Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow.

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E(2) (PGE(2)) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA(+) activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions.

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact-dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34(+) progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP-protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P(1), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S1P(1) in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P(1) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

B-cell depletion reactivates B lymphopoiesis in the BM and rejuvenates the B lineage in aging.

Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. These changes decrease the body's ability to mount protective antibody responses. We show here that age-related changes in the B lineage are mediated by the accumulating long-lived B cells. Thus, depletion of B cells in old mice was followed by expansion of multipotent primitive progenitors and common lymphoid progenitors, a revival of B-lymphopoiesis in the bone marrow, and generation of a rejuvenated peripheral compartment that enhanced the animal's immune responsiveness to antigenic stimulation. Collectively, our results suggest that immunosenescence in the B-lineage is not irreversible and that depletion of the long-lived B cells in old mice rejuvenates the B-lineage and enhances immune competence.

Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Chronic B cell deficiency from birth prevents age-related alterations in the B lineage.

Aging is accompanied by a decline in B lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. The mechanisms underlying these changes are unclear. To explore whether aging in the B lineage is subjected to homeostatic regulation, we used mutant mice bearing chronic B cell deficiency from birth. We show that chronic B cell deficiency from birth, resulting from impaired maturation (CD19(-/-) and CD74(-/-)) or reduced survival (baff-r(-/-)), prevents age-related changes in the B lineage. Thus, frequencies of early and late hematopoietic stem cells, B lymphopoiesis, and the rate of B cell production do not substantially change with age in these mice, as opposed to wild-type mice where kinetic experiments indicate that the output from the bone marrow is impaired. Further, we found that long-lived B cells did not accumulate and peripheral repertoire was not altered with age in these mice. Collectively, our results suggest that aging in the B lineage is not autonomously progressing but subjected to homeostatic regulation.

Heparanase regulates retention and proliferation of primitive Sca-1+/c-Kit+/Lin- cells via modulation of the bone marrow microenvironment.

Heparanase is involved in tumor growth and metastasis. Because of its unique cleavage of heparan sulfate, which binds cytokines, chemokines and proteases, we hypothesized that heparanase is also involved in regulation of early stages of hematopoiesis. We report reduced numbers of maturing leukocytes but elevated levels of undifferentiated Sca-1(+)/c-Kit(+)/Lin(-) cells in the bone marrow (BM) of mice overexpressing heparanase (hpa-Tg). This resulted from increased proliferation and retention of the primitive cells in the BM microenvironment, manifested in increased SDF-1 turnover. Furthermore, heparanase overexpression in mice was accompanied by reduced protease activity of MMP-9, elastase, and cathepsin K, which regulate stem and progenitor cell mobilization. Moreover, increased retention of the progenitor cells also resulted from up-regulated levels of stem cell factor (SCF) in the BM, in particular in the stem cell-rich endosteum and endothelial regions. Increased SCF-induced adhesion of primitive Sca-1(+)/c-Kit(+)/Lin(-) cells to osteoblasts was also the result of elevation of the receptor c-Kit. Regulation of these phenomena is mediated by hyperphosphorylation of c-Myc in hematopoietic progenitors of hpa-Tg mice or after exogenous heparanase addition to wildtype BM cells in vitro. Altogether, our data suggest that heparanase modification of the BM microenvironment regulates the retention and proliferation of hematopoietic progenitor cells.

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

Daily light and darkness onset and circadian rhythms metabolically synchronize hematopoietic stem cell differentiation and maintenance: The role of bone marrow norepinephrine, tumor necrosis factor, and melatonin cycles.

Hematopoietic stem and progenitor cells (HSPCs) are essential for daily mature blood cell production, host immunity, and osteoclast-mediated bone turnover. The timing at which stem cells give rise to mature blood and immune cells while maintaining the bone marrow (BM) reservoir of undifferentiated HSPCs and how these opposite tasks are synchronized are poorly understood. Previous studies revealed that daily light onset activates norepinephrine (NE)-induced BM CXCL12 downregulation, followed by CXCR4+ HSPC release to the circulation. Recently, we reported that daily light onset induces transient elevations of BM NE and tumor necrosis factor (TNF), which metabolically program BM HSPC differentiation and recruitment to replenish the blood. In contrast, darkness onset induces lower elevations of BM NE and TNF, activating melatonin production, which metabolically reprograms HSPCs, increasing their short- and long-term repopulation potential, and BM maintenance. How the functions of BM-retained HSPCs are influenced by daily light and darkness cycles and their clinical potential are further discussed.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

An MTCH2 pathway repressing mitochondria metabolism regulates haematopoietic stem cell fate.

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2's ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor-positive (EPCR(+)) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α-converting enzyme (TACE), enhanced CXCL12-CXCR4-induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR(+) LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NO(low) EPCR(+) LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR(+) LT-HSCs, with potential clinical relevance for stem cell transplantation.

Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment.

SIGNIFICANCE: Blood forming, hematopoietic stem cells (HSCs) mostly reside in the bone marrow in a quiescent, nonmotile state via adhesion interactions with stromal cells and macrophages. Quiescent, proliferating, and differentiating stem cells have different metabolism, and accordingly different amounts of intracellular reactive oxygen species (ROS). Importantly, ROS is not just a byproduct of metabolism, but also plays a role in stem cell state and function. RECENT ADVANCES: ROS levels are dynamic and reversibly dictate enhanced cycling and myeloid bias in ROS(high) short-term repopulating stem cells, and ROS(low) quiescent long-term repopulating stem cells. Low levels of ROS, regulated by intrinsic factors such as cell respiration or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity, or extrinsic factors such as stem cell factor or prostaglandin E2 are required for maintaining stem cell self-renewal. High ROS levels, due to stress and inflammation, induce stem cell differentiation and enhanced motility. CRITICAL ISSUES: Stem cells need to be protected from high ROS levels to avoid stem cell exhaustion, insufficient host immunity, and leukemic transformation that may occur during chronic inflammation. However, continuous low ROS production will lead to lack of stem cell function and opportunistic infections. Ultimately, balanced ROS levels are crucial for maintaining the small stem cell pool and host immunity, both in homeostasis and during stress situations. FUTURE DIRECTIONS: Deciphering the signaling pathway of ROS in HSC will provide a better understanding of ROS roles in switching HSC from quiescence to activation and vice versa, and will also shed light on the possible roles of ROS in leukemia initiation and development.

Dynamic Cross Talk between S1P and CXCL12 Regulates Hematopoietic Stem Cells Migration, Development and Bone Remodeling.

Hematopoietic stem cells (HSCs) are mostly retained in a quiescent non-motile mode in their bone marrow (BM) niches, shifting to a migratory cycling and differentiating state to replenish the blood with mature leukocytes on demand. The balance between the major chemo-attractants CXCL12, predominantly in the BM, and S1P, mainly in the blood, dynamically regulates HSC recruitment to the circulation versus their retention in the BM. During alarm situations, stress-signals induce a decrease in CXCL12 levels in the BM, while S1P levels are rapidly and transiently increased in the circulation, thus favoring mobilization of stem cells as part of host defense and repair mechanisms. Myeloid cytokines, including G-CSF, up-regulate S1P signaling in the BM via the PI3K pathway. Induced CXCL12 secretion from stromal cells via reactive oxygen species (ROS) generation and increased S1P1 expression and ROS signaling in HSCs, all facilitate mobilization. Bone turnover is also modulated by both CXCL12 and S1P, regulating the dynamic BM stromal microenvironment, osteoclasts and stem cell niches which all functionally express CXCL12 and S1P receptors. Overall, CXCL12 and S1P levels in the BM and circulation are synchronized to mutually control HSC motility, leukocyte production and osteoclast/osteoblast bone turnover during homeostasis and stress situations.

GSK3ß regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement.

Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3ß (GSK3ß) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3ß in the BM. Nevertheless, GSK3ß signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3ß compared with mature cells was selectively enhanced by stem cell factor-induced activation of GSK3ß. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3ß expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3ß signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3ß in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.

MT1-MMP and RECK: opposite and essential roles in hematopoietic stem and progenitor cell retention and migration.

Migratory capacity is a fundamental property of hematopoietic stem and progenitor cells (HSPCs). This feature is employed in clinical mobilization of HSPCs to the circulation and constitutes the basis for modern bone marrow (BM) transplantation procedures which are routinely used to treat hematological malignancies. Therefore, characterization of new players in the complex process of HSPC motility in steady-state conditions as well as during stress situations is a major challenge. We report that while the metalloproteinase membrane type 1-metalloprotease (MT1-MMP) has an essential role in human HSPC trafficking during granulocyte colony-stimulating factor (G-CSF)-induced mobilization, its inhibitor reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and the adhesion molecule CD44 are required for HSPC retention to the BM in steady-state conditions. The nervous system via Wnt signaling along with HGF/c-Met signaling and the complement cascade play a major role in regulating MT1-MMP increased activity, CD44 cleavage, and RECK-reduced expression during G-CSF-induced mobilization. This review will elaborate on the opposite roles of MT1-MMP and RECK in HSPC migration and retention and suggest targeting them in order to facilitate HSPC mobilization and engraftment upon BM transplantation in patients.