“Emergency” granulopoiesis in G-CSF–deficient mice in response to Candida albicans infection

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3725-3733 ◽  
Author(s):  
Sunanda Basu ◽  
George Hodgson ◽  
Hui-Hua Zhang ◽  
Melissa Katz ◽  
Cathy Quilici ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Candida Albicans ◽  
Emergency Response ◽  
Emergency Situation ◽  
Wild Type ◽  
Gm Csf ◽  
Stimulating Factor ◽  
Physiologic Role ◽  
Deficient Mice

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein believed to play an important role in regulating granulopoiesis both at steady state and during an “emergency” situation. Generation of G-CSF and G-CSF receptor–deficient mice by gene targeting has demonstrated unequivocally the importance of G-CSF in the regulation of baseline granulopoiesis. This study attempted to define the physiologic role of G-CSF during an emergency situation by challenging a cohort of wild-type and G-CSF–deficient mice with Candida albicans. Interestingly, after infection, G-CSF–deficient mice developed an absolute neutrophilia that was observed both in blood and bone marrow. In addition, 3 days after Candida infection increased numbers of granulocyte-macrophage (GM) and macrophage (M) progenitors were observed in the bone marrow of G-CSF–deficient mice. Of the cytokines surveyed, interleukin (IL)-6 levels in serum were elevated; interestingly, levels of IL-6 were higher and more sustained in G-CSF–deficient mice infected with C albicans than similarly infected wild-type mice. Despite the higher levels of serum IL-6, this cytokine is dispensable for the observed neutrophilia because candida-infected IL-6–deficient mice, or mice simultaneously deficient in G-CSF and IL-6, developed neutrophilia. Similarly, mice lacking both G-CSF and GM-CSF developed absolute neutrophilia and had elevated numbers of GM and M progenitors in the bone marrow; thus, G-CSF and GM-CSF are dispensable for promoting the emergency response to candidal infection.

“Emergency” granulopoiesis in G-CSF–deficient mice in response to Candida albicans infection

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3725-3733 ◽  
Author(s):  
Sunanda Basu ◽  
George Hodgson ◽  
Hui-Hua Zhang ◽  
Melissa Katz ◽  
Cathy Quilici ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Candida Albicans ◽  
Emergency Response ◽  
Emergency Situation ◽  
Wild Type ◽  
Gm Csf ◽  
Stimulating Factor ◽  
Physiologic Role ◽  
Deficient Mice

Abstract Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein believed to play an important role in regulating granulopoiesis both at steady state and during an “emergency” situation. Generation of G-CSF and G-CSF receptor–deficient mice by gene targeting has demonstrated unequivocally the importance of G-CSF in the regulation of baseline granulopoiesis. This study attempted to define the physiologic role of G-CSF during an emergency situation by challenging a cohort of wild-type and G-CSF–deficient mice with Candida albicans. Interestingly, after infection, G-CSF–deficient mice developed an absolute neutrophilia that was observed both in blood and bone marrow. In addition, 3 days after Candida infection increased numbers of granulocyte-macrophage (GM) and macrophage (M) progenitors were observed in the bone marrow of G-CSF–deficient mice. Of the cytokines surveyed, interleukin (IL)-6 levels in serum were elevated; interestingly, levels of IL-6 were higher and more sustained in G-CSF–deficient mice infected with C albicans than similarly infected wild-type mice. Despite the higher levels of serum IL-6, this cytokine is dispensable for the observed neutrophilia because candida-infected IL-6–deficient mice, or mice simultaneously deficient in G-CSF and IL-6, developed neutrophilia. Similarly, mice lacking both G-CSF and GM-CSF developed absolute neutrophilia and had elevated numbers of GM and M progenitors in the bone marrow; thus, G-CSF and GM-CSF are dispensable for promoting the emergency response to candidal infection.

scholarly journals Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

2008 ◽  
Vol 295 (1) ◽  
pp. L114-L122 ◽  
Author(s):  
Megan N. Ballinger ◽  
Leah L. N. Hubbard ◽  
Tracy R. McMillan ◽  
Galen B. Toews ◽  
Marc Peters-Golden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Host Defense ◽  
Receptor Expression ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Bacterial Killing ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E2(PGE2) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF−/− donors (GM-CSF−/− BMT) with untransplanted mice. GM-CSF−/− BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF−/− BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF−/− BMT mice overproduced PGE2, but expression of the inhibitory EP2receptor was diminished. As a consequence of decreased EP2receptor expression, we found diminished accumulation of cAMP in response to PGE2stimulation in GM-CSF−/− BMT AMs compared with control BMT AMs. In addition, GM-CSF−/− BMT AMs retained cysteinyl leukotriene production and normal TNF-α response compared with AMs from control BMT mice. GM-CSF−/− BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF−/− recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.

scholarly journals Generation of a Mouse Model for Arginase II Deficiency by Targeted Disruption of the Arginase II Gene

2001 ◽  
Vol 21 (3) ◽  
pp. 811-813 ◽  
Author(s):  
Ou Shi ◽  
Sidney M. Morris ◽  
Huda Zoghbi ◽  
Carl W. Porter ◽  
William E. O'Brien
Keyword(s):  
Urea Cycle ◽  
Elevated Plasma ◽  
Wild Type ◽  
Targeted Disruption ◽  
Arginase Ii ◽  
Plasma Arginine ◽  
Physiologic Role ◽  
Deficient Mice ◽  
Arginase I

ABSTRACT Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.

scholarly journals Pivotal role of IL-6 in the hyperinflammatory responses to subacute ozone in adiponectin-deficient mice

2014 ◽  
Vol 306 (6) ◽  
pp. L508-L520 ◽  
Author(s):  
David I. Kasahara ◽  
Hye Y. Kim ◽  
Joel A. Mathews ◽  
Norah G. Verbout ◽  
Alison S. Williams ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Γδ T Cells ◽  
Pivotal Role ◽  
Ozone Exposure ◽  
Granulocyte Colony Stimulating Factor ◽  
Wild Type ◽  
Neutrophilic Inflammation ◽  
Stimulating Factor ◽  
Deficient Mice

Adiponectin is an adipose-derived hormone with anti-inflammatory activity. Following subacute ozone exposure (0.3 ppm for 24–72 h), neutrophilic inflammation and IL-6 are augmented in adiponectin-deficient ( Adipo−/−) mice. The IL-17/granulocyte colony-stimulating factor (G-CSF) axis is required for this increased neutrophilia. We hypothesized that elevated IL-6 in Adipo−/−mice contributes to their augmented responses to ozone via effects on IL-17A expression. Therefore, we generated mice deficient in both adiponectin and IL-6 ( Adipo−/−/IL-6−/−) and exposed them to ozone or air. In ozone-exposed mice, bronchoalveolar lavage (BAL) neutrophils, IL-6, and G-CSF, and pulmonary Il17a mRNA expression were greater in Adipo−/−vs. wild-type mice, but reduced in Adipo−/−/IL-6−/−vs. Adipo−/−mice. IL-17A+F4/80+cells and IL-17A+γδ T cells were also reduced in Adipo−/−/IL-6−/−vs. Adipo−/−mice exposed to ozone. Only BAL neutrophils were reduced in IL-6−/−vs. wild-type mice. In wild-type mice, IL-6 was expressed in Gr-1+F4/80−CD11c−cells, whereas in Adipo−/−mice F4/80+CD11c+cells also expressed IL-6, suggesting that IL-6 is regulated by adiponectin in these alveolar macrophages. Transcriptomic analysis identified serum amyloid A3 ( Saa3), which promotes IL-17A expression, as the gene most differentially augmented by ozone in Adipo−/−vs. wild-type mice. After ozone, Saa3 mRNA expression was markedly greater in Adipo−/−vs. wild-type mice but reduced in Adipo−/−/IL-6−/−vs. Adipo−/−mice. In conclusion, our data support a pivotal role of IL-6 in the hyperinflammatory condition observed in Adipo−/−mice after ozone exposure and suggest that this role of IL-6 involves its ability to induce Saa3, IL-17A, and G-CSF.

scholarly journals 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

1997 ◽  
Vol 186 (12) ◽  
pp. 1997-2004 ◽  
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.

Podocalyxin Negatively Regulates Stress Myelopoiesis, Directly Binds Rap-1A and Modulates Its G-CSF-Induced Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1184-1184
Author(s):  
Pan Li ◽  
Rose McGlauflin ◽  
Amanda J Favreau ◽  
Edward Jachimowicz ◽  
Calvin Vary ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Functional Role ◽  
Blood Analysis ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Peripheral Blood Analysis ◽  
Specific Deletion

Abstract Podocalyxin (PodxL) is a CD34 family member previously identified to mark hematopoietic stem cells (HSCs) and other progenitor cells. Previously, we discovered PodxL as a potent erythropoietin (EPO) response gene and demonstrated to promote egression of immature reticulocytes from bone marrow into circulation. PodxL is upregulated in several cancers, including myeloid and lymphoid leukemia. Herein, we aim to define the functional role of PodxL in hematopoiesis - specifically myelopoiesis - by employing conditional PodxL knock out (KO) mouse models. Hematopoietic-specific deletion was achieved using Cre mice with a Vav1 driver and myeloid-specific deletion was achieved with Lyzm2 - Cre driver. We confirmed the deletion of exons 3-7 at the gene, transcript and protein levels using PCR, RT-qPCR and western blotting, respectively. Peripheral blood analysis revealed no difference in blood cell lineages for either KO mouse strain. At steady state, colony forming unit-granulocyte/macrophage (CFU-GM) assay also showed no difference between the KO strains and wild type. In order to examine the functional role of PodxL during stress myelopoiesis, PodxL-/- ; Vav1-Cre mice were treated with 5-Fluorouracil (5FU), a chemotherapeutic agent induces myeloablation. Notably, during rebound of neutrophils, the PodxL-/- ; Vav1-Cre mice showed a sharp increase in neutrophil counts at day 12.5, which at later time points reverted to normal levels comparable to wild type mice. Previously, our in silico analyses combined with outcomes from truncated EpoR knock-in alleles had revealed that PodxL is a potential STAT5 transcriptional target. Here, we tested if G-CSF induces PodxL expression in hematopoietic progenitors. In vivo, G-CSF significantly induced PodxL expression four fold. We then tested the role of PodxL in G-CSF induced neutrophil formation in vivo. Both KO strains (Podxl-/-;Vav1-Cre and Podxl-/-;Lyzm2-Cre) and wild type were treated with G-CSF (125ug/kg/day) for 5 days. Peripheral blood analysis revealed increased neutrophil and monocyte levels in the PodxL-/-;Vav1-Cremice. In order to then determine a possible role of PodxL at the progenitor level, CFU-GM assays were performed. PodxL-/- ; lyzm2-Cre mice had increased colony forming capabilities but there was no difference in PodxL-/-;Vav1-Cre mice compared to wild type. Our results imply that PodxL is playing a negative regulatory role in stress myelopoiesis. Interestingly, the deletion of PodxL in hematopoietic progenitors (Vav1-Cre) resulted in enhanced migration of neutrophils, whereas deletion of PodxL in myeloid compartment (Lyzm2-Cre) resulted in decreased neutrophil migration. This may be in part due to a compensatory effect by CD34 in the hematopoietic compartment. To dissect the molecular mechanism of PodxL during stress myelopoiesis, upon in vivo G-CSF treatment, bone marrow derived hematopoietic progenitors were isolated and PodxL protein was immunoprecipitated. LC-MS/MS proteomic analysis was performed to identify the interacting partners with PodxL. Rap-1A, a small GTPase and member of the RAS family, was among the top interacting proteins. Rap-1A has been shown to promote adhesion and migration of myeloid cells. The association of PodxL with Rap-1A was further confirmed in hematopoietic progenitors by immunoprecipitation and western blotting. To determine if the interaction of PodxL directly regulates Rap-1A activity, a GTP-Rap-1A activity assay was performed in response to G-CSF, GM-CSF and IL-3. Rap-1A activity was significantly elevated in hematopoietic progenitors upon G-CSF treatment in PodxL-/-:Vav1-Cre mice compared to wild type, followed by IL3; however, GM-CSF did not affect Rap-1A activity. In conclusion, our results indicate an important functional role for PodxL in stress myelopoiesis, a function likely mediated via Rap-1A. A complete understanding of the PodxL/Rap-1A axis may reveal important molecular insights into G-CSF-induced mobilization of neutrophils and provide mechanistic understanding into the pathological role of PodxL in aggressive cancers, including leukemia, which in turn may facilitate identification of novel therapeutic targets in PodxL associated cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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

Blood ◽  
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.

Bone Marrow Failure in RPS19-Deficient Mice Is Partly Caused by p53 Activation and Responds to L-Leucine Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 727-727
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Johan Flygare ◽  
Stefan Karlsson
Keyword(s):  
Bone Marrow ◽  
Preliminary Data ◽  
Bone Marrow Failure ◽  
Mtor Pathway ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
P53 Activation ◽  
Deficient Mice

Abstract Abstract 727 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al. Blood. 2010;116:193. ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. In the current study we have analyzed the role of p53 in RPS19-deficient hematopoiesis by crossing the transgenic mice into Trp53 null background. To isolate the hematopoietic phenotype we transplanted bone marrow cells from these mice into lethally irradiated wild-type recipients. We have previously shown that the severity of the hematopoietic phenotype in transplanted recipients is highly dependent on the level of RPS19 downregulation, and the recipients with low RPS19 expression die 2–3 weeks after induction because of bone marrow failure. Remarkably, the inactivation of Trp53 rescued the early mortality in these recipients. However, although the inactivation of Trp53 completely reversed the erythrocyte and leukocyte numbers in the recipients with intermediate RPS19 downregulation, the recipients with low RPS19 expression still developed a mild anemia and macrocytosis. p53 activation is known to inhibit the AKT/mTOR pathway, a central regulator of cell growth and survival. Although the role of this pathway in DBA pathogenesis remains poorly defined, some patients positively respond to treatment with amino acid L-leucine, a nutrient signal that stimulates mTOR activity. Currently we are studying the role of L-leucine in RPS19-deficient hematopoiesis both in vitro and in vivo. Our preliminary data confirm that L-leucine modestly enhances the proliferation of RPS19-deficient c-Kit -enriched hematopoietic progenitors in vitro (1.2 fold in 8 days), while there is no effect on wild-type cultures. Interestingly, the proliferative response in RPS19-deficient cultures appears more pronounced when cells are cultured in low cytokine concentration (1.6 fold in 8 days). Since primary cells from DBA patients are highly responsive to stem cell factor (SCF), which also mediates its effect partly via PI3K/AKT/mTOR pathway, we are studying whether L-leucine has a synergistic role with SCF enhancing the proliferation of hematopoietic progenitors. Finally, a 15% L-leucine supplement in drinking water partly rescues the erythrocyte and leukocyte number in RPS19-deficient mice. In summary, our results demonstrate a key role of p53 activation in RPS19-deficient DBA, although they also suggest that p53-independent pathways may contribute towards phenotype upon severe RPS19 deficiency. Furthermore, our preliminary data supports the role of L-leucine as a therapeutic agent in the treatment of DBA. Disclosures: No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

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