Hematopoietic Stem/progenitor Cells Trafficking Research Articles

The Different Responsiveness of C3- and C5-deficient Murine BM Cells to Oxidative Stress Explains Why C3 Deficiency, in Contrast to C5 Deficiency, Correlates with Better Pharmacological Mobilization and Engraftment of Hematopoietic Cells.

The liver-derived circulating in peripheral blood and intrinsic cell-expressed complement known as complosome orchestrate the trafficking of hematopoietic stem/progenitor cells (HSPCs) both during pharmacological mobilization and homing/engraftment after transplantation. Our previous research demonstrated that C3 deficient mice are easy mobilizers, and their HSPCs engraft properly in normal mice. In contrast, C5 deficiency correlates with poor mobilization and defects in HSPCs' homing and engraftment. The trafficking of HSPCs during mobilization and homing/engraftment follows the sterile inflammation cues in the BM microenvironment caused by stress induced by pro-mobilizing drugs or myeloablative conditioning for transplantation. Therefore, to explain deficiencies in HSPC trafficking between C3-KO and C5-KO mice, we evaluated the responsiveness of C3 and C5 deficient cells to low oxidative stress. As reported, oxidative stress in BM is mediated by the activation of purinergic signaling, which is triggered by the elevated level of extracellular adenosine triphosphate (eATP) and by the activation of the complement cascade (ComC). In the current work, we noticed that BM lineage negative cells (lin-) isolated from C3-KO mice display several mitochondrial defects reflected by an impaired ability to adapt to oxidative stress. In contrast, C5-KO-derived BM cells show a high level of adaptation to this challenge. To support this data, C3-KO BM lin- cells were highly responsive to eATP stimulation, which correlates with enhanced levels of reactive oxygen species (ROS) generation and more efficient activation of intracellular Nlrp3 inflammasome. We conclude that the enhanced sensitivity of C3-KO mice cells to oxidative stress and better activation of the Nox2-ROS-Nlrp3 inflammasome signaling axis explains the molecular level differences in trafficking between C3- and C5-deficient HSPCs.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P<0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) by Downregulating Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) Activity.

Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide anevidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Novel Evidence That Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) By Inhibiting Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) - Implications to Understand Better Diurnal Trafficking of HSPCs and Anti-Inflammatory Effects of Melatonin

Background. Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and pick at early morning hours. We have reported that the level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) (Stem Cell Rev Rep. 2018;14(5):677-685) that drives activation of Nlrp3 inflammasome (Stem Cell Rev Rep. 2020;16(2):335-343). Therefore, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Cell migration is also inhibited by the anti-inflammatory enzyme heme oxygenase-1 (HO-1), which inhibits Nlrp3 inflammasome (Leukemia. 2022;36(1):23-32). It is well known that circadian rhythm is regulated by the release of melatonin from the pineal gland, which also has some anti-inflammatory effects (J. Pineal Res. 2018;65:e12525). Aim of the study.Since stem cell trafficking is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from bone marrow (BM) into PB by inhibiting Nlrp3 inflammasome activation. Materials and Methods. To test this hypothesis, first, we tested the expression of melatonin receptors in murine and human HSPCs. We employed Transwell system to see the effect of melatonin on the migration of human and murine HSPCs to the SDF-1 gradient. Next, we performed pharmacological mobilization of normal mice with G-CSF or AMD3100 in the presence of melatonin. Finally, employing Glow assay, we evaluated the effect of melatonin on activation of Nlrp3 inflammasome in HSPCs stimulated by BM chemotactic factors and the effect of melatonin on expression of migration inhibitory HO-1. Since membrane lipid rafts (MLRs) are crucial for the proper response of cells to chemoattractants, we also evaluated the effect of melatonin on the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in the synthesis of cholesterol and sphingolipids that are major constituents of MLRs. Results. We noticed that HSPCs express melatonin receptors, and melatonin inhibits in vitro Transwell assays migration of HSPCs and negatively affects mobilization of these cells into PB in response to G-CSF and AMD3100 administration. We also observed that melatonin inhibits Nlrp3 inflammasome activation and the formation of MLRs. This is explained by a decrease in the expression of enzymes such as SREBP2, HMGCs, and HMGCR involved in cholesterol synthesis leading to impaired formation of MLRs. Moreover, melatonin increases in HSPCs expression of heme oxygenase-1 (HO-1), that is a known inhibitor of Nlrp3 inflammasome and a negative regulator of cell migration. Conclusions. We provide for the first-time evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs in vitro assays and in vivo their release from BM in response to G-CSF and AMD3100. This is correlated with inhibition of cholesterol synthesis, which results in defective assembly of MLRs, as well as a decrease in Nlrp3 inflammasome activity and an increase in expression of HO-1. Since melatonin is a commonly used shelf drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more light on molecular mechanisms of melatonin involvement in the diurnal circulation of HSPCs.

Novel evidence that the P2X1 purinergic receptor-Nlrp3 inflammasome axis orchestrates optimal trafficking of hematopoietic stem progenitors cells.

Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking. We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking. We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499. This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs.

The P2X4 purinergic receptor has emerged as a potent regulator of hematopoietic stem/progenitor cell mobilization and homing-a novel view of P2X4 and P2X7 receptor interaction in orchestrating stem cell trafficking.

Recent evidence indicates that extracellular adenosine triphosphate (eATP), as a major mediator of purinergic signaling, plays an important role in regulating the mobilization and homing of hematopoietic stem progenitor cells (HSPCs). In our previous work we demonstrated that eATP activates the P2X7 ion channel receptor in HSPCs and that its deficiency impairs stem cell trafficking. To learn more about the role of the P2X purinergic receptor family in hematopoiesis, we phenotyped murine and human HSPCs with respect to the seven P2X receptors and observed that, these cells also highly express P2X4 receptors, which shows ~50% sequence similarity to P2X7 subtypes, but that P2X4 cells are more sensitive to eATP and signal much more rapidly. Using the selective P2X4 receptor antagonist PSB12054 as well as P2X4-KO mice, we found that the P2X4 receptor, similar to P2X7 receptor, promotes trafficking of HSPCs in that its deficiency leads to impaired chemotaxis of HSPCs in response to a stromal-derived factor 1 (SDF-1) gradient, less effective pharmacological mobilization, and defective homing and engraftment of HSPCs after transplantation into myeloablated hosts. This correlated with a decrease in SDF-1 expression in the BM microenvironment. Overall, our results confirm the proposed cooperative dependence of both receptors in response to eATP signaling. In G-CSF-induced mobilization, a lack of one receptor is not compensated by the presence of the other one, which supports their mutual dependence in regulating HSPC trafficking.

Danger-associated molecular pattern molecules take unexpectedly a central stage in Nlrp3 inflammasome\u2013caspase-1-mediated trafficking of hematopoietic stem/progenitor cells

Like their homing after transplantation to bone marrow (BM), the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not fully understood, and several overlapping pathways are involved. Several years ago our group proposed that sterile inflammation in the BM microenvironment induced by pro-mobilizing agents is a driving force in this process. In favor of our proposal, both complement cascade (ComC)-deficient and Nlrp3 inflammasome-deficient mice are poor G-CSF and AMD3100 mobilizers. It is also known that the Nlrp3 inflammasome mediates its effects by activating caspase-1, which is responsible for proteolytic activation of interleukin-1β (IL-1β) and interleukin-18 (IL-18) and their release from cells along with several danger-associated molecular pattern molecules (DAMPs). We observed in the past that IL-1β and IL-18 independently promote mobilization of HSPCs. In the current work we demonstrated that caspase-1-KO mice are poor mobilizers, and, to our surprise, administration of IL-1β or IL-18, as in the case of Nlrp3-KO animals, does not correct this defect. Moreover, neither Caspase-1-KO nor Nlrp3-KO mice properly activated the ComC to execute the mobilization process. Interestingly, mobilization in these animals and activation of the ComC were both restored after injection of the DAMP cocktail eATP+HGMB1+S100A9, the components of which are normally released from cells in an Nlrp3 inflammasome–caspase-1-dependent manner. In addition, we report that caspase-1-deficient HSPCs show a decrease in migration in response to BM homing factors and engraft more poorly after transplantation. These results for the first time identify caspase-1 as an orchestrator of HSPC trafficking.

Extracellular Adenosine Triphosphate (eATP) and Its Metabolite, Extracellular Adenosine (eAdo), as Opposing "Yin-Yang" Regulators of Nlrp3 Inflammasome in the Trafficking of Hematopoietic Stem/Progenitor Cells.

Nlrp3 inflammasome plays a pleiotropic role in hematopoietic cells. On the one hand, physiological activation of this intracellular protein complex is crucial to maintaining normal hematopoiesis and the trafficking of hematopoietic stem progenitor cells (HSPCs). On the other hand, its hyperactivation may lead to cell death by pyroptosis, and prolonged activity is associated with sterile inflammation of the BM and, as a consequence, with the HSPCs aging and origination of myelodysplasia and leukemia. Thus, we need to understand better this protein complex’s actions to define the boundaries of its safety window and study the transition from being beneficial to being detrimental. As demonstrated, the Nlrp3 inflammasome is expressed and active both in HSPCs and in the non-hematopoietic cells that are constituents of the bone marrow (BM) microenvironment. Importantly, the Nlrp3 inflammasome responds to mediators of purinergic signaling, and while extracellular adenosine triphosphate (eATP) activates this protein complex, its metabolite extracellular adenosine (eAdo) has the opposite effect. In this review, we will discuss and focus on the physiological consequences of the balance between eATP and eAdo in regulating the trafficking of HSPCs in an Nlrp3 inflammasome-dependent manner, as seen during pharmacological mobilization from BM into peripheral blood (PB) and in the reverse mechanism of homing from PB to BM and engraftment. We propose that both mediators of purinergic signaling and the Nlrp3 inflammasome itself may become important therapeutic targets in optimizing the trafficking of HSPCs in clinical settings.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)-P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1-7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs.Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP-P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP-P2X4 axis is, like the eATP-P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP-P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes. Disclosures No relevant conflicts of interest to declare.

Pannexin-1 channel \u201cfuels\u201d by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

Novel Evidence That the Ectonucleotidases CD39 and CD73, Which Are Expressed on Hematopoietic Stem/Progenitor Cells (HSPCs), Regulate Mobilization and Homing - Studies in CD39-/- and CD73-/- Mice and with Small-Molecule CD39 and CD73 Inhibitors

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by extracellular nucleotides (EXNs), including most importantly the purine ATP and its nucleoside metabolite, adenosine. Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. ATP, the most important EXN, is secreted from cells through pannexin and connexin channels and is processed in the extracellular space to ADP, AMP and finally adenosine by the cell-surface-expressed ectonucleotidases CD39 and CD73. We recently reported that the ATP-adenosine axis regulates the mobilization of hematopoietic stem progenitor cells (HSPCs) (Leukemia 2018, in press,doi: 10.1038/s41375-018-0122-0). Hypothesis. We hypothesized that the ectonucleotidases CD39 and CD73 are important modulators of the ATP-adenosine axis and are new and underappreciated modulators, not only of mobilization but also of bone marrow (BM) homing of HSPCs. Materials and Methods. To shed more light on the role of purinergic signaling in the trafficking of HSPCs, we i) phenotyped murine and human HSPCs for expression of CD39 and CD73 ectonucleotidases by FACS, ii) performed mobilization and homing studies in CD39-/- and CD73-/- mice, and iii) blocked CD39 and CD73 activity by employing the small-molecule inhibitors ARL 67156 and AMP-CP, respectively. CD39- and CD73-deficient mice were mobilized with G-CSF or AMD3100, and wild type (WT) animals were exposed to CD39 and CD73 inhibitors in addition to mobilizing agents. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. To address the involvement of CD39 and CD73 expressed on the surface of HSPCs or in the hematopoietic microenvironment, we created irradiation chimeras that were subsequently mobilized with G-CSF or AMD3100. Bone marrow (BM) transplantation studies were performed by transplanting WT mice with WT BM cells or BM cells from ectonucleotidase-deficient mice. Homing efficiency was evaluated by determining i) the number of fluorochrome-labeled cells in BM 24 hours after transplantation ii) the number of CFU-S and CFU-GM progenitors that had engrafted in BM on day 12, and iii) the recovery of peripheral blood counts. Results. We found that CD39 and CD73 are expressed by murine and human HSPCs and process ATP to adenosine, which as we observed, is a novel and potent inhibitor of HSPC trafficking. In support of this finding, CD73-/- and CD39-/- mice, with reduced extracellular adenosine levels, mobilize HSPCs much better than their normal control littermates. A similar effect was observed in WT mice exposed to CD39 and CD73 small-molecule inhibitors during mobilization. Studies in irradiation chimeras revealed that this effect depended on expression of CD39 and CD73 on the surface of HSPCs and not in the BM microenvironment. Moreover, adenosine also inhibited the homing of HSPCs to BM, as cells from CD39-/- or CD73-/- mice engrafted better than WT cells. Furthermore, homing was also inhibited in the presence of exogenously added adenosine. That ectonucleotidase-deficient mice engrafted BM cells from WT littermates as efficiently as did WT recipients indicates the involvement of HSPC-expressed CD39 and CD73 in regulating the homing process. Conclusions. We demonstrate for the first time that HSPC-expressed CD39 and CD39 ectonucleotidases modulate the trafficking of HSPCs. By processing ATP to adenosine, these enzymes negatively affect both the mobilization and the homing processes. Based on this finding, future strategies employing small-molecule inhibitors of both ectonucleotidases could find practical application in improving mobilization in poor mobilizers and accelerating the engraftment of HSPCs after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Poor Mobilization in T-Cell-Deficient Nude Mice is Explained by Defective Activation of Granulocytes and Monocytes

It has been reported that both SCID mice and SCID patients poorly mobilize hematopoietic stem/progenitor cells (HSPCs) in response to granulocyte colony-stimulating factor (G-CSF). This defect has been proposed to result from a lack of naturally occurring IgM immunoglobulins to trigger activation of the complement cascade (ComC) and release of C5 cleavage fragments crucial in the mobilization process. However, SCID individuals also have T-cell deficiency, and T cells have been shown to modulate trafficking of HSPCs. To learn more about the role of T lymphocytes, we performed mobilization studies in T-lymphocyte-deficient nude mice and found that these mice respond poorly to G-CSF and zymosan but are normal mobilizers in response to AMD3100. Since nude mice have normal levels of IgM immunoglobulins in peripheral blood and may activate the ComC, we focused on the potential involvement of Gr1+ granulocytes and monocytes, which show defective maturation in these animals. Using a nude mouse mobilization model, we found further support for the proposition that proper function of Gr1+ cells is crucial for optimal mobilization of HSPCs.

Novel Evidence That Hematopoietic-Specific PLC-β2 Is Required for Normal Homing and Engraftment of Hematopoietic Stem Cells

Background. We recently identified phospholipase C-β2 (PLC-β2) as the first lipolytic enzyme known to be involved in the mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) (Leukemia 2016, 4:919-28). The phospholipase C (PLC) family of enzymes consists of 13 members in six subfamilies, including the PLC-δ (1, 3, 4), -β (1-4), -γ (1, 2), -ε, -ζ, and -η isoforms. PLC enzymes are associated with cell surface receptors that convert phosphatidyloinositol-4,5-biphosphate into two important second messengers, diacylglycerol (DAG) and inositol-1,4.5-triphosphate (IP3). Among these isoforms, PLC-β2 is somewhat unique in being a hematopoietic-specific enzyme. Moreover, PLC-β2, when released extracellularly from granulocytes and HSPCs upon stimulation, cleaves glycolipid glycosylphosphatidylinositol anchor (GPI-A) in cell membranes and thus disrupts the structure of membrane lipid rafts, which are important in the retention of HSPCs in BM niches. It is well known that the major BM-retention receptors for HSPCs, CXCR4 and VLA-4, are membrane lipid raft-associated receptors. What is important for the current study, while performing mobilization studies, we previously found that BM cells from these animals show somewhat reduced chemotaxis in response to several chemoattractants involved in BM homing.Aim of the Study. Based on this latter observation, we became interested in the potential role of PLC-β2 in regulating the migration of HSPCs, as this enzyme could be potentially involved in BM homing and engraftment of HSPCs after transplantation. We also explored the potential relationship between PLC-β2 and heme oxygenase 1 (HO-1), which is, as we recently demonstrated, a negative regulator of HSPC trafficking (Cell Transplant. 2016 epub).Materials and Methods. We performed i) homing, ii) short-term engraftment, and iii) long-term competitive engraftment studies in WT mice transplanted with BM cells derived from PLC-β2-/- mice and control (WT) animals. Chemotaxis and adhesion of PLC-β2-/- HSPCs was evaluated in Transwell dishes in response to SDF-1, S1P, C1P, and ATP. The expression of HO-1 was evaluated by real-time PCR and western blotting. Finally, both HSPCs and Gr-1+ cells were evaluated for release of Ca2+ upon stimulation by homing factors.Results. We report here for first time that, besides its role in the release of HSPCs from BM niches, PLC-β2 also regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including stromal derived factor-1 (SDF-1), sphingosine-1 phosphate (S1P), ceramide-1 phosphate C1P, and adenosine triphosphate (ATP). Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release and calcium signaling in cells from PLC-β2-KO mice and a high baseline level of HO-1, an enzyme that negatively regulates cell migration, in PLC-β2-KO mouse cells. Downregulation of HO-1 activity in HSPCs improved homing of PLC-β2-/- BM cells.Conclusions. PLC-β2 plays an important role in homing and engraftment of HSPCs. The decrease in migration of HSPCs in PLC-β2-KO mice can be explained by impaired calcium release and a high baseline level of HO-1, an enzyme that negatively regulates cell migration and our data provides evidence that downregulation of HO-1 activity in HSPCs by small molecular inhibitors could become a new strategy to improve homing and engraftment after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Inducible Nitric Oxide Synthase (iNOS) Is a Novel Negative Regulator of Hematopoietic Stem/Progenitor Cell Trafficking

Nitric oxide (NO) is a gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. Based on reports that NO inhibits migration of granulocytes and monocytes, we became interested in the role of inducible NO synthetase (iNOS) in pharmacological mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). To address the role of NO in HSPC trafficking, we upregulated or downregulated iNOS expression in hematopoietic cell lines. Next, we performed mobilization studies in iNOS−/− mice and evaluated engraftment of iNOS−/− HSPCs in wild type (control) animals. Our results indicate that iNOS is a novel negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during mobilization. At the molecular level, downregulation of iNOS resulted in downregulation of heme oxygenase 1 (HO-1), and, conversely, upregulation of iNOS enhanced HO-1 activity. Since HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS identified by us can be at least partially explained by its enhancing the HO-1 level in BM cells.

Novel evidence that crosstalk between the complement, coagulation and fibrinolysis proteolytic cascades is involved in mobilization of hematopoietic stem/progenitor cells (HSPCs)

The role of blood proteinases in the mobilization of hematopoietic stem/progenitor cells (HSPCs) is still not well understood. As previously reported, activation of the complement cascade (ComC) and cleavage of C5 by C5 convertase are enabling events in the release of C5a that plays a crucial role in the egress of HSPCs from bone marrow (BM) into peripheral blood (PB) and explains why C5-deficient mice are poor mobilizers. Here we provide evidence that during granulocyte colony-stimulating factor- and AMD3100-induced mobilization, not only the ComC but also two other evolutionarily ancient proteolytic enzyme cascades, the coagulation cascade (CoaC) and the fibrynolytic cascade (FibC), become activated. Activation of all three cascades was measured by generation of C5a, decrease in prothrombin time and activated partial thromboplastin time as well as an increase in the concentrations of plasmin/antiplasmin and thrombin/antithrombin. More importantly, the CoaC and FibC, by generating thrombin and plasmin, respectively, provide C5 convertase activity, explaining why mobilization of HSPCs in C3-deficient mice, which do not generate ComC-generated C5a convertase, is not impaired. Our observations shed more light on how the CoaC and FibC modulate stem cell mobilization and may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, as it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (for example, infections, tissue/organ damage or strenuous exercise) and show a circadian rhythm of activation, they must be involved in both stress-induced and circadian changes in HSPC trafficking in PB.

Novel Evidence That Crosstalk Between Three Evolutionarily Ancient Proteolytic Enzyme Cascades (coagulation, fibrinolysis, and complement) Plays An Important Role In Mobilization Of Hematopoietic Stem/Progenitor Cells (HSPCs)

BackgroundHematopoietic stem/progenitor cells (HSPCs) residing in BM are released from their niches and circulate under steady-state conditions at detectable levels in the peripheral blood (PB), and their number increases in response to i) systemic or local inflammation, ii) strenuous exercise, iii) stress, iv) tissue/organ injury, and v) pharmacological agents. All these processes involve activation of the complement cascade (ComC), and while mice deficient in the complement protein C3, which is an upstream component of the ComC, are easy mobilizers (Blood 2004; 103:2071), mice deficient in a downstream component of ComC, complement protein 5 (C5), are very poor mobilizers (Leukemia 2009; 23:2052). To explain these observations, it has been suggested that during the mobilization process the C5a cleavage fragment stimulates release of proteolytic enzymes from BM-residing myeloid cells, which attenuates SDF-1–CXCR4 and VLA4–VCAM-1 retention signals in BM niches. In addition, C5a generated in BM sinusoids is a potent chemoattractant for granulocytes and monocytes, which, as the first cells egressing from the BM, play an important role in permeabilization of the BM–PB barrier and thus facilitate the subsequent egress of HSPCs. It is also known that activation of ComC is based on stepwise activation of the cascade of proteolytic pro-enzymes, and thus the lack of upstream C3 should theoretically affect generation of C5 convertase, which is a proteolytic enzyme activating a downstream component of ComC (C5). However, surprisingly, C3–/– mice are easy mobilizers (Blood 2004; 103:2071). HypothesisTo explain how C5 can be activated during the mobilization process even when C3 is missing, we hypothesized that other proteases that are products of the activated coagulation cascade (CoaC) and fibrynolytic cascade (FibC) compensate for the lack of proteolytic activity of ComC-derived C5 convertase. Materials and MethodsIn our experiments 2-month-old C3-deficient mice (C3–/–) and normal wild type (WT) littermates were mobilized for 6 days by G-CSF in the presence or absence of selected CoaC and FibC inhibitors such as refludan (a direct inhibitor of thrombin) and tranexamic acid (an inhibitor of plasminogen activation). Following mobilization, we measured in PB i) the total number of white blood cells (WBC), ii) the number of circulating clonogenic CFU-GM, and iii) the number of Sca-1+c-kit+lineage– (SKL) cells. In parallel, we measured the activation of C5 by measuring the level of C5a and evaluated activation of CoaC by measuring prothrombin (PT) and activated partial thromboplastin times (APTT) as well as thrombin/antithrombin (TAT) and plasmin/antiplasmin (PAP) complexes. ResultsWe observed that G-CSF-induced mobilization of HSPCs was significantly reduced in easy-mobilizing C3–/– mice if the mice were treated during mobilization with refludan (a CoaC inhibitor) or tranexamic acid (an FibC inhibitor). This reduction correlated with significant inhibition of C5 activation/cleavage. More importantly, we also noticed that inhibitors of CoaC and FibC had a negative effect on mobilization of HSPCs in normal WT animals. The activation of ComC, CoaC, and FibC in mice mobilized with G-CSF was confirmed by an increase in C5a level (Figure 1A) and by measuring PT and APTT time (Figure 1B) as well as TAT and PAP complexes (Figure 1C). ConclusionsThe data presented in this work demonstrate, for the first time, the existence of crosstalk between all three evolutionarily ancient proteolytic enzyme cascades, ComC, CoaC, and FibC, in the mobilization process of HSPCs. These results also confirm that C5, which plays an important role in egress of HSPCs from the BM, can be activated/cleaved during mobilization not only by ComC-generated C5 convertase but in addition by proteolytic enzymes of CoaC and FibC. Our observations of crosstalk between ComC, CoaC, and FibC may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, since it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (e.g., infections, tissue/organ damage, or strenuous exercise) and show a circadian rhythm of activation due to a drop in blood pH during deep sleep at night, they are involved both in stress-induced as well as in circadian changes in HSPC trafficking in PB. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expansion of bone marrow neutrophils following G-CSF administration in mice results in osteolineage cell apoptosis and mobilization of hematopoietic stem and progenitor cells

Proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) within bone marrow (BM) niches are regulated by adhesion molecules and cytokines produced by mesenchymal stem/progenitor cells (MPC) and osteoblasts (OB). HSPCs that egresses to peripheral blood are widely used for transplant and granulocyte-colony stimulating factor_(G-CSF) is used clinically to induce mobilization. The mechanisms, through which G-CSF regulates HSPC trafficking however, are not completely understood. Herein we show that G-CSF driven neutrophil expansion alters the BM niche that leads to HSPC mobilization. Alcam−Sca-1+MPC and Alcam+Sca-1− OB are reduced coincident with mobilization, which correlates inversely with BM neutrophil expansion. In mice made neutropenic by the neutrophil specific anti-Ly6G antibody, G-CSF mediated reduction in MPC and OB is attenuated and mobilization reduced without an effect on monocytes/macrophages. Neutrophils, expanded in response to G-CSF induce MPC and OB apoptosis leading to reduced production of BM HSPC retention factors including stromal cell derived factor-1 (SDF-1), stem cell factor (SCF) and vascular cell adhesion molecule-1(VCAM-1). Blockade of neutrophil reactive oxygen species (ROS) attenuates G-CSF mediated MPC and OB apoptosis. These data show that the expansion of BM neutrophils by G-CSF contributes to the transient degradation of retention mechanisms within the BM niche, facilitating enhanced HSPC egress/mobilization.