The microenvironment, or niche, surrounding a stem cell largely governs its cellular fate. Two anatomical niches for hematopoietic stem cells (HSCs) have been reported in the bone marrow, but a distinct function for each of these niches remains unclear. Here we report a new role for the adhesion molecule E-selectin expressed exclusively by bone marrow endothelial cells in the vascular HSC niche. HSC quiescence was enhanced and self-renewal potential was increased in E-selectin knockout (Sele(-/-)) mice or after administration of an E-selectin antagonist, demonstrating that E-selectin promotes HSC proliferation and is a crucial component of the vascular niche. These effects are not mediated by canonical E-selectin ligands. Deletion or blockade of E-selectin enhances HSC survival threefold to sixfold after treatment of mice with chemotherapeutic agents or irradiation and accelerates blood neutrophil recovery. As bone marrow suppression is a severe side effect of high-dose chemotherapy, transient blockade of E-selectin is potentially a promising treatment for the protection of HSCs during chemotherapy or irradiation.

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colonystimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM.In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.

ABSTRACTinflammation on bone, hematopoietic and erythropoietic maintenance and agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene (-eGFP) to dissect the persistence of bone marrow and splenic macrophage subsets following lethal irradiation and autologous hematopoietic stem cell transplantation in a mouse model. Multiple recipient bone marrow and splenic macrophage subsets survived after autologous hematopoietic stem cell transplantation with organ-specific persistence kinetics. Short-term persistence (5 weeks) of recipient resident macrophages in spleen paralleled the duration of extramedullary hematopoiesis. In bone marrow, radiation-resistant recipient CD169 resident macrophages and erythroid-island macrophages self-repopulated long-term after transplantation via autonomous cell division. Posttransplant peak expansion of recipient CD169 resident macrophage number in bone marrow aligned with the persistent engraftment of phenotypic long-term reconstituting hematopoietic stem cells within bone marrow. Selective depletion of recipient CD169 macrophages significantly compromised the engraftment of phenotypic long-term reconstituting hematopoietic stem cells and consequently impaired hematopoietic reconstitution. Recipient bone marrow resident macrophages are essential for optimal hematopoietic stem cell transplantation outcomes and could be an important consideration in the development of pretransplant conditioning therapies and/or chemoresistance approaches.

Prospective isolation of mouse erythroblastic island macrophages together with new genetic mouse models to specifically target erythroblastic island macrophages will enable molecular studies to better define their role in controlling erythroblast maturation. These studies have revealed the key role of erythroblastic island macrophages in regulating normal erythropoiesis and could be interesting targets to treat β-thalassemia or polycythemia vera.

Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.

The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans and mice. We have recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. Herein we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused a whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However unlike G-CSF, this effect of Flt3-L on EI macrophages was indirect as Flt3 was not detected at the surface of EI macrophages or erythroid progenitors.

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.