Evaluation of role of G-CSF in the production, survival, and release of neutrophils from bone marrow into circulation

Abstract In steady-state hematopoiesis, G-CSF (granulocyte-colony stimulating factor) regulates the level of neutrophils in the bone marrow and blood. In this study, we have exploited the availability of G-CSF–deficient mice to evaluate the role of G-CSF in steady-state granulopoiesis and the release of granulocytes from marrow into circulation. The thymidine analogue bromodeoxyuridine (BrdU) was used to label dividing bone marrow cells, allowing us to follow the release of granulocytes into circulation. Interestingly, the labeling index and the amount of BrdU incorporated by blast cells in bone marrow was greater in G-CSF–deficient mice than in wild-type mice. In blood, 2 different populations of BrdU-positive granulocytes, BrdUbright and BrdUdim, could be detected. The kinetics of release of the BrdUbright granulocytes from bone marrow into blood was similar in wild-type and G-CSF–deficient mice; however, BrdUdim granulocytes peaked earlier in G-CSF–deficient mice. Our findings suggest that the mean transit time of granulocytes through the postmitotic pool is similar in G-CSF–deficient and control mice, although the transit time through the mitotic pool is reduced in G-CSF–deficient mice. Moreover, the reduced numbers of granulocytes that characterize G-CSF–deficient mice is primarily due to increased apoptosis in cells within the granulocytic lineage. Collectively, our data suggest that at steady state, G-CSF is critical for the survival of granulocytic cells; however, it is dispensable for trafficking of granulocytes from bone marrow into circulation.

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Protective Role of Connexin 32 in Experimental Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.2239.2239 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2239-2239

Author(s):

Yoko Hirabayashi ◽

Byung-Il Yoon ◽

Isao Tsuboi ◽

Yan Huo ◽

Yukio Kodama ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Pcr Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Competitive Assay ◽

Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.

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Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽

10.1182/blood.v110.11.2221.2221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2221-2221

Author(s):

Cyrus Khandanpour ◽

Ulrich Duehrsen ◽

Tarik Möröy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

T Cell ◽

Bone Marrow Cells ◽

Cell Lymphoma ◽

Wild Type ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

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27-Hydroxycholesterol Induces Hematopoietic Stem Cell Mobilization and Extramedullary Hematopoiesis Mediated By Estrogen Receptor Alpha

Blood ◽

10.1182/blood.v130.suppl_1.636.636 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 636-636

Author(s):

Hideyuki Oguro ◽

Jeffrey McDonald ◽

Sean Morrison

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Steady State ◽

Sex Hormones ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Extramedullary Hematopoiesis ◽

Wild Type ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract In adults, hematopoietic stem cells (HSCs) reside primarily in the bone marrow and their number is tightly regulated under steady state conditions. However, acute demands on the hematopoietic system promote HSC division and mobilization to extramedullary tissues such as the spleen, to increase production of blood cells. While the mechanisms that regulate HSC numbers and residence in the bone marrow under steady-state conditions have been extensively characterized, the mechanisms that activate HSCs in response to acute hematopoietic demands are less well understood. We have previously reported that extramedullary hematopoiesis (EMH) is induced during pregnancy when maternal blood volume expands rapidly. This requires HSC division and mobilization, processes that depend upon estrogen receptor α (ERα) in HSCs. Signaling through this nuclear hormone receptor can be triggered by sex hormones, such as 17β-estradiol (E2), as well as 27-hydroxycholesterol (27HC), which is the first identified endogenous ER ligand other than sex hormones. However, it has been unclear whether 27HC has a physiological role that is effected through ERα signaling in normal mice. Here we show that treatment of mice with E2, which increases during pregnancy, induced HSC division in the bone marrow but did not increase HSC number in the spleen, indicating that E2 treatment does not induce HSC mobilization. In contrast, treatment with the alternative endogenous ERα ligand, 27HC, increased HSC number in the spleen and induced EMH, but not HSC division in the bone marrow, indicating a role in inducing HSC mobilization. The effect of 27HC on HSC mobilization was nullified by deletion of Esr1 (the gene that encodes ERα) in hematopoietic cells using Vav1-icre ; Esr1fl/fl mice, indicating that 27HC-induced HSC mobilization is dependent on ERα. To test whether 27HC acts directly on HSCs, we competitively transplanted Vav1-icre ; Esr1fl/fl donor bone marrow cells along with wild-type competitor bone marrow cells and treated the recipient mice with 27HC four months after the transplantation. 27HC treated mice had significantly lower frequencies of donor-derived (Esr1- deficient) HSCs in the spleen as compared to vehicle-treated mice. This indicates that Esr1- deficient HSCs were at a disadvantage compared to wild-type HSCs in the same mice for mobilization in response to 27HC. ERα thus acts cell-autonomously within HSCs to promote mobilization in response to 27HC. 27HC is generated directly from cholesterol by the sterol hydroxylase, Cyp27a1, and plasma 27HC levels correlate with total cholesterol levels. It has been reported that mice with defects in cholesterol efflux exhibit increased mobilization of hematopoietic stem and progenitor cells (HSPCs) associated with increased serum granulocyte colony-stimulating factor (G-CSF) levels. However, we observed that G-CSF deficiency using Csf3-/- mice did not affect the magnitude of the increase in mobilized HSCs in response to 27HC treatment. Together, 27HC and G-CSF co-treatment additively increased the numbers of colony-forming HSPCs in the blood. Therefore, 27HC and G-CSF likely act through distinct mechanisms. During pregnancy, 27HC levels increased in HSPCs as a result of Cyp27a1. Cyp27a1 -deficient mice had significantly reduced 27HC levels but, under steady-state conditions, Cyp27a1 deficiency did not affect the numbers of HSCs and hematopoietic cells in both bone marrow and spleen. However, during pregnancy, Cyp27a1 -deficient mice had significantly reduced HSC mobilization and EMH, while the increased rate of HSC division and hematopoiesis in the bone marrow was not affected. In contrast, Cyp27a1 deficiency did not affect HSC mobilization and EMH in response to blood loss or G-CSF treatment. Distinct hematopoietic stresses thus induce EMH through different mechanisms. Taken together, these results indicate that two different endogenous ERα ligands, E2 and 27HC, work together to promote EMH during pregnancy, revealing a collaboration of hormone and lipid signaling as well as a physiological function for 27HC in normal mice. Disclosures No relevant conflicts of interest to declare.

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The critical role of T cells in glucocorticoid-induced osteoporosis

Cell Death and Disease ◽

10.1038/s41419-020-03249-4 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Lin Song ◽

Lijuan Cao ◽

Rui Liu ◽

Hui Ma ◽

Yanan Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Steady State ◽

Side Effects ◽

Critical Role ◽

Wild Type ◽

Receptor Activator ◽

Steady State Levels ◽

Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

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Selected Contribution: Differential role of nitric oxide synthase isoforms in fever of different etiologies: studies using Nosgene-deficient mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.01042.2002 ◽

2003 ◽

Vol 94 (6) ◽

pp. 2534-2544 ◽

Cited By ~ 19

Author(s):

Wieslaw Kozak ◽

Anna Kozak

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Corn Oil ◽

Normal Male ◽

Wild Type ◽

Oxide Synthase ◽

And Control ◽

Nos Isoforms ◽

Deficient Mice

Male C57BL/6J mice deficient in nitric oxide synthase (NOS) genes (knockout) and control (wild-type) mice were implanted intra-abdominally with battery-operated miniature biotelemeters (model VMFH MiniMitter, Sunriver, OR) to monitor changes in body temperature. Intravenous injection of lipopolysaccharide (LPS; 50 μg/kg) was used to trigger fever in response to systemic inflammation in mice. To induce a febrile response to localized inflammation, the mice were injected subcutaneously with pure turpentine oil (30 μl/animal) into the left hindlimb. Oral administration (gavage) of N G-monomethyl-l-arginine (l-NMMA) for 3 days (80 mg · kg−1 · day−1in corn oil) before injection of pyrogens was used to inhibit all three NOSs ( N G-monomethyl-d-arginine acetate salt and corn oil were used as control). In normal male C57BL/6J mice, l-NMMA inhibited the LPS-induced fever by ∼60%, whereas it augmented fever by ∼65% in mice injected with turpentine. Challenging the respective NOS knockout mice with LPS and with l-NMMA revealed that inducible NOS and neuronal NOS isoforms are responsible for the induction of fever to LPS, whereas endothelial NOS (eNOS) is not involved. In contrast, none of the NOS isoforms appeared to trigger fever to turpentine. Inhibition of eNOS, however, exacerbates fever in mice treated with l-NMMA and turpentine, indicating that eNOS participates in the antipyretic mechanism. These data support the hypothesis that nitric oxide is a regulator of fever. Its action differs, however, depending on the pyrogen used and the NOS isoform.

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Distinct Roles of Lymphotoxin α and the Type I Tumor Necrosis Factor (TNF) Receptor in the Establishment of Follicular Dendritic Cells from Non–Bone Marrow–derived Cells

Journal of Experimental Medicine ◽

10.1084/jem.186.12.1997 ◽

1997 ◽

Vol 186 (12) ◽

pp. 1997-2004 ◽

Cited By ~ 92

Author(s):

Mitsuru Matsumoto ◽

Yang-Xin Fu ◽

Hector Molina ◽

Guangming Huang ◽

Jinho Kim ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Tumor Necrosis ◽

Type I ◽

Follicular Dendritic Cells ◽

Wild Type ◽

Lymphotoxin Α ◽

Deficient Mice ◽

Necrosis Factor

In mice deficient in either lymphotoxin α (LT-α) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-α and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-α–deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-α–expressing cells required to establish organized FDC are derived from BM. The role of LT-α in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)–deficient BM cells into LT-α–deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-α–deficient recipient. Thus, expression of LT-α in the BM-derived cells, but not in the non–BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I–deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non–BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I–deficient BM was able to restore FDC organization and GC formation in LT-α–deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-α–expressing BM-derived cells and by TNFR-I-expressing non–BM-derived cells.

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Stat3β Promotes Basal Granulopoiesis and Inhibits Emergency Granulopoiesis, While Stat3α Inhibits Basal Granulopoiesis and Promotes Emergency Granulopoiesis

Blood ◽

10.1182/blood.v112.11.3871.3871 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3871-3871

Author(s):

Michele Redell ◽

S. Wen-Wen Chen ◽

Marcos J. Ruiz ◽

David J. Tweardy

Keyword(s):

Bone Marrow ◽

B Lymphocyte ◽

Inflammatory Responses ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Lymphoid Cells ◽

Transactivation Domain ◽

Wild Type ◽

Alternative Mrna Splicing ◽

Deficient Mice

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key signaling intermediate that is activated by several cytokines that regulate hematopoiesis, including granulocyte-colony stimulating factor (G-CSF), interleukin 6, and stem cell factor (SCF). Studies using mice with Stat3 deletion targeted to hematopoietic cells have shown that Stat3 negatively regulates basal granulopoiesis but positively regulates emergency granulopoiesis. Stat3 also has been reported to promote B lymphocyte differentiation. Defining the hematopoietic functions of Stat3 is further complicated by the existence of two isoforms: full-length Stat3α (p92), and truncated Stat3β (p83). Stat3β is derived from alternative mRNA splicing resulting in replacement of the C-terminal transactivation domain with 7 unique amino acids (CT7), which have been demonstrated to confer markedly prolonged nuclear retention. Homozygous Stat3α-deficient mice are not viable, whereas Stat3β-deficient mice survive to adulthood and are fertile, but have increased inflammatory responses compared to wild-type mice. We compared basal granulopoiesis and lymphopoiesis, as well as emergency granulopoiesis, in homozygous Stat3β-deficient mice (βΔ/βΔ), which express only Stat3α, vs. their wild-type (+/+) littermates. We found that βΔ/βΔ mice were significantly leukopenic (2880 ± 1260/ml v. 4600 ± 1670/ml; p<0.05), with lower absolute neutrophil counts (ANC, 360 ± 180/ml v. 800 ± 380/ml, p<0.05) and B lymphocyte counts (780 ± 470/ml v. 1830 ± 1260/ml, p<0.05), compared to +/+ mice. Within the circulating B-lymphocyte population, the mature B220hi/IgM− cells were most dramatically reduced (170 ± 70/ml v. 480 ± 350/ml, p<0.05). Percentages of myeloid and lymphoid cells in the spleen and bone marrow were not significantly different between βΔ/βΔ and +/+ mice. Bone marrow from βΔ/βΔ mice generated significantly fewer myeloid colonies (CFU-GM) compared to wild-type marrow (28 ± 9 v. 42 ± 8 colonies per 20,000 cells, p<0.05). Additionally, βΔ/βΔ lineage-depleted bone marrow cells cultured in G-CSF and SCF produced significantly fewer CD11b+/Gr1+ myeloid cells compared to +/+ cells (52.8 ± 6.5% v. 68.3 ± 2.6%, p<0.05). In contrast, bone marrow from βΔ/βΔ and +/+ mice produced equal numbers of pro-B colonies in CFU assays containing the lymphopoietic cytokine IL-7. Finally, as a test of emergency granulopoiesis, we administered a single dose of G-CSF (250 μg/kg subcutaneously) or an equal volume of PBS, and 24 hr later measured the ANC, percentage of CD11b+/Gr1+ myeloid cells in the bone marrow, and CFU-GM generation. Mice of both genotypes responded to G-CSF stimulation with increases in ANC, percent of myeloid cells within the marrow, and CFU-GM. Bone marrow from βΔ/βΔ mice showed a larger G-CSF-induced increase in CFU-GM (PBS: 22 ± 5 v. G-CSF: 39 ± 1, p<0.05) compared to +/+ marrow (PBS: 24 ± 14 v. G-CSF: 31 ± 14, NS). Thus, Stat3β positively regulates basal granulopoiesis in the bone marrow, and may negatively regulate emergency granulopoiesis. This pattern is the opposite of that seen with deletion of both Stat3 isoforms, indicating that Stat3α’s function is to negatively regulate basal granulopoiesis and positively regulate emergency granulopoiesis. Stat3β also positively regulates circulating B lymphocyte numbers, via a mechanism other than B lymphocyte production in the bone marrow.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽

10.1182/blood.v122.21.1169.1169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1169-1169

Author(s):

Maren Weisser ◽

Kerstin B. Kaufmann ◽

Tomer Itkin ◽

Linping Chen-Wichmann ◽

Tsvee Lapidot ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Stem Cell ◽

Steady State ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Deficient Mice ◽

Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽

10.1182/blood-2018-99-110836 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 941-941

Author(s):

Brian Wadugu ◽

Amanda Heard ◽

Joseph Bradley ◽

Matthew Ndonwi ◽

Jin J Shao ◽

...

Keyword(s):

Bone Marrow ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Poly I:C ◽

Wild Type ◽

Hematopoietic Stem ◽

Mutant Cells ◽

Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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