stress. Because the integration of these stresses is critical to the pathogenesis of diabetes, agents and cellular molecules that can modulate these stress responses are emerging as potential targets for intervention and treatment of diabetic diseases. It has been recognized that heme oxygenase-1 (HO-1) plays an important role in cellular protection. Because HO-1 can reduce oxidative stress, inflammatory stress, and ER stress, in part by exerting antioxidant, anti-inflammatory, and antiapoptotic effects, HO-1 has been suggested to play important roles in pathogenesis of diabetes. In the present review, we will explore our current understanding of the protective mechanisms of HO-1 in diabetes and present some emerging therapeutic options for HO-1 expression in treating diabetic diseases, together with the therapeutic potential of curcumin analogues that have their ability to induce HO-1 expression.

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The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.70.9.4968-4976.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4968-4976 ◽

Cited By ~ 65

Author(s):

Susanna Ricci ◽

Robert Janulczyk ◽

Lars Björck

Keyword(s):

Oxidative Stress ◽

Streptococcus Pyogenes ◽

Stress Responses ◽

Metal Binding ◽

Iron Homeostasis ◽

Bacterial Species ◽

Transcriptional Analysis ◽

Wild Type ◽

Allelic Replacement

ABSTRACT Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

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Hematopoietic stem cells fail to regenerate following inflammatory challenge

10.1101/2020.08.01.230433 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ruzhica Bogeska ◽

Paul Kaschutnig ◽

Malak Fawaz ◽

Ana-Matea Mikecin ◽

Marleen Büchler-Schäff ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Recovery Period ◽

Cumulative Impact ◽

Self Renewal ◽

Hematopoietic Stem ◽

Inflammatory Stress ◽

Kinetics Of

AbstractHematopoietic stem cells (HSCs) are canonically defined by their capacity to maintain the HSC pool via self-renewal divisions. However, accumulating evidence suggests that HSC function is instead preserved by sustaining long-term quiescence. Here, we study the kinetics of HSC recovery in mice, following an inflammatory challenge that induces HSCs to exit dormancy. Repeated inflammatory challenge resulted in a progressive depletion of functional HSCs, with no sign of later recovery. Underlying this observation, label retention experiments demonstrated that self-renewal divisions were absent or extremely rare during challenge, as well as during any subsequent recovery period. While depletion of functional HSCs held no immediate consequences, young mice exposed to inflammatory challenge developed blood and bone marrow hypocellularity in old age, similar to elderly humans. The progressive, irreversible attrition of HSC function demonstrates that discreet instances of inflammatory stress can have an irreversible and therefore cumulative impact on HSC function, even when separated by several months. These findings have important implications for our understanding of the role of inflammation as a mediator of dysfunctional tissue maintenance and regeneration during ageing.

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The Effects of Galactic Cosmic Radiation Exposure on Hematopoietic Stem Cell Dysfunction and Oncogenesis

Blood ◽

10.1182/blood.v128.22.5297.5297 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5297-5297

Author(s):

Rutulkumar Patel ◽

Scott Welford ◽

Stanton L. Gerson

Keyword(s):

Stem Cell ◽

Mismatch Repair ◽

Hematopoietic Stem Cell ◽

Cosmic Radiation ◽

Whole Body ◽

Wild Type ◽

Radiation Quality ◽

Hematopoietic Stem ◽

Dna Mismatch

Abstract Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles (SPE) and trapped energetic particles in a planetary magnetic field. These different sources of space radiation consist of protons of various energies, particle nuclei of high energy and charge (HZE) and neutrons of different energies. These sources are difficult to shield because of their high energies and dense ionization patterns, thus posing significant health risks to astronauts on long term inter-planetary missions. Efforts to protect astronauts from harmful cosmic radiation require a deeper understanding of the effects of GCR on human health. In particular, very little is known about the effects of GCR exposure on the hematopoietic stem cell (HSC) population and whether disruptions in genetic stability in HSCs could result in the development of hematopoietic malignancies in astronauts on deep space missions. The average age of shuttle crew has risen above 46 years, and our work and others have shown that HSCs display diminished function with age. Recent data from our group has demonstrated that middle-aged individuals show frequent defects in DNA mismatch repair (MMR) in HSCs. MMR corrects DNA mismatches generated by DNA polymerase during replication which prevents mutations from becoming permanent in dividing cells. Thus, MMR plays a crucial role in the DNA damage response pathway to prevent short-term mutagenesis and long-term tumorigenesis. Several human MMR proteins have been identified as MutS and MutL homologues consisting of MSH2 and MLH1 heterodimers that functions in DNA mismatch/damage recognition, endonuclease activity and termination of mismatch-provoked excision. Our group has shown that humans accumulate microsatellite instability (MSI) with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Therefore, we employed a DNA mismatch repair deficient mouse model (MLH1+/- and MLH1-/-) to study the effects of different radiation sources including 56Fe, 28Si, 4He, 1H and ᵞ-rays on HSCs to examine HSCs of potential astronaut population under GCR conditions. The complete blood count (CBC) data after 5 months and 9 months of whole body irradiation with different ions showed a slight dose-dependent decrease in all blood counts but absence of any significant difference in CBC of MLH1+/+ vs MLH1+/- mice. In addition, CFU and competitive repopulation data demonstrated a radiation quality effects on HSC function, but not an MLH1 effect. These results demonstrate that hematopoietic stem cell function is normal and that a MLH1 defect does not differentiate progenitor and mature effector cells following HZE radiation. To study long term effects of different ions on the potential for disease progression in a MLH1 dependent manner, we performed whole body irradiation with 56Fe, 28Si, 1H and ᵞ-rays on MLH1+/+ and MLH1+/- mice and followed them up to 18 months post exposure. We observed that MLH1+/- mice show dramatic increases in lymphomagenesis 10-12 months after 56Fe irradiation compared to wild type mice, with greater than 60 % of MLH1+/- mice developed lymphomas at doses 10 cGy and 100 cGy compared to less than 10 % of wild type. For comparison, roughly 10 % of MLH1+/- mice developed lymphomas when mice were treated with whole body sparsely ionizing ᵞ-rays at 100 cGy compared to none of the control. Thus the date show that MMR defects in HSCs lead to sensitization to radiation induced hematopoietic malignancy and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening, as well as lead to important questions regarding safety of ion therapy and development of second malignancies for cancer patients who remain on Earth. Disclosures No relevant conflicts of interest to declare.

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Transferrin Receptor 1 Up-Regulation Plays a Role in Iron Accumulation in a Murine Model of Sideroblastic Anemia.

Blood ◽

10.1182/blood.v106.11.3585.3585 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3585-3585

Author(s):

Florent M. Martin ◽

Timothy J. Gilmartin ◽

Gabriela Bydlon ◽

Megan L. Welsh ◽

Jeffrey S. Friedman

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Stem Cells ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Oxygen Species ◽

Wild Type ◽

Sideroblastic Anemia ◽

Hematopoietic Stem ◽

Transferrin Receptor 1

Abstract Manganese superoxide dismutase (SOD2) detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). While SOD2-deficiency is lethal, SOD2-deficient (SOD2−/−) hematopoietic stem cells can rescue lethally irradiated wild-type mice. SOD2−/− hematopoietic chimeras show a persistent hemolytic anemia similar to human sideroblastic anemia (Friedman et al. J. Exp. Med. 2001). SOD2−/− erythroid progenitor cells have increased mitochondrial mass, and reticulocytes show mitochondrial iron deposition. Mature RBC show abundant siderotic granules, evidence of a defect in iron incorporation into heme, and shortened lifespan. SOD2−/− progenitors and mature RBC show both enhanced reactive oxygen species production and protein oxidative damage (Friedman et al. Blood 2004; Martin et al. Submitted). To define early events in the pathogenesis of the SOD2-deficiency anemia and, in particular to identify genes involved in the response of erythroid progenitors to oxidative stress, we compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2−/−versus wild-type hematopoietic stem cells recipients. Using cDNA microarrays and class comparison analysis, we identified 600 transcripts as significantly discriminant between genotypes. Analysis showed that eleven transcripts encoding different subunits of the mitochondrial oxidative phosphorylation, ATP synthase, and TCA cycle were down-regulated in SOD2−/− erythroblasts. Previous work showed similar results at the protein level in SOD2−/− RBC (Friedman et al. Blood 2004) and at the activity level in specific tissues of SOD2−/− neonates prior to death (Melov et al. PNAS 1999). One interpretation is that SOD2−/− erythroblasts show metabolic decline. Of interest, a single transcript involved in iron homeostasis, Trfr, was found to be expressed at twice the levels found in wild-type erythroblasts (p<0.0007, parametric p value). Trfr encodes transferrin receptor 1; two-fold up-regulation of transferrin receptor 1 was also shown at the protein level by flow cytometry analysis of SOD2−/− erythroblasts (p<0.0001, unpaired two-tailed t-test). Transferrin receptor 1 is the cellular gatekeeper for iron uptake whose genetic inactivation induces abnormal erythropoiesis and iron homeostasis (Levy et al. Nat. Genet. 1999). The stability of the Trfr transcript is highly regulated by iron regulatory proteins (IRPs), that are known to be controlled by numerous effectors including reactive oxygen species (Hentze et al. Cell 2004, for review). We focus our current work on investigating, in vitro and in vivo, the role that up-regulation of transferrin receptor 1, likely through oxidative stress-mediated IRPs activity regulation, plays in iron overload in our SOD2-deficiency model. Taken together, our findings raise the possibility that increased iron delivery may be sufficient to cause sideroblastic anemia or may contribute to a self-reinforcing cycle where oxidative stress favors increased iron, and increased iron results in enhanced oxidative damage.

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HDAC8 Regulates Long-Term Hematopoietic Stem Cell Quiescence and Maintenance

Blood ◽

10.1182/blood.v128.22.1468.1468 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1468-1468

Author(s):

Wei-Kai Hua ◽

Jing Qi ◽

Qi Cai ◽

Emily Carnahan ◽

Ling Li ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Poly I:C ◽

Control Group ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Polycytidylic Acid ◽

Deficient Mice

Abstract Long-term (LT) hematopoietic stem cells (HSC) are responsible for life-long production of mature blood cells of all lineages through tightly concerted cell fate decisions including quiescence, self-renewal, differentiation and apoptosis. Histone deacetylase 8 (HDAC8) is a member of class I HDAC enzymes that remove acetyl moieties from lysine residues on histones and a variety of non-histone proteins. Specifically, HDAC8 has been shown to modulate the acetylation cycle of cohesin complex protein SMC3. Loss-of-function mutations in HDAC8, located on the X chromosome q13, have been found in patients with Cornelia de Lange Syndrome (CdLS) and those with CdLS-like features. These HDAC8 mutations are associated with severely skewed X-inactivation (100% wild type allele) in the peripheral blood of female patients, possibly due to selection against the mutant alleles. However, the expression and function of HDAC8 in normal HSCs and hematopoiesis remain unknown. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8f/f(y)) andconfirmed that Hdac8 is successfully deleted by polyinosinic-polycytidylic acid [poly (I:C)] treatment. Phenotypic analysis of Hdac8 deficient mice showed increased LT-HSC population compared to similarly treated control mice. However, largely normal steady state hematopoietic profile was found in Hdac8 deficient mice at 6 weeks and 1 year after induction. To further track Hdac8-deleted cells, we generated Cre/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/mG (mT/mG) Cre reporter allele, which expresses dTomato prior to Cre induction and becomes GFP+ after Cre-mediated recombination. We assessed hematopoietic repopulation by transplanting bone marrow cells from Cre/Hdac8f/f(y)/mTmG+mice (2 x 105) together with wild type support cells (2 x 105) into lethally irradiated CD45.1+ congenic recipients. Hdac8 deletion was induced by treating the recipients with 7 does (14 m▢g/kg/dose) of poly (I:C). We found that Hdac8 deletion did not affect CD45.2+ or GFP+ donor-derived overall engraftment or lineage repopulation up to 16 weeks. There was also no change in the frequency or number of GFP+ donor-derived phenotypic LT-HSCs in the bone marrow. Serial transplantation was performed to further assess long-term repopulating activity of HSCs. Hdac8 deficient cells were significantly (p=0.019; n=3) compromised in multi-lineage repopulation in secondary transplant recipients. Except a modest reduction in Pre-GM, there was no change in the overall composition of Hdac8 deficient CD45.2+-derived populations. Upon tertiary transplantation, no donor engraftment was observed for Hdac8 deficient cells (0 out of 4) compared to 50% positive engraftment in control group (4 out of 8). These results indicate that HDAC8 is crucial for maintaining long-term serial-repopulating activity over time. Cell cycle analysis revealed that Hdac8 deficient LT-HSCs display reduced quiescence and increased cycling, consistent with the increased number of phenotypic LT-HSC seen in Hdac8 deleted mice. Therefore, we further tested the sensitivity of Hdac8 deficient mice to serial ablation with 5-fluorouracil (5FU), an S phase-specific cytotoxic chemotherapeutic agent. Impaired hematopoietic recovery and increased lethality (p<0.001; n=23) was seen in Hdac8 deficient mice treated with 5-FU (100 mg/kg) every 7 days, indicating that Hdac8 deletion renders hypersensitivity to serial ablation. There were significnatly less phenotypic LT-HSCs in Hdac8 deficient mice 6 days after 5-FU treatment (p<0.01; n=4). In parallel, we observed increased DNA strand beaks as indicated by γ-H2AX staining and comet assays (p<0.001; n>100 cells). Analysis of p53 activation, cell cycle regulators and DNA dmage response are ongoing. Collectively, our study indicates that HDAC8 plays a pivotal role in LT-HSC quiescence and maintenance. Disclosures No relevant conflicts of interest to declare.

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