ALBERT

Description: Erythropoiesis is a highly controlled process partly regulated in erythroblastic islands by the central erythroblastic island macrophage (EI MΦ) which provides iron, growth factors and mediates enucleation of the maturing erythroblasts. As macrophages are key effectors of inflammation, we investigated the effect of bacterial LPS in vivo on erythropoiesis and EI MΦ defined as CD11b+ F4/80+ VCAM1+ CD169+Ly6G+ in mice. C57BL/6 mice were injected i.p. with 2.5 mg/kg/day LPS from E. coli for 2 days and the effect on medullar erythropoiesis examined at various time-points after this. LPS administration caused a marked whitening of the bone marrow (BM) with decreased numbers of basophilic (9-fold), polychromatic (3.7-fold), orthochromatic erythroblasts (2.2-fold) and reticulocytes (2.5-fold) 48hrs after LPS challenge. Those remained significantly reduced up to 6 days post-LPS. Likewise, EI MΦ were suppressed in the BM 24-48hrs after LPS challenge and remained significantly reduced 6 days post-LPS. This loss of medullar erythropoiesis was compensated by increased number of EI MΦ (13.6-fold), pro-erythroblasts (1.5-fold), polychromatic (1.9-fold), orthochromatic (3.2-fold) and reticulocytes (2.3-fold) in the spleen. As this phenotype resembled suppression of medullar erythropoiesis following G-CSF treatment, we examined whether the mechanism could be indirect via endogenous G-CSF release. LPS induced a transient 80-fold increase in G-CSF concentration in the blood from 123pg/mL (n=6) to 10ng/ml (n=6) 2 days post-LPS. LPS was administered to TLR4 KO and G-CSF receptor (GCSFR) KO mice. These LPS-mediated responses were abrogated in TLR4 KO mice demonstrating that erythropoiesis suppression in response to LPS is fully TLR4-dependant. However responses in GCSFR KO mice were more contrasted. EI MΦ numbers did not change in GCSFR KO mice in response to LPS demonstrating that suppression of EI MΦ in response to LPS is an indirect effect of endogenous G-CSF release. In contrast, medullar erythropoiesis was still suppressed in GCSFR KO mice with significantly reduced numbers of basophilic, polychromatic and orthochromatic erythroblasts demonstrating that medullar erythroblast suppression 1) persists despite the presence of EI MΦ, and 2) is not G-CSF-dependent. Unexpectedly, the BM from GCSFR KO mice treated with LPS was not whitened with high numbers of reticulocytes/erythrocytes. To further understand how BM erythrocytes could be increased whilst erythropoiesis is suppressed in LPS-treated GCSFR KO mice, we measured vascular leakage by injecting Evans Blue i.v. Blood plasma volume in the BM of LPS-treated GCSFR KO mice was 2.9-fold higher compared to LPS-treated wild-type mice and untreated WT and KO mice (6.0±2.0 µL vs 2.1±0.9 µL blood plasma/femur, p=0.005) suggesting that GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS. In conclusion LPS-mediated medullar erythropoiesis suppression involves at least two different TLR4-dependent mechanisms in regards to their requirement for GCSFR: 1) GCSFR-dependent suppression of EI MΦ, 2) GCSFR-independent and EI MΦ-independent suppression of maturing erythroblasts. We are currently investigating whether the latter mechanism involves hepcidin. Finally we also discovered that GCSFR-mediated signaling is necessary to maintain the BM vasculature integrity following LPS challenge. Disclosures Winkler: GlycoMimetics: Research Funding. Levesque:GlycoMimetics: Equity Ownership.

Description: Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported: endosteal niches in close contact with osteoblasts, and endothelial niches near vascular sinuses. Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E−/ −), P-selectin (P−/ −) or both (PE−/ −) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT+ Sca-1+ CD34− (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P−/ − mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E−/ − and PE−/ − double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E−/ − mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E−/ − mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin− Sca1+ CD41− CD48− CD150+ long-term reconstituting HSC. We found HSC recovery to be enhanced in E−/ − mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E−/ − HSC, BrdU incorporation remained significantly lower in E−/ − HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44−/ − PSGL-1−/ − double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo, endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.

Description: Hypoxia and hypoxia-inducible factors (HIFs) are implicated in the regulation of normal and malignant hematopoiesis. HIF-1α stabilization makes leukemia stem cells and normal HSC dormant and is necessary to maintain their self-renewal potential. In sharp contrast, HIF-2α, which shares 60% homology with HIF-1a, promotes proliferation of renal clear carcinoma and embryonic stem cells by enhancing expression of oct-4, sox2 and activating c-myc. In this study, we investigated the role of hypoxia and HIF-2α in leukemia. In normal mouse and human bone marrow (BM), HIF-2α mRNA expression was observed predominantly in non-hematopoietic stromal cells, while hematopoietic cells displayed low to undetectable levels. In contrast, HIF-2α mRNA and protein were detected in the BM of moribund NOD/SCID mice engrafted with 3 different human ALL, and in cultured human ALL and AML cell lines, suggesting that HIF-2α is abnormally expressed in leukemic cells. To investigate the potential roles of HIF-2α in leukemic cells, we cloned human HIF-2α cDNA into the MXIE retroviral vector. In a 1st model the GM-CSF-dependent mouse pre-leukemic cell line FDCP1, which does not express HIF-2α, was retrovirally transduced with HIF-2α. HIF-2α provided a significant proliferative advantage to FDCP1 cells in hypoxic or normoxic cultures and reduced GM-CSF dependency. We next transplanted retrovirally transduced FDCP1 cells into non-irradiated syngeneic DBA/2 mice. All recipients of FDCP1 transduced with HIF-2α-MXIE vector succumbed to leukemia by week 28 post-transplantation. In sharp contrast, mice receiving FDCP1 transduced with empty MXIE vector, displayed a leukemia penetrance of only 15% by week 45 (Fig. 1a; p=0.0001 log rank, hazard ratio = 12.28).Fig. 1Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors.Fig. 1. Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors. In a 2nd model, HSC from vavBcl2 transgenic mice were transduced with human HIF-2α-containing or empty MXIE retroviral vectors and subsequently transplanted into lethally irradiated wild-type recipients. Transduction of vavBcl2 HSC with HIF-2α resulted in the outgrowth of HIF-2α-expressing B cells which was not observed in recipients of vavBcl2 HSC transduced with empty vector. Consequently recipients of HIF-2α transduced vavBcl2 HSC succumbed more rapidly to spontaneous lymphoma compared to controls (Fig. 1b; p=0.036 log rank, hazard ratio = 2.971, MXIE median survival = 56 weeks, HIF2α median survival = 41 weeks). Finally, HIF-2α was knocked-down in human leukemia cell lines U937 and HL60 using a shRNA lentiviral vector. HIF-2α knock-down resulted in a 2-fold decrease in proliferation in vitro. We next transplanted HL60-HIF-2a shRNA and HL60-scrambled shRNA cells into NOD/SCID/ IL2Rγ-/- (NSG) mice for each group. Notably, all recipients of HL60-HIF-2a shRNA cells succumbed to leukemia significantly later than recipients of HL60-scrambled shRNA cells (Fig. 1c; p=

Description: In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.

Description: Abstract 216 Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization. HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes. In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p

Description: Haematopoietic stem cells (HSCs) are regulated by their immediate microenvironment or niche. The most potent functional HSCs are enriched at the endosteum near the bone, which comprises ~10% of total bone marrow (BM). To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes that were 〉2-fold overexpressed in the endosteal BM relative to the central BM. In this screen, we uncovered known essential HSC niche factors overexpressed in the endosteal BM such as Scf, Cxcl12, and Angpt1, which validated our approach. Among the genes overexpressed in the endosteal BM, prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteum (〉10-fold by qRT-PCR, p

Description: 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.

Description: Key Points HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.