scholarly journals TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 568-568
Author(s):  
Jing Fang ◽  
Lyndsey Bolanos ◽  
Juana Serrano-Lopez ◽  
Susanne Christie ◽  
Jose A Cancelas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Gene Dosage ◽  
Critical Role ◽  
Myeloid Cells ◽  
Physiological Role ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem

Abstract Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function. To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis. To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells). Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. Disclosures No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 143-143
Author(s):  
Jarrod A Dudakov ◽  
Alan M Hanash ◽  
Lauren F. Young ◽  
Natalie V Singer ◽  
Mallory L West ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Thymic Epithelial Cells ◽  
Immune Competence ◽  
Strong Negative Correlation ◽  
Hematopoietic Stem ◽  
Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4335-4335
Author(s):  
Fan LIU ◽  
Guoyan Cheng ◽  
Fabiana Perna ◽  
Xu Haiming ◽  
Pierre-Jacques Hamard ◽  
...  
Keyword(s):  
Cell Biology ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Conditional Knockout ◽  
Major Type ◽  
Cytokine Signaling ◽  
Methyltransferase Activity ◽  
Hematopoietic Stem ◽  
Arginine Residues

Abstract Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology. Disclosures No relevant conflicts of interest to declare.

p19INK4d Modulates Human Terminal Erythroid Differentiation By Post-Transcriptionally Regulating GATA1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 697-697
Author(s):  
Xu Han ◽  
Jieying Zhang ◽  
Yuanliang Peng ◽  
Huiyong Chen ◽  
Xiao Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Late Stage ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Erk Activation ◽  
Protein Levels ◽  
Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process during which hematopoietic stem cell (HSCs) are first committed to erythroid progenitors, which subsequently undergo terminal erythroid differentiation to produce mature red blood cells. During terminal erythroid differentiation, proerythroblasts undergo 4-5 mitoses to sequentially generate basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts that expel their nuclei to produce enucleated reticulocytes. Terminal erythropoiesis is a tightly regulated process. The most well studied regulatory mechanisms include EPO/EPOR mediated signal transduction and transcription factors among which GATA1 plays critical role. Terminal erythroid differentiation is also tightly coordinated with cell cycle exit, which is regulated by cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain largely undefined. Our RNA-seq of human terminal erythroid differentiation shows that of seven CDKI members, only three of them, p18INK4c, p19INK4d and p27KIP1, are abundantly expressed in erythroid cells and their expressions are significantly upregulated in late stage erythroblasts, which were further confirmed by western blotting analysis. In contrast to demonstrated roles of p18INK4c and p27KIP1 in terminal erythroid differentiation, the function of p19INK4d this process has not been studied. To explore the role of p19INK4d during human erythropoiesis, we employed a shRNA-mediated knockdown approach in CD34+ cells and found that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, led to increased apoptosis and generation of abnormally nucleated late stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather it led to decreased GATA1 protein levels. Importantly, the differentiation and nucleus defects were rescued by ectopic expression of GATA1. As GATA1 protein is protected by nuclear HSP70, to explore the mechanism for the decreased GATA1 protein levels, we examined the effects of p19INK4d knockdown on HSP70 and found p19INK4d knockdown led to decreased nuclear localization of HSP70 due to reduced ERK activation. Further biochemical analysis revealed that p19INK4d directly binds to Ras kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1 which in turn led to reduced ERK activation. These results demonstrate that p19INK4d maintains GATA1 protein levels through PEBP1-pERK-HSP70-GATA1 pathway. Our findings identify previously unknown and unexpected roles for p19INK4d in human terminal erythroid differentiation via a novel pathway. Disclosures No relevant conflicts of interest to declare.

The Interplay Between Microrna-223 and E2F1 Regulates Cell Cycle Control During Granulopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 255-255
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Philomina Sona Peramangalam ◽  
Abdul A. Peer Zada ◽  
Carsten Müller tidow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromatin Immunoprecipitation ◽  
Cell Cycle Progression ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Granulocytic Differentiation ◽  
Cycle Progression ◽  
Protein Levels ◽  
Cell Cycle Inhibition

Abstract Abstract 255 Transcription factor CCAAT enhancer binding protein α (C/EBPα) functions as a master regulator of granulocyte development by co-ordinating cell cycle inhibition and differentiation. Recent findings demonstrate that deregulation of C/EBPα is a critical step in the development of acute myeloid leukemia (AML). Inhibition of E2F1, the key regulator of cell cycle progression by C/EBPα is essential for granulopoiesis and disruption of this function of C/EBPα leads to leukemia. The mechanism with which C/EBPα inhibits E2F1 in granulopoiesis is poorly understood. Recent advances in our understanding about microRNAs suggest that these molecules have profound impact in gene expression programmes. Also, deregulation of microRNAs has been shown as a hall mark of many cancers including leukemia. microRNA-223 (miR-223) is upregulated by C/EBPα during granulopoiesis. The pivotal role of miR-223 in granulopoiesis is shown by the finding that mice deficient for miR-223 display defects in granulopoiesis. In this study, we explored the role of miR-223 in the cell cycle inhibition function of C/EBPα. Computational analysis by using programmes such as Target Scan suggests that E2F1 is a putative target of miR-223. Luciferase assays using 3'UTR of E2F1 suggest E2F1 is a potential target of miR-223. Western blot analysis using bone marrow cells isolated from miR-223 null mice shows accumulation of E2F1 protein levels. Interestingly, E2F1 protein levels were downregulated during miR-223 overexpression in myeloid cells. Analysis of miR-223 by quantitative Real-Time RT-PCR in AML patient samples shows that miR-223 is downregulated in different subtypes of AML. Proliferation assays, cell cycle analysis and BrdU assays show that miR-223 functions as an inhibitor of myeloid cell cycle progression. Several studies have reported the ability of E2F1 to block granulocytic differentiation. We next analysed whether E2F1 is inhibiting myeloid differentiation through miR-223. Promoter assays show that E2F1 inhibits the miR-223 promoter activity. By using Chromatin immunoprecipitation assays, we found that E2F1 binds to miR-223 promoter in leukemia derived cell lines and this binding is reversed during granulocytic differentiation. We also observed that E2F1 is bound to the miR-223 promoter in blast cells isolated from AML patients as analysed by chromatin immunoprecipitation assays. In addition, we show that overexpression of E2F1 leads to down regulation of miR-223 levels in myeloid cells. All these data suggest that E2F1 functions as a transcriptional repressor of the miR-223 gene. Taken together, our data suggest that granulopoiesis is regulated by the interplay between miR-223 and E2F1 and deregulation of this interplay may lead to the development of AML. Overexpression of miR-223 could be a potential strategy in the treatment of AML patients in which E2F1 inhibition by C/EBPα is deregulated. Disclosures: No relevant conflicts of interest to declare.

Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 219-219
Author(s):  
Daniela S. Krause ◽  
Keertik Fulzele ◽  
Kevin Barry ◽  
Sutada Lotinun ◽  
Roland Baron ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Remodeling ◽  
Conflicts Of Interest ◽  
Bone Matrix ◽  
Myeloid Cells ◽  
Hematopoietic Stem ◽  
Inflammatory Protein ◽  
G Protein Coupled

Abstract Abstract 219 Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated. We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected. In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls. In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling. Disclosures: No relevant conflicts of interest to declare.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 641-641
Author(s):  
Steffen Boettcher ◽  
Rahel Gerosa ◽  
Ramin Radpour ◽  
Markus G. Manz
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Clinical Signs ◽  
Dependent Manner ◽  
Perivascular Cells ◽  
Hematopoietic Stem ◽  
Emergency Myelopoiesis

Abstract Abstract 641 Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as 'emergency myelopoiesis'. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4−/−→WT mice) and WT hematopoiesis on a TLR4−/− nonhematopoietic background (WT→TLR4−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date. We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88−/−→Myd88−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, Tek-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b+Gr-1low immature myeloid precursors accompanied by a decrease of BM CD11b+Gr-1high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

A Critical Role for the Retinoblastoma Tumor Suppressor Gene in Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2548-2548
Author(s):  
Hartmut Geiger ◽  
Marie-Dominique Filippi ◽  
Theodosia A. Kalfa ◽  
Deidre Daria
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Function ◽  
Fetal Liver ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

Abstract The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell cycle, DNA-damage checkpoint, differentiation and apoptosis. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. Positive as well as inhibitory signals are integrated into the phosphorylation of the RB protein to regulate the G1 to S-phase progression of the cell cycle. Despite the importance of RB in leukemia, the consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are still not clear and have been controversially discussed. Using Cre-enzyme expression driven by the hematopoietic specific Vav1-promotor, we generated mice that are constitutively deficient in RB (hemRb−/− animals) in HSPCs. HemRb−/− mice showed anemia with an increased number of reticulocytes in PB, consistent with a published role of RB in erythroid differentiation. In addition, the frequency of Mac-1 positive cells in BM was increased to 67% compared to 47% in control animals, whereas the frequency of B220 positive B-lymphoid cells was almost 10-fold reduced, without affecting the T-lymphoid compartment. HemRb−/− mice possessed a 3-fold enlarged spleen with a 5-fold increased number of colony-forming cells (CFCs) and severe extramedullary hematopoiesis, a phenotype also reported for animals transplanted with Rb−/− fetal liver cells. BM of hemRb−/− mice showed an almost 3-fold reduction of HSC frequency, measured by the cobblestone-area forming cell assay (CAFC) assay, but not a decrease in the number of HSCs determined by cell surface staining and flow cytometry. Upon transplantation into NOD/SCID animals or upon competitive transplantation into C57BL/6. CD45.1 animals, HSPCs from hemRb−/− mice contributed 4 to 6-fold less to hematopoiesis. HSPCs from hemRb−/− animals were neither impaired in their ability to home to the BM, nor did they show increased apoptosis. Finally, we detected a significant 4-fold decrease in stem cell function/numbers upon stress caused by 5-FU treatment in hemRB−/− mice compared to control animals. We conclude that upon transplantation/stress, HSPCs from hemRb−/− animals are impaired in their self-renewal function. HemRb−/− animals also showed a 2-fold increase in the frequency of CFCs in peripheral blood. As we detected no increased leukemia incidence in the hemRb−/− animals (now up to 1 year of age), loss of the tumor suppressor RB in hematopoietic cells might be regarded as necessary, but not sufficient for causing early onset leukemia. In summary, loss of RB results in context/localization dependent phenotypes in the hematopoietic hierarchy, influencing stem and progenitor cells in function, localization and differentiation ability.

PU.1 Is Indispensable for Development of Mature Dendritic Cells and Their Function That Plays a Critical Role in Maintenance of Normal T Cell Pool.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1471-1471
Author(s):  
Tadafumi Iino ◽  
Yong Jeong ◽  
Shin-ichi Mizuno ◽  
Kentaro Kohno ◽  
Kyoko Ito ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
Deficient Mice ◽  
Wild Type Control

Abstract Abstract 1471 Poster Board I-494 PU.1, a hematopoietic transcription factor, is indispensable for development of myelo-lymphoid cells from hematopoietic stem cells (HSCs). PU.1-deficient mice fail to develop common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. By disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool and their functions. We crossed PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC. In these mice, PU.1 gene was disrupted in all conventional DCs but not in other hematopoietic cells, including lymphoid cells, myeloid cells and their progenitors. Development of DC precursors such as Lin−c-KitloFLT3+MCSFR+, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <40%, <25%, <10% and <5% as compared with the wild-type control, respectively. Moreover, the number of mature T cells reduced to ∼60% in the spleen as compared to the control. PU.1-deficient DCs displayed impaired functions to induce antigen-driven T cell proliferation, and to produce inflammatory cytokines (TNFa, IL-6, IL-12) in response to Toll like receptor (TLR) stimulation. These results clearly show that PU.1 is required for development of the peripheral DC pool and for maintenance of their immunological functions, which might be required for maintenance of the peripheral T cell pool. Disclosures: No relevant conflicts of interest to declare.

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