Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice

Romosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost−/−) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost−/− mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT®Sost−/− chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost−/− BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost−/− mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.

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Sclerostin depletion induces inflammation in the bone marrow of mice

10.1101/2020.11.01.364158 ◽

2020 ◽

Author(s):

Cristine Donham ◽

Betsabel Chicana ◽

Alexander Robling ◽

Asmaa Mohamed ◽

Sonny Elizaldi ◽

...

Keyword(s):

Bone Marrow ◽

Animal Studies ◽

Immune Cell ◽

Extramedullary Hematopoiesis ◽

Phase Iii ◽

Bone Homeostasis ◽

Mineral Density ◽

Hematopoietic Stem ◽

Phase Iii Clinical Trials

ABSTRACTRomosozumab, a humanized monoclonal antibody specific for sclerostin, has been approved for treatment of post-menopausal women with osteoporosis at high risk for fracture. In several Phase III clinical trials, romosozumab decreased the risk of vertebral fractures up to 73% and increased total hip area bone mineral density by 3.2%. Previous work in 12 to 15-week-old sclerostin-knockout (Sost-/-) mice indicated that changes in immune cell development occur in the bone marrow (BM), which could be a possible side effect to follow in human patients. Our overall goal was to define the mechanisms that guide behavior of long-term hematopoietic stem cells (LT-HSCs) after exposure to an irregular BM microenvironment. SOST plays an important role in maintaining bone homeostasis, as demonstrated by the increased ratio of bone volume to total volume observed in Sost-/- mice. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young (8 week-old) mice receiving sclerostin-antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost-deficiency on wild-type (WT) LT-HSCs transplanted into older (16-22 week-old) cohorts of Sost-/- mice. Our analyses revealed an increased frequency of granulocytes and decreased frequency of lymphocytes in the BM of Scl-Ab treated mice and WT→Sost-/- hematopoietic chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α and MCP-1 in the serum of the Sost-/- BM. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost-/- mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM. Our animal studies strongly recommend tracking of hematopoietic function in patients treated with romosozumab.

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Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽

10.1182/blood.v90.11.4354 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4354-4362 ◽

Cited By ~ 25

Author(s):

Nobuko Uchida ◽

Annabelle M. Friera ◽

Dongping He ◽

Michael J. Reitsma ◽

Ann S. Tsukamoto ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Number ◽

Short Term ◽

Synthesis Inhibitor ◽

Hematopoietic Stem ◽

Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

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In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Blood ◽

10.1182/blood.v120.21.213.213 ◽

2012 ◽

Vol 120 (21) ◽

pp. 213-213

Author(s):

Nicola Vannini ◽

Olaia M. Naveiras ◽

Vasco Campos ◽

Eija Pirinen ◽

Riekelt Houtkooper ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

High Fat Diet ◽

Mitochondrial Activity ◽

High Fat ◽

Short Term ◽

Hematopoietic Stem ◽

Post Transplant ◽

Progenitor Compartment

Abstract Abstract 213 Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1–4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

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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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Identification of Lin–Sca1+kit+CD34+Flt3– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients

Blood ◽

10.1182/blood-2004-06-2159 ◽

2005 ◽

Vol 105 (7) ◽

pp. 2717-2723 ◽

Cited By ~ 271

Author(s):

Liping Yang ◽

David Bryder ◽

Jörgen Adolfsson ◽

Jens Nygren ◽

Robert Månsson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Transplant Recipients ◽

Short Term ◽

Hematopoietic Stem ◽

Regulatory Pathways ◽

Stem And Progenitor Cells ◽

Bone Marrow Ablation

AbstractIn clinical bone marrow transplantation, the severe cytopenias induced by bone marrow ablation translate into high risks of developing fatal infections and bleedings, until transplanted hematopoietic stem and progenitor cells have replaced sufficient myeloerythroid offspring. Although adult long-term hematopoietic stem cells (LT-HSCs) are absolutely required and at the single-cell level sufficient for sustained reconstitution of all blood cell lineages, they have been suggested to be less efficient at rapidly reconstituting the hematopoietic system and rescuing myeloablated recipients. Such a function has been proposed to rather be mediated by less well-defined short-term hematopoietic stem cells (ST-HSCs). Herein, we demonstrate that Lin–Sca1+kithiCD34+ short-term reconstituting cells contain 2 phenotypically and functionally distinct subpopulations: Lin–Sca1+kithiCD34+flt3– cells fulfilling all criteria of ST-HSCs, capable of rapidly reconstituting myelopoiesis, rescuing myeloablated mice, and generating Lin–Sca1+kithiCD34+flt3+ cells, responsible primarily for rapid lymphoid reconstitution. Representing the first commitment steps from Lin–Sca1+kithi CD34–flt3– LT-HSCs, their identification will greatly facilitate delineation of regulatory pathways controlling HSC fate decisions and identification of human ST-HSCs responsible for rapid reconstitution following HSC transplantations.

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Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽

10.1182/blood.v124.21.1591.1591 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1591-1591

Author(s):

Juliana M. Xavier ◽

Lauremilia Ricon ◽

Karla Priscila Vieira ◽

Longhini Ana Leda ◽

Carolina Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Progenitor Cells ◽

Bone Marrow Niche ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (meanÂ± SD) (2.07 Â± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 Â± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 Â±1.39; Arhgap21+/-: 2.17 Â± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 Â± 3.85; Arhgap21+/-: 14.07 Â±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 Â±0.03; Arhgap21+/-0.12Â±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 Â±1.1; Arhgap21+/-94.3Â±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means Â±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

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CCL3 Regulates Normal Hematopoiesis but Is Not Essential for the Maintenance of a Long-Term Engrafting Hematopoietic Stem Cell

Blood ◽

10.1182/blood.v128.22.1482.1482 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1482-1482

Author(s):

Rhonda J. Staversky ◽

Lila Yang ◽

Alexandra N. Goodman ◽

Mary A Georger ◽

Michael W. Becker ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Hematologic Malignancies ◽

Myelogenous Leukemia ◽

Short Term ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Background/Rationale: Hematologic malignancies are known to remodel the bone marrow microenvironment, reducing support for normal hematopoiesis while increasing support for the malignant clone. The chemokine CCL3 has been demonstrated to play a role in microenvironmental dysfunction in multiple malignancies including myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome. In addition, CCL3 has been shown to be critical for the progression of chronic mylogenous leukemia in murine models. However, to consider anti-CCL3 therapy as an option for hematologic malignancies we must first understand its role in the regulation of normal hematopoiesis. To date the role of CCL3 in this process is poorly understood. Methods/Results: In these experiments we utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. Peripheral blood counts revealed that monocytes, granulocytes, and red blood cells were all significantly decreased in the peripheral blood of CCL3KO mice as compared to WT controls at 12 weeks of age (9.78 ± 0.3 vs. 8.06 ± 0.2 RBCs*106/μl, WT vs. CCL3KO p≤0.001 n=8 mice/group). CCL3KO mice also demonstrate a 2-fold increase in the frequency and number of phenotypic long-term hematopoietic stem cells (LT-HSCs: Lin-sca1+ckit+flt3-CD150+CD48-) at 12 weeks of age in the bone marrow by flow cytometric analysis (0.0053 ± 0.0005 vs. 0.0106 ± 0.0007 % of cells, WT vs. CCL3KO p≤0.0001 n=8 mice/group). A significant increase was also seen in short-term HSCs (ST-HSCs), but not in multipotent progenitor (MPP) populations (data not shown), suggesting that CCL3 regulates the most immature hematopoietic cells. To quantify functional hematopoietic stem cells in the marrow of CCL3KO mice competitive transplants were performed using whole bone marrow cells. In primary transplants CCL3KO mice demonstrated a small but significant decrease in engraftment over 22 weeks when compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Decreased engraftment was seen in B cells, T cells, and myeloid cells in the peripheral blood. Upon secondary transplantation the decrease in engraftment of HSCs from CCL3KO donor mice was much more profound. At 16 weeks post-transplant HSCs from CCL3KO donors contributed to hematopoiesis at a rate 5 times lower than WT littermate controls (64.67 ± 1.967 vs. 11.97 ± 5.322 % of cells, WT vs. CCL3KO p≤0.0001 n=10 mice/group). These results were seen in both male and female mice and suggest that, although phenotypic HSCs were increased in the bone marrow of CCL3KO mice, those HSCs were defective. To test this hypothesis we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for LT-HSCs in order to establish stem cell activity on a per cell basis through competitive transplantation. As with the whole bone marrow transplants, primary transplant of sorted Flt3-LSK cells resulted in reduced engraftment of CCL3KO cells as compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Surprisingly, upon secondary transplantation, CCL3KO Flt3-LSK donor cells performed better than the WT littermate controls (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). This result suggests that a transplantable population of cells excluded by the Flt3-LSK sorting parameters is responsible for repression of long-term engraftment capacity of marrow from CCL3KO mice. In establishing the mechanism by which CCL3 regulates hematopoiesis we investigated the rate of apoptosis by quantification of caspase 3 activation, as well as cell cycle status by quantification of Ki67 positivity and DNA content by flow cytometric analysis. We found no difference in the rate of apoptosis, however there was a significant decrease in the fraction of short term HSCs (ST-HSCs) (Flt3-CD48-CD150-LSK) that were actively cycling (2.06 ± 0.43 vs. 1.23±0.44 % of ST-HSCs WT vs. CCL3KO p<0.05 n=3 mice/group). This suggests that CCL3 regulates the proliferation of hematopoietic progenitor cells downstream of the LT-HSC. Conclusions: These results highlight a role for the chemokine CCL3 in the maintenance of the hematopoietic system under benign, physiologic conditions. However, a long-term engrafting HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well tolerated by the hematopoietic system. Disclosures No relevant conflicts of interest to declare.

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Short-Term Fasting Induces Cell Cycle Arrest of Immature Hematopoietic Cells and Increases the Number of Naive T Cells in the Bone Marrow of Mice

Blood ◽

10.1182/blood.v128.22.5047.5047 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5047-5047

Author(s):

Teruhito Takakuwa ◽

Yasuhiro Nakashima ◽

Hideo Koh ◽

Takahiko Nakane ◽

Hirohisa Nakamae ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Research Funding ◽

Bone Marrow Cells ◽

Short Term ◽

Hematopoietic Stem ◽

And Control

Abstract Calorie restriction has long been studied not only as a way to prolong longevity but also as an approach to improve cancer prevention and treatment. Dietary restriction may prevent senescence of the immune and hematopoietic systems. In addition, short-term fasting before chemotherapy can reduce hematological toxicity in cancer patients. We studied the influence of fasting on immune cells, hematopoietic stem cells, and progenitor cells in the bone marrow and spleen of mice. Six-to-twelve-week old C57BL/6 mice were starved for 48 hours before analysis. We collected bone marrow and splenic cells from starved and control mice. After 48 hours of fasting, the body weight significantly decreased by an average of 24.1% compared to that of normal control mice. Calorie restriction caused a significant decrease in peripheral white blood cell count by an average of 48.3%, but hemoglobin level and platelet count were less affected. The averaged total number of bone marrow cells in the fasting group was significantly lower than that in the normal control group (2.45×107 versus 3.14×107, P < 0.01). In fasted mice there was a significant reduction in the hematopoietic stem cell count, using detection based on the lineage- c-kit+ Sca-1+, compared to control mice (0.83×105 versus 1.24×105, P < 0.05). In contrast, there was no significant difference for progenitor cells detected based on the lineage- c-kit+ Sca-1- (6.81×105 versus 7.75×105, P = 0.11). We performed colony assays with bone marrow cells from fasted and control mice. There was no difference between the two groups for not only the primary colony assay but also for the secondary and tertiary assays. Annexin V and 7-AAD analysis by flow cytometry showed that the rates of early and late apoptosis were almost identical in hematopoietic stem cells and progenitor cells, on comparing fasted and control mice. Furthermore, DNA cell cycle analysis of progenitor cells showed that short-term fasting caused a significant increase in the percentage in G0/G1 phase (83.1% versus 70.7%, P < 0.05) and decreases in the S and G2/M phases. These results imply that immature bone marrow cells retained their proliferative capacity by maintaining cell cycle arrest after short-term fasting, a finding that may account for the protective effect of starvation against chemotherapy in cancer patients. Calorie restriction caused a significant decrease in B cells in bone marrow (5.38×106 versus 8.1×106, P < 0.05) and especially in the spleen (6.65×106 versus 33.0×106, P < 0.001), and also significantly decreased T cells in the spleen (3.91×106 versus 14.5×106, P < 0.01). To our surprise, we detected a remarkable increase in the number of T cells in the bone marrow of fasted mice (1.25×106 versus 0.91×106, P < 0.01). Of greatest significance CD44- naive CD8+ T cells were dramatically increased in fasted bone marrow (1.74×106 versus 0.47×106, P < 0.01), and CD44- naive CD4+ T cells were also increased (0.23×106 versus 0.07×106, P < 0.05). In contrast, the numbers of CD62L- CD44+ effector memory and CD62L+ CD44+central memory T cells were not substantially changed after starvation. The increased naive T cells had no activated markers and appear to have migrated into bone marrow in a resting state without proliferating there. Short-term fasting decreased the number of hematopoietic stem cells but progenitor cells remained in a relatively quiescent state, with a prolonged DNA cell cycle and retention of colony-forming capabilities. The number of T cells in the bone marrow of fasted mice also increased dramatically, especially naive CD8+ T cells which probably migrated in from other lymphoid tissues. These findings imply that immature hematopoietic cells and some lymphoid cells can survive starvation while maintaining their function. The mechanisms by which T lymphoid cells promptly accumulate in bone marrow during starvation are under investigation. Disclosures Koh: Pfizer: Consultancy, Honoraria. Nakane:Mundipharma KK: Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Hino:Nippon Shinyaku: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Alexion: Honoraria, Research Funding.

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The Trithorax-Group Protein ASH1L Regulates Hematopoietic Stem Cell Homeostasis Independently of Its Histone Methyltransferase Activity

Blood ◽

10.1182/blood-2018-99-117367 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1270-1270 ◽

Cited By ~ 1

Author(s):

Gloria Jih ◽

Jennifer Chase ◽

Yile Zhou ◽

Ann Friedman ◽

Xiaomin Feng ◽

...

Keyword(s):

Bone Marrow ◽

Wild Type ◽

Set Domain ◽

Hematopoietic Stem ◽

Trithorax Group ◽

Multipotent Progenitors ◽

Donor Bone Marrow ◽

A Cell ◽

Group Protein

Abstract Absent, small or homeotic discs 1-like (Ash1l) encodes the mammalian homolog of a Trithorax-group protein with a conserved SET domain that harbors histone H3 lysine 36 dimethyltransferase activity. Using mice with constitutive knockdown of Ash1l due to the insertion of a "gene trap" cassette in the first intron (Ash1lGT/GT mice), we previously reported that Ash1l is a critical regulator of quiescence and self-renewal in adult hematopoietic stem cells (HSCs). While wild-type HSCs could readily establish long-term bone marrow reconstitution after transplantation into irradiated recipients, Ash1l-deficient HSCs had markedly decreased quiescence and failed to establish long-term bone marrow reconstitution (Jones, Chase et al., JCI 2015). Here, we addressed three important questions to better understand the role of Ash1l in hematopoiesis: 1) What is the impact of complete, as opposed to partial, Ash1l loss on adult hematopoiesis? 2) Does Ash1l regulate HSC homeostasis in a cell-autonomous manner? 3) Is the catalytic activity of ASH1L required for its function? To move beyond the limitations of the constitutive knockdown Ash1lGT allele and address the first two questions, we studied newly generated mice carrying conditional knockout (cKO) Ash1l alleles (exon 13 flanked by loxP sites) along with the Mx1-Cre transgene. Induction of Mx1-Cre expression with poly(I:C) in Mx1-Cre+Ash1lf/f mice resulted in nonsense-mediated decay of Ash1l mRNA in hematopoietic cells, thereby completely knocking out Ash1l in adult hematopoietic stem and progenitor cells. Four weeks after inducing Ash1l inactivation in the hematopoietic compartment, we observed a profound depletion of HSCs and multipotent progenitors in Ash1l cKO mice similar to the phenotype of Ash1lGT/GTmice, indicating that conditional Ash1l knockout has a comparable impact on adult hematopoiesis to that of constitutive Ash1l knockdown. Of note, overt hematopoietic failure in steady-state conditions was not observed in either model. To address the second question, we transplanted a mixture of wild-type and Mx1-Cre+Ash1lf/f bone marrow into irradiated wild-type hosts, allowed donor bone marrow to occupy the wild-type niche and establish hematopoiesis, then induced Cre-mediated excision to inactivate Ash1l in Mx1-Cre+Ash1lf/f hematopoietic cells. Upon Cre induction, donor-derived Ash1lΔ/Δ HSCs and myeloid progeny were depleted or outcompeted by wild-type cells, consistent with the model that Ash1l regulates HSC homeostasis in a cell-autonomous manner. Given that Ash1l encodes a SET domain, we next sought to directly examine whether its catalytic activity is required for its role in regulating HSCs. We studied an Ash1l allele with an in-frame deletion of exon 11 and 12, resulting in preserved expression of ASH1L with internally deleted SET domain (ΔSET) (Miyazaki et al., PLOS Genetics 2013). Homozygous ΔSET mice were viable, and phenotypic analysis of adult ΔSET mice revealed normal frequencies of HSCs and multipotent progenitors, in contrast to our observations in Ash1lGT/GTand Ash1l cKO mice. Furthermore, transplanting ΔSET donor bone marrow into irradiated wild-type hosts resulted in sustained long-term reconstitution throughout primary, secondary and tertiary competitive transplantation assays, consistent with preserved HSC function and no alterations in cell cycle regulation. These findings establish that Ash1l regulates HSC homeostasis independently of its SET domain and histone methyltransferase activity. As ASH1L is a very large protein encoding multiple chromatin binding domains, we speculate that ASH1L may serve as a platform to recruit other partners to form novel protein complex(es) that regulate genes critical for HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

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An Early Myelosuppression in the Acute Mouse Sepsis Is Partly Outcome-Dependent

Frontiers in Immunology ◽

10.3389/fimmu.2021.708670 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tomasz Skirecki ◽

Susanne Drechsler ◽

Aldona Jeznach ◽

Grażyna Hoser ◽

Mohammad Jafarmadar ◽

...

Keyword(s):

Bone Marrow ◽

Bacterial Infections ◽

Cecal Ligation ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Stem And Progenitor Cells ◽

Short And Long Term ◽

The Common ◽

And Control

Adult hematopoietic stem and progenitor cells (HSPCs) respond to bacterial infections by expansion to myeloid cells. Sepsis impairs this process by suppressing differentiation of stem cells subsequently contributing to an ineffective immune response. Whether the magnitude of HSPCs impairment in sepsis is severity-dependent remains unknown. This study investigated dynamics of the HSPC immune-inflammatory response in the bone marrow, splenic, and blood compartments in moribund and surviving septic mice. The 12-week-old outbred CD-1 female mice (n=65) were subjected to a cecal ligation and puncture (CLP) sepsis, treated with antibiotics and fluid resuscitation, and stratified into predicted-to-die (P-DIE) and predicted-to-survive (P-SUR) cohorts for analysis. CLP strongly reduced the common myeloid and multipotent progenitors, short- and long-term hematopoietic stem cell (HSC) counts in the bone marrow; lineage−ckit+Sca-1+ and short-term HSC suppression was greater in P-DIE versus P-SUR mice. A profound depletion of the common myeloid progenitors occurred in the blood (by 75%) and spleen (by 77%) of P-DIE. In P-SUR, most common circulating HSPCs subpopulations recovered to baseline by 72 h post-CLP. Analysis of activated caspase-1/-3/-7 revealed an increased apoptotic (by 30%) but not pyroptotic signaling in the bone marrow HSCs of P-DIE mice. The bone marrow from P-DIE mice revealed spikes of IL-6 (by 5-fold), CXCL1/KC (15-fold), CCL3/MIP-1α (1.7-fold), and CCL2/MCP-1 (2.8-fold) versus P-SUR and control (TNF, IFN-γ, IL-1β, -5, -10 remained unaltered). Summarizing, our findings demonstrate that an early sepsis-induced impairment of myelopoiesis is strongly outcome-dependent but varies among compartments. It is suggestive that the HSCPC loss is at least partly due to an increased apoptosis but not pyroptosis.

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