Essential Role of Gab2 in CSF-1 Dependent Macrophage Development in the Bone Marrow.

Abstract Mononuclear phagocytes (MNPs) are critical in health to maintain tissue homeostasis and in disease as major effectors of innate immunity. In the adult, MNPs develop from bone marrow (BM) progenitors that differentiate to monocytes, tissue macrophages (Mϕs), and specialized cells (dendritic cells, microglia and osteoclasts). Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of MNPs. GAB2, a member of the GAB family of scaffolding proteins (GAB1-3), modulates and amplifies signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and Shp2 phosphatase. Knockdown studies in the 32D myeloid cell line from our lab showed that GAB2 is required for CSF-1 induced mitogenesis and activation of Akt, a PI3K effector. To test the hypothesis that the GAB2-PI3K axis is important for MNP development in vivo, we examined Mϕ development in GAB2 +/+ and −/− mice (gift of Josef Penninger). GAB2 is upregulated 14-fold during CSF-1-induced differentiation of primary BM cells from GAB2+/+ mice. A significant difference is detected in the steady state percentage of F4/80+ BM cells (F4/80 is a mature Mϕ marker): 17.5 ± 1.6 (GAB2+/+, n=8) vs. 11.4 ± 1.6 (GAB2–/−, n=6) (p=0.025, 2-sided t-test). Using the CFU-C progenitor assay with CSF-1 as the only growth factor, primary BM cells from GAB2 −/− mice show a striking 7-fold reduction in colony numbers compared to those from GAB2 +/+ mice (p=0.004) and the colonies were much smaller. Thus GAB2 is essential for optimal CSF-1-dependent Mϕ colony formation. We then used CD31 and Ly6C and flow cytometry to follow the kinetics of Mϕ formation during BM differentiation. These markers monitor sequential stages of Mϕ development: CD31highLy6C– -> CD31+Ly6C+ -> CD31-Ly6Chigh (Eur. J. Immunol.24:2279). As early as 2 days after differentiation induction, GAB2−/− BM cells show a 2-fold reduction in the CD31+Ly6C+ subset (p=6×10−6) and a 6-fold increase in the CD31-Ly6Chigh subset (p=1×10−4), indicating that in the absence of GAB2, CSF-1 promotes a smaller increase in myeloid progenitors and an earlier appearance of more mature cells. To assess proliferation in the progenitor population, day 2 BM cells were labeled with CFSE. Consistent with decreased cell division during early stages of Mϕ development in the absence of GAB2, we observed a 66% reduction in CFSE intensity in GAB2+/+ compared to −/− cells after 3 days in culture. A 2-fold reduction in proliferation by the MTS assay is similarly observed during late Mϕ development (days 5-7) (p=10−4). No difference in viability or expression of CSF-1R or CD11b is found in day 7 Mϕs from GAB2+/+ and −/− mice, excluding increased cell death or arrested differentiation as causes. To investigate the role of GAB2-PI3K, we transduced BM cells with viruses expressing WT-GAB2, 3YF-GAB2 (defective in PI3K binding), both in MSCV-IRES-GFP, or empty MSCV. WT- and 3YF-GAB2 expression in GAB2−/− cells increase the numbers of CFU-Cs by 5- and 2-fold respectively and by 8- and 2.4-fold in GFP+ colonies ≥ 500 μ. Conversely, 3YF-GAB2 exerts a dominant-negative effect on GAB2+/+ cells (a decrease of 30% and 76% in unsorted cells and GFP+ colonies ≥ 500 μ respectively). Therefore PI3K recruitment by GAB2 is required for CSF-1-induced Mϕ colony formation but other GAB2 effector pathways are also important. Our findings support the conclusion that GAB2 is crucial for CSF-1 mediated Mϕ development in the BM, by regulating monocyte/Mϕ progenitor expansion and Mϕ proliferation, in part through PI3K.

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The Role of Dominant-Negative IKAROS Mutations In the Pathogenesis and Treatment Responsiveness of BCR-ABL1 positive Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.540.540 ◽

2010 ◽

Vol 116 (21) ◽

pp. 540-540 ◽

Cited By ~ 2

Author(s):

J. Racquel Collins-Underwood ◽

Nidal Boulos ◽

Debbie Payne-Turner ◽

Shann-Ching Chen ◽

Richard Williams ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Lymphoblastic Leukemia ◽

Marrow Transplant ◽

Dominant Negative ◽

Empty Vector ◽

Transplant Model ◽

Treatment Responsiveness

Abstract Abstract 540 Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p<0.001), suggesting that expression of the dominant-negative Ikaros isoform Ik6 may play a key role in resistance to therapy and poor outcome in human BCR-ABL1 positive ALL. These results indicate that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 positive ALL, and that expression of dominant negative IKZF1 isoforms influences tumor responsiveness. Disclosures: No relevant conflicts of interest to declare.

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HIF-1α Is Not Essential For The Establishment Of MLL-Leukaemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3767.3767 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3767-3767

Author(s):

Deniz Gezer ◽

Amelie V Guitart ◽

Milica Vukovic ◽

Chithra Subramani ◽

Karen Dunn ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Myeloid Leukaemia ◽

Progenitor Cells ◽

Board Of Directors ◽

Advisory Committees ◽

Hypoxic Conditions ◽

Significant Difference

Abstract Haematopoietic stem cells (HSCs) reside in hypoxic niches in the bone marrow (BM) and sustain long-life haematopoiesis. HSCs are largely quiescent, self-renew, undergo apoptosis and generate progenitor cells, which differentiate to multiple blood lineages. The strict regulation of the balance between these fate decisions is essential for haematopoiesis and their dysregulation in HSCs and progenitor cells can result in leukaemic transformation. HSCs and leukemic stem cells (LSCs) are suggested to share the same niche and are in need to adapt to hypoxic conditions. Hypoxia-inducible-factor-1α (HIF-1α) is a key mediator of cellular responses to hypoxia and is important for the maintenance of HSC functions under stressful conditions. Furthermore, in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) HIF-1α is essential for LSC maintenance and ablation or knockdown of HIF-1α leads to exhaustion of established LSCs. The aim of this study was to investigate the requirement for HIF-1α in the generation of pre-LSCs and the establishment of LSCs. To investigate the role of HIF-1α in the generation of pre-LSCs we retrovirally transduced haematopoietic stem and progenitor cells (HSPCs) from either WT or HIF1-αfl/fl Vav-iCre with MLL-ENL retroviruses. Next we performed serial re-plating assays under normoxic and hypoxic conditions to generate pre-LSCs. Surprisingly, WT and HIF-1α deficient HSPCs generated comparable numbers of colonies in normoxia and hypoxia (Fig. 1a). In addition no significant difference was found in the immunophenotypic profile of colonies (Figure 1b). Furthermore, microscopic examination indicated that colonies of all genotypes were dense consistent with their transformed shape (Fig. 1c). WT and HIF-1α-deficient pre-LSCs cultured under normoxia and hypoxia had similar cloning efficiency, which is known to directly correlate with the numbers of LSCs in vivo (Fig. 2). These results indicate that HIF-1α is dispensable for the generation of pre-LSCs. To test the role of HIF-1α in establishment of LSCs from pre-LSCs we transplanted pre-LSCs into lethally irradiated mice together with support BM and monitored the mice for disease development. No significant difference was found in disease latency (Fig. 3a) or frequency of LSCs in peripheral blood, bone marrow or spleens (Fig. 3b) indicating that pre-LSCs lacking HIF-1α can efficiently generate LSCs that cause aggressive AML. In conclusion, we provide genetic evidence that HIF-1α is dispensable for the generation of pre-LSCs and the establishment of LSCs from pre-LSCs. These surprising findings, together with published results indicating that HIF-1α is essential for maintenance of LSCs, imply that HIF-1α has different roles at different stages of leukaemic transformation. Further studies are required to explain the distinct roles of HIF-1α in different stages of leukaemogenesis. Disclosures: Ratcliffe: RedOx: Founder Other. Holyoake:Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees.

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Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

Biological Chemistry ◽

10.1515/bc.2011.064 ◽

2011 ◽

Vol 392 (7) ◽

Cited By ~ 7

Author(s):

Sandra Petrovic ◽

Andreja Leskovac ◽

Jelena Kotur-Stevuljevic ◽

Jelena Joksic ◽

Marija Guc-Scekic ◽

...

Keyword(s):

Bone Marrow ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Significant Difference ◽

Heterozygous Carriers ◽

Male Carriers ◽

Female Carriers

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.

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Osteal Macrophages and Megakaryocytes Increase Adipogenesis

Proceedings of IMPRS ◽

10.18060/25739 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Nikhil Tewari ◽

Deepa Kanagasabapathy ◽

Rachel J. Blosser ◽

Edward F. Srour ◽

Angela Bruzzaniti ◽

...

Keyword(s):

Bone Marrow ◽

Cell Sorting ◽

Fetal Liver ◽

Fold Increase ◽

Wild Type ◽

Bone Marrow Adipose Tissue ◽

New Treatments

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified. We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

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Interleukin-3 (IL-3) stimulates the clonal growth of pulmonary alveolar macrophage of the mouse: role of IL-3 in the regulation of macrophage production outside the bone marrow

Blood ◽

10.1182/blood.v72.2.685.685 ◽

1988 ◽

Vol 72 (2) ◽

pp. 685-690 ◽

Cited By ~ 17

Author(s):

BD Chen ◽

M Mueller ◽

T Olencki

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Alveolar Macrophage ◽

Colony Formation ◽

Clonal Growth ◽

Mononuclear Phagocytes ◽

Pulmonary Alveolar Macrophage ◽

Interleukin 3 ◽

Short Term Exposure

Abstract Interleukin-3 (IL-3) is one of the hematopoietic growth factors that regulates the growth and differentiation of pluripotent stem cells, thereby leading to the production of all the major blood cell types. The role of IL-3 in the regulation of pulmonary alveolar macrophage (PAM) production was investigated. IL-3 stimulated the proliferation and clonal growth of murine PAM with a dose-response curve similar to that of bone marrow granulocyte-macrophage colony-forming cells. The IL- 3-induced colony formation by cells outside the bone marrow appeared to be unique to PAM; IL-3 failed to cause colony formation by both peritoneal exudate macrophages (PEM) and blood monocytes. Unlike bone marrow stem cells, PAM are unipotential and in vitro gave rise to only mononuclear phagocytes under the influence of IL-3. Nevertheless, cells derived from PAM cultures in media containing IL-3 displayed a high degree of heterogeneity in terms of their Fc receptor-mediated phagocytic activity. At low concentrations, IL-3 induced a synergistic response with colony-stimulating factor 1 (CSF-1), which resulted in an enhanced proliferative capacity of PAM. A synergistic effect was also observed by short-term exposure of PAM to IL-3 followed by incubating with CSF-1 alone. This study shows that IL-3 exhibited a macrophage growth factor activity unique to PAM.

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The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

Biochemical Journal ◽

10.1042/bj20111542 ◽

2012 ◽

Vol 443 (2) ◽

pp. 397-405 ◽

Cited By ~ 15

Author(s):

Julia Knöckel ◽

Ingrid B. Müller ◽

Sabine Butzloff ◽

Bärbel Bergmann ◽

Rolf D. Walter ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vitamin B6 ◽

De Novo ◽

Active Form ◽

Cellular Level ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Transcription Analysis

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5′-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

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Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system

Blood ◽

10.1182/blood-2003-05-1479 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3938-3946 ◽

Cited By ~ 279

Author(s):

Jing Zhang ◽

Merav Socolovsky ◽

Alec W. Gross ◽

Harvey F. Lodish

Keyword(s):

Flow Cytometry ◽

Colony Formation ◽

Culture System ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Dominant Negative ◽

Ras Signaling ◽

Proliferation And Differentiation

Abstract Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

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CXCR4 Has a CXCL12-Independent Essential Role in MLL-AF9 Driven Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-116137 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 774-774

Author(s):

Ramprasad Ramakrishnan ◽

Pablo Peña-Martínez ◽

Puneet Agarwal ◽

Carl Högberg ◽

Marion Chapellier ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Fold Increase ◽

Drop Out ◽

Leukemic Cells ◽

Leukemia Cells ◽

Cxcl12 Expression

Abstract Acute Myeloid Leukemia (AML) is a clonal hematological disorder associated with poor prognosis, and there is a strong need to develop new therapeutic strategies. AML is propagated by a small population of leukemic cells in the bone marrow with self-renewal capacity, a population termed leukemia stem cells (LSC). To identify in vivo dependencies of LSCs, we conducted a CRISPR/Cas9 drop-out screen targeting cell surface receptors on murine LSCs. To this end, we generated a pooled lentiviral CRISPR library targeting 96 cell surface receptors that are upregulated on murine MLL-AF9 LSC compared to normal granulocyte and macrophage progenitor (GMP) cells based on global gene expression data. The CRISPR library was transduced into Cas9-expressing murine c-Kit+MLL-AF9 AML cells and subsequently injected into sublethally irradiated (600 cGy) C57BL/6 mice. Two weeks later when the mice had developed leukemia, the bone marrow was harvested, and sgRNAs sequenced. A high reproducibility in sgRNA representation was observed between five biological replicates, demonstrating that the screen was robust. The screen ranked CXCR4 as the most critical cell surface receptor on AML stem cells with all five sgRNAs targeting Cxcr4 depleting more than 4-fold in vivo. Cxcr4 has previously been shown to be critical in several types of cancers including hematological malignancies. Moreover, CXCR4 has been shown to be important for homing of normal and leukemia stem cells in the bone marrow. To validate the role of CXCR4 in a microenvironment and cell intrinsic manner in AML, we disrupted Cxcr4 in c-Kit+MLL-AF9 AML cells using lentiviral vectors expressing sgRNAs targeting Cxcr4 and GFP as a marker gene. Whereas Cxcr4 disruption did not affect the growth and survival of the AML cells in in vitro cultures with standard cytokines, a 15-fold depletion (p = 0.006) of Cxcr4 sgRNA-expressing AML cells was observed 12 days post-transplantation in vivo. We next studied whether the loss of CXCR4 on the surface of leukemic cells would translate into a survival difference in vivo. Notably, the mice receiving sorted leukemia cells expressing Cxcr4 sgRNAs did not develop AML, demonstrating that CXCR4 is essential for LSCs in vivo. To investigate if this effect was due to a homing defect, we transplanted Cxcr4 sgRNA-expressing cells into sublethally irradiated mice and measured the number of AML cells in the bone marrow after 24 hours. A 2.5-fold reduction (p = 0.03) of leukemia cells expressing Cxcr4 sgRNA compared to the control sgRNA was observed in the bone marrow, suggesting that the depletion of leukemic cells in vivo was partially due to a homing defect. CXCL12, the most studied ligand of CXCR4, is a homeostatic chemokine widely expressed by several cell types in the bone marrow and exists both in a membrane and soluble form. Although the CXCR4-CXCL12 interaction is critical for the retention of normal hematopoietic stem and progenitor cells in the bone marrow, the role of CXCL12 in AML is less characterised. To study the role of different CXCL12-expressing bone marrow cell populations in AML development, we first transplanted c-Kit+MLL-AF9 leukemia cells into Cxcl12f/f-Tek-Cre+ mice, which are devoid of Cxcl12 expression in endothelial cells, and in Cxcl12f/f-Prx1-Cre+ mice, which lack expression of Cxcl12 in mesenchymal stem cells. No reduction in leukemia levels in the blood and bone marrow was observed with either of these mouse strains as recipients, indicating that Cxcl12 expression by endothelial cells and MSC is not critical for AML development. Next, we repeated the experiment using Cxcl12f/f-Ubc-Cre+mice, which lack Cxcl12 expression in all cells. Intriguingly, there was no reduction in leukemia levels in these mice. Instead, a significant 2-fold increase in leukemia levels in the peripheral blood (p=0.011) and a 1.5-fold increase in leukemia levels in the bone marrow (p=0.043) were observed 12 days after transplantation. These findings suggest that the CXCR4-CXCL12 interaction is dispensable for AML development and progression. Taken together, we here established an in vivo pooled CRISPR/Cas9 drop-out screen using murine LSCs and identified Cxcr4 as an essential regulator for LSCs. Further validations identified that CXCR4 has a previously uncharacterized CXCL12-independent essential role for MLL-AF9 AML cells. Ongoing studies are aimed at identifying the ligand that is essential for CXCR4 in AML. Disclosures No relevant conflicts of interest to declare.

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PTEN Regulates Natural Killer Cell Trafficking in Vivo

Blood ◽

10.1182/blood.v124.21.753.753 ◽

2014 ◽

Vol 124 (21) ◽

pp. 753-753

Author(s):

Jeffrey W Leong ◽

Stephanie E Schneider ◽

Ryan P Sullivan ◽

Anvita Singh ◽

Todd A Fehniger

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Fold Increase ◽

Cell Trafficking ◽

Pten Loss ◽

Cell Compartments

Abstract Introduction Phosphatase and tensin homolog (PTEN) is the principal negative regulator of the PI3-kinase pathway, members of which are essential for regulating natural killer (NK) cell activation and effector functions (Tassi et al Immunity 2007, Kim et al Blood 2007). However, the role of PTEN in NK cell biology remains unknown, in part hampered by the embryonic lethality of global PTEN loss. Thus, we hypothesized that disruption of PTEN would uniquely impact NK cell developmental and functional pathways. To evaluate whether PTEN was required for normal NK cell functions, we generated and evaluated a mouse model of NK cell-specific PTEN deficiency (Ncr1iCre knockin x PTENflox; PTENΔ/Δ). Results In contrast to T and B lymphocytes with conditional PTEN loss, we discovered that PTEN primarily acts to regulate NK cell distribution, but not their development. PTEN deletion resulted in a significant loss of NK cells (Fig. 1) in the bone marrow (48% reduction, p<0.001), spleen (56% reduction, p<0.001), and other lymphoid tissues, but markedly increased numbers within the peripheral blood (3.4-fold increase, p<0.001) and lung (3.8-fold increase, p<0.05). Surprisingly, we observed unaltered NK cell maturation (defined by CD27 and CD11b expression) within the peripheral organs of PTENΔ/Δ mice, indicating that PTEN operates to re-distribute NK cells without effects on terminal maturation. To determine whether this aberrant localization could be attributed to dysregulated migration, we examined NK cell trafficking between lymphoid organs. PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments (mean sinusoidal fraction: 19% [control] vs. 34% [PTENΔ/Δ], p<0.01). Following combined in vivo inhibition of CXCL12 and VLA-4, the peripheral blood of control mice phenocopied the blood NK cell expansion observed in PTENΔ/Δ mice, suggesting that insensitivity to these retention signals is one contributing mechanism. Short-term, i.v. adoptive transfer of control and PTENΔ/Δ NK cells revealed that PTENΔ/Δ NK cells are strongly retained once in the blood (2.8-fold greater retention of blood PTENΔ/Δ vs. control NK cells, p<0.05) (Fig. 2). Furthermore, the loss of PTEN results in increased NK cell homeostatic proliferation in vivo (mean 3-day bone marrow NK cell BrdU incorporation: 20% [control] vs. 52% [PTENΔ/Δ], p=0.0008). Thus, PTEN regulates multiple factors that contribute to the normal steady-state distribution in the naïve mouse. Given the inappropriate localization without PTEN, we further evaluated the NK cell requirement for PTEN during an anti-lymphoma response. PTENΔ/Δ mice challenged i.p. with the NK-sensitive RMA/S lymphoma had defective expansion of the peritoneal compartment (absolute peritoneal NK cells at 48 hours: 1.9x105 vs. 7.2x104, p=0.04). Furthermore, using an adoptive transfer model that requires NK cell trafficking to distal sites of lymphoma challenge, we found that PTEN-deficient NK cells had significant defects in their recruitment to localized tumors (18.4-fold vs 1.5-fold increase in recruited NK cells, p<0.001) (Fig. 3). Conclusions In this study, we describe the first report of NK-cell intrinsic PTEN loss in vivo. Collectively, our data suggests that unopposed PI3K signaling in NK cells dominantly affects key events responsible for appropriate cell trafficking and distribution, which is distinct from the role of PTEN in related lymphocyte lineages. These data implicate PTEN as a critical mediator of NK cell recruitment to sites of lymphoma and suggest that PTEN dysregulation, as in the case of PTEN loss-of-function mutations and hamartoma tumor syndromes, may result in defective NK cell-mediated immunity. Figure 1 NK-specific PTEN-deficient mice re-distribute NK cells among NK cell compartments. Figure 1. NK-specific PTEN-deficient mice re-distribute NK cells among NK cell compartments. Figure 2 Inappropriate NK cell retention in the blood contributes to the NK cell re-distribution observed in PTENΔ/Δ mice. Blood mononuclear cells were isolated, i.v. transferred into WT recipients and sacrificed after 16 hours. Figure 2. Inappropriate NK cell retention in the blood contributes to the NK cell re-distribution observed in PTENΔ/Δ mice. Blood mononuclear cells were isolated, i.v. transferred into WT recipients and sacrificed after 16 hours. Figure 3 Intravenous adoptively transferred PTENΔ/Δ NK cells are unable to migrate to peritoneal lymphoma. Control or PTENΔ/Δ NK cells were i.v. transferred with RMA/S challenge i.p. into RAG2-/-γc-/- hosts. Peritoneal exudate cells (PECs) were isolated after 48 hours. Figure 3. Intravenous adoptively transferred PTENΔ/Δ NK cells are unable to migrate to peritoneal lymphoma. Control or PTENΔ/Δ NK cells were i.v. transferred with RMA/S challenge i.p. into RAG2-/-γc-/- hosts. Peritoneal exudate cells (PECs) were isolated after 48 hours. Disclosures No relevant conflicts of interest to declare.

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CD169+ Macrophages Regulate Erythropoiesis Under Homeostasis, Recovery From Erythron Injury and in JAK2V617F-Induced Polycythemia Vera

Blood ◽

10.1182/blood.v120.21.80.80 ◽

2012 ◽

Vol 120 (21) ◽

pp. 80-80

Author(s):

Andrew Chow ◽

Matthew Huggins ◽

Jalal Ahmed ◽

Daniel Lucas ◽

Daigo Hashimoto ◽

...

Keyword(s):

Bone Marrow ◽

Polycythemia Vera ◽

Conflicts Of Interest ◽

Critical Role ◽

Iron Chelation ◽

Mononuclear Phagocytes ◽

Novel Strategy

Abstract Abstract 80 The role of macrophages (MΦ) in erythropoiesis was suggested several decades ago with the description of “erythroblastic islands” in the bone marrow (BM) composed of a central MΦ surrounded by developing erythroblasts. This hypothesis was strengthened by in vitro observations using cell culture systems showing that MΦ promote erythroblast proliferation and survival. However, the in vivo role of MΦ in erythropoiesis under homeostasis or disease remains unclear. Central MΦ reportedly express CD169 (or Sialoadhesin), an antigen that specifically marks tissue resident MΦ among mononuclear phagocytes of the bone marrow and spleen. Specific depletion of CD169+ MΦ markedly reduced erythroblasts in the BM (40.4+1.8%) but did not result in overt anemia under homeostasis, likely due to concomitant compensatory splenic erythropoiesis and alterations in RBC clearance. However, MΦ depletion significantly impaired erythroid recovery from PHZ-induced hemolytic anemia (reticulocytes: 8.2-fold lower, p<0.01 and hematocrit: 2-fold lower, p<0.01 on day 6 post-PHZ challenge) and acute blood loss (reticulocytes: 3.2-fold lower, p<0.001 and hematocrit: 1.6-fold lower, p<0.001 on day 4 post-phlebotomy). Furthermore, depletion of CD169+ MΦ in the BM and spleen impaired erythroblast recovery seven days after bone marrow transplantation (BM: 8.2-fold lower, p<0.01 and spleen: 120-fold lower, p<0.05 on day 7 post-BMT) and delayed recovery of reticulocyte numbers (4-fold lower, p<0.001 on day 10 post-BMT) and hematocrit (1.1-fold lower, p<0.05 on day 14 post-BMT). Mechanistically, we observed a rapid drop in reticulocyte hemoglobin content (CHr) in CD169+ MΦ-depleted animals starting four days post-BMT, but iron supplementation was unable to correct the impaired expansion of erythroblasts, suggesting other mechanisms. We determined that VCAM-1 expressed by BM CD169+ MΦ and BMP4 derived from splenic red pulp macrophages were critical for the efficient recovery of the erythron after BMT. Moreover, depletion of host-derived, radioresistant macrophages shortly after transplantation was sufficient to delay erythroblast recovery, implicating a critical role for this population until donor-derived macrophages can repopulate post-BMT. In addition, we characterized a CD169+ VCAM1+ MΦ population in human BM aspirates that represents the first step in clinically targeting the analogous BM resident macrophage population in humans. Since CD169+ MΦ support recovery after erythropoietic injury, we hypothesized that MΦ depletion could potentially normalize the erythron in a JAK2V617F-driven murine model of polycythemia vera (PV). Indeed, we observed that MΦ depletion in PV mice reduced erythroblasts in the BM (1.6-fold lower, p<0.05 after 4 weeks of depletion) and spleen (14-fold lower, p<0.01 after 4 weeks of depletion). This reduction of the expanded PV erythron was associated with an efficient (within 20 days of MΦ depletion) and durable (up to 40 days after last depletion) normalization of the hematocrit. A rapid and durable reduction in CHr was observed after MΦ depletion in PV mice, but systemic iron chelation did not produce the same effect as MΦ depletion, further confirming the contribution of additional mechanisms. MΦ depletion abrogated the induction of BMP4 (3.4-fold lower, p<0.001) and stress erythropoiesis (stress BFU-E: 790-fold reduction, p<0.05) in the spleen. Importantly, MΦ depletion reduced the number of erythropoietin-independent colonies in the spleen of PV mice (endogenous BFU-E: 29-fold lower, p<0.05 and endogenous CFU-E: 1400-fold lower, p<0.05), indicating that erythropoiesis in PV, unexpectedly, remains under the control of MΦ in the BM and splenic microenvironments. Altogether, these studies strongly support the notion that CD169+ MΦ promote erythrocyte development and that modulation of the MΦ compartment represents a novel strategy to treat erythropoietic disorders. Disclosures: No relevant conflicts of interest to declare.

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