Mobilizing Doses Of G-CSF Stop Medullary Erythropoiesis By Depleting F4/80+ VCAM1+ ER-HR3+ CD169+ Erythroid-Island Macrophages

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 309-309
Author(s):  
Rebecca Jacobsen ◽  
Allison R Pettit ◽  
Liza J Raggatt ◽  
Bianca Nowlan ◽  
Valerie Barbier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Central Component ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Diphtheria Toxin Receptor ◽  
Hsc Niche ◽  
Selective Depletion ◽  
Toxin Receptor

Abstract G-CSF mobilizes hematopoietic stem cells (HSCs) from the bone marrow (BM) into the blood by suppressing a subset of HSC niche supportive macrophages. As macrophages are the central component of erythropoietic islands in BM, spleen and liver, we examined the effect of G-CSF on erythropoiesis in C57BL/6 mice. Mobilizing doses of G-CSF caused a marked whitening of the BM, a 15-fold decrease in the number of phenotypic erythroblasts, a 1.5-fold decrease in polychromatic and orthochromatic erythroblasts, and a 4.5-fold reduction in reticulocytes in the BM. Conversely, more immature pro-erythroblasts increased 4.4-fold. As the cell surface antigen ER-HR3 identifies erythroid island macrophages in mouse liver and spleen, while VCAM-1 and CD169 on macrophages have been independently reported to be critical for erythropoiesis, we followed the expression on these antigens on BM macrophages during mobilization. G-CSF treatment caused a 35-fold reduction in the number of CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages that paralleled the loss of erythroblasts. As a result, splenic erythropoiesis was up-regulated to compensate for the loss of medullary erythropoiesis with a 4-5 fold increase in pro-erythroblasts, all erythroblast subsets and reticulocytes. In another set of experiments, we quantified medullar erythropoiesis and macrophages during recovery after a 4 day G-CSF treatment. Erythroblasts and supportive macrophages significantly recovered as early as 24 hours after cessation of G-CSF but it took 7 days to normalize to pre-mobilization values. This suggests that mobilizing doses of G-CSF transiently block erythroblast differentiation specifically in the BM (but not the spleen) by affecting central macrophages in erythroid islands. To confirm that CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages are critical to the maturation of pro-erythroblasts into erythroblasts, we next performed a broad macrophage depletion by injecting clodronate-loaded liposomes, or a selective depletion of CD169+ macrophages in mice knocked-in with diphtheria toxin receptor into the Siglec1 (CD169) gene. Both clodronate liposome treatment in wild-type mice, and diphtheria toxin treatment in Siglec1DTR/+ mice caused a concomitant depletion of CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ macrophages, loss of erythroblasts and accumulation of pro-erythroblasts. Unlike G-CSF, these two treatments also blocked splenic erythropoiesis. In conclusion, we propose that 1) CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages include nursing macrophages at the centre of erythroid islands and are essential for the maturation of pro-erythroblasts to erythroblasts and 2) mobilizing doses of G-CSF transiently stop medullary erythropoiesis by depleting CD11b+ F4/80+ VCAM1+ ER-HR3+ CD169+ Ly6G+ macrophages in erythropoietic islands in the BM, but not in the spleen. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Osteoblast Ablation Causes an Imbalance of the HSC Population

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4367-4367
Author(s):  
Mayra Garcia
Keyword(s):  
Diphtheria Toxin ◽  
Conflicts Of Interest ◽  
Long Term Effects ◽  
Hematopoietic Stem ◽  
Mature Osteoblast ◽  
Cell Ablation ◽  
Diphtheria Toxin Receptor ◽  
Toxin Receptor

Abstract The hematopoietic stem cell (HSC) niche is composed of many different types of support cells including endothelial cells, stromal cells, and osteoblast. Studies that ablated mature osteoblast showed that they might be important to support maintenance of HSCs. These studies used Col2.3ΔTK to ablate differentiated osteoblast with daily treatments of ganciclovir after which the samples were analyzed immediately. Two opposing studies have been done using this system one showing a decrease in HSC and another showing an increase thus the role of osteoblast in HSC maintenance is still unclear. We have used a different method of ablating cells using Osteocalcin (Oc) and Osterix (Osx) Cre to drive expression of a diphtheria toxin receptor (iDTR), in mature osteoblast and osteoprogenitors, respectively. We injected eight week old mice with three doses of diphtheria toxin and analyzed the LSK and LT-HSC populations a month after cell ablation. There was no effect when the osteoprogenitors were ablated using Osx-Cre; suggesting ablation at postnatal stages is likely necessary to see an effect using Oxs-Cre. On the other hand, we found that a month after ablation of the Oc expressing cells the osteoblast population had recovered but the LSK and LT-HSC populations were still affected with an increase in both populations, suggesting that being exposed to a defective niche might have long-term effects on the HSC population. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yosuke Ono ◽  
Osamu Yoshino ◽  
Takehiro Hiraoka ◽  
Erina Sato ◽  
Akiko Furue ◽  
...  
Keyword(s):  
Mouse Model ◽  
Diphtheria Toxin ◽  
Immunohistochemical Study ◽  
Mrna Levels ◽  
Cytokines And Chemokines ◽  
Diphtheria Toxin Receptor ◽  
Endothelial Cell Marker ◽  
Toxin Receptor ◽  
Group A

AbstractIn endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is unclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines and chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable to deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion of CD206+ MΦ decreased the total weight of endometriotic-like lesions significantly (p < 0.05). In the endometriotic-like lesions in the DT group, a lower proliferation of endometriotic cells and the decrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFβ1, angiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, respectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell marker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number of TGFβ1 expressing MΦ was significantly reduced compared to control. These data suggest that CD206+ MΦ promotes the formation of endometriotic-like lesions by inducing angiogenesis around the lesions.

Expression cloning of a diphtheria toxin receptor: Identity with a heparin-binding EGF-like growth factor precursor

Cell ◽  
1992 ◽  
Vol 69 (6) ◽  
pp. 1051-1061 ◽  
Author(s):  
Joseph G. Naglich ◽  
James E. Metherall ◽  
David W. Russell ◽  
Leon Eidels
Keyword(s):  
Growth Factor ◽  
Diphtheria Toxin ◽  
Expression Cloning ◽  
Heparin Binding ◽  
Diphtheria Toxin Receptor ◽  
Toxin Receptor

Diphtheria toxin receptor–mediated conditional and targeted cell ablation in transgenic mice

10.1038/90795 ◽  
2001 ◽  
Vol 19 (8) ◽  
pp. 746-750 ◽  
Author(s):  
Michiko Saito ◽  
Takao Iwawaki ◽  
Choji Taya ◽  
Hiromichi Yonekawa ◽  
Munehiro Noda ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Diphtheria Toxin ◽  
Cell Ablation ◽  
Diphtheria Toxin Receptor ◽  
Toxin Receptor

Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor

1999 ◽  
Vol 112 (22) ◽  
pp. 3899-3909 ◽  
Author(s):  
G. Skretting ◽  
M.L. Torgersen ◽  
B. van Deurs ◽  
K. Sandvig
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Hela Cells ◽  
Diphtheria Toxin ◽  
Surface Receptors ◽  
Caveolin 1 ◽  
Ultrastructural Studies ◽  
Diphtheria Toxin Receptor ◽  
Early Endosomes ◽  
Toxin Receptor

We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.

scholarly journals Diphtheria toxin receptor.

1993 ◽  
Vol 48 (5) ◽  
pp. 685-692 ◽  
Author(s):  
Eisuke MEKADA
Keyword(s):  
Diphtheria Toxin ◽  
Diphtheria Toxin Receptor ◽  
Toxin Receptor

Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 721-721 ◽  
Author(s):  
Noboru Asada ◽  
Yoshio Katayama ◽  
Mari Sato ◽  
Kentaro Minagawa ◽  
Kanako Wakahashi ◽  
...  
Keyword(s):  
Bone Cells ◽  
Conflicts Of Interest ◽  
Morphological Changes ◽  
Fibroblast Growth Factor 23 ◽  
Bone Matrix ◽  
Fold Increase ◽  
Nerve Fibers ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Endosteal Niche

Abstract Abstract 721 Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3669-3669
Author(s):  
Stephan Emmrich ◽  
Franziska Schmidt ◽  
Ramesh Chandra Pandey ◽  
Aliaksandra Maroz ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Gene Set Enrichment Analysis ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Lncrna Expression ◽  
Fold Reduction ◽  
Non Coding Rnas

Abstract Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs. The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis. Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs. Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA). For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays. Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures: No relevant conflicts of interest to declare.

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