Regulation of CXCR1, CXCR2 and CXCR4 in Human Neutrophils: Potential Role in the Release from the Bone Marrow, Clearance of Senescent Cells, and Cell Function at Sites of Inflammation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3068-3068
Author(s):  
Sedat Yildirim ◽  
Frank Bautz ◽  
Andreas M. Boehmler ◽  
Lothar Kanz ◽  
Robert Möhle
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Northern Blot Analysis ◽  
Cell Function ◽  
Ex Vivo ◽  
Cxcr4 Expression ◽  
Human Neutrophils ◽  
Similar Time ◽  
Cxcr4 Mrna ◽  
Ex Vivo Culture

Abstract In the mouse model, it has been shown that the interleukin-8 (IL-8) receptor CXCR2 is involved in the release of mature neutrophils from the bone marrow into the circulation. When neutrophils age, upregulation of CXCR4 and downmodulation of CXCR2 result in homing and subsequent sequestration of senescent cells in the bone marrow. In our study, we observed a similar time-dependent (starting at 3 hrs., maximum at 12–18 hrs.) downregulation of CXCR2 in human neutrophils during aging in ex vivo culture, while expression of the second IL-8 receptor CXCR1, which is mainly responsible for the IL-8-induced chemotaxis, was unchanged. Furthermore, strong upregulation of CXCR4 was noted on the cell surface which could not solely be attributed to re-expression of internalized, intracellular receptors, as an increased amount of CXCR4 mRNA was detected by Northern blot analysis in these cells. The increase in CXCR4 expression was not influenced by inflammatory cytokines such as TNF and IL-1, as well as by IL-8 or G-CSF. Accordingly, SDF-1-induced transendothelial migration of aged neutrophils was 6-fold increased and even exceeded migration in response to IL-8. We conclude that also in human neutrophils, loss of CXCR2 and gain of CXCR4 expression on the cell surface may favor homing and sequestration of senescent cells in the bone marrow. At sites of inflammation however, retained expression of CXCR1 and increased expression of CXCR4 still allow a response of aged, pre-apoptotic neutrophils to the chemotactic mediators IL-8 and SDF-1, as the latter is not only released in the bone marrow, but also at sites of tissue damage and necrosis.

Enhanced Homing of HSPCs After Treatment with Prostaglandin E2 (PGE2) May Be An Effect of Transcriptional Regulation of CXC Chemokine Receptor 4 (CXCR4) Through Hypoxia Inducible Factor 1α (HIF1α)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 918-918 ◽  
Author(s):  
Jennifer M Speth ◽  
Jonathan Hoggatt ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Surface ◽  
Transcriptional Activity ◽  
Ex Vivo ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Cxcr4 Expression ◽  
Hematopoietic Stem ◽  
Hypoxia Inducible Factor 1Α

Abstract Abstract 918 Hematopoietic stem cell (HSC) transplantation is widely used to treat a number of hematological malignancies. However, to be effective, transplanted HSCs must efficiently “home” to their supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to impaired immune function and bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation outcomes. We have previously shown that treatment of mouse or human HSCs with 1μM 16–16 dimethyl PGE2 (dmPGE2) ex vivo increases their bone marrow homing efficiency and engraftment, coordinate with an increase in surface CXCR4 expression. The transcription factor Hypoxia Inducible Factor 1α (HIF1α) has been shown to up-regulate CXCR4 in certain cancer models. In addition, PGE2 has been shown to stabilize HIF1α under normoxic conditions. We hypothesized that the effects of PGE2 on CXCR4 expression and homing might result from stabilization of HIF1α and its subsequent transcriptional activity in the transplanted HSCs. Treatment of lineageneg bone marrow cells with 1μM dmPGE2 resulted in a 33 ± 8.6% increase in HIF1α protein levels over vehicle (p<0.05) measured by Western Blot analysis. In addition, treatment of HSC with the hypoxia mimetic dimethyloxallyl glycine (DMOG) also resulted in an increase in CXCR4 cell surface expression in mouse LinnegSca-1posc-kitpos (SKL) cells (11.3 ± 3.9% MFI over vehicle, p<0.05) and human CD34+ umbilical cord blood cells (20.1 ± 3.6% MFI over vehicle), which was comparable to treatment with dmPGE2. This increase in CXCR4 expression translated to a functional increase in SKL migration to SDF-1 (46.7 ± 1.5% migration compared to 35 ± 3.1% migration in vehicle, p<0.05). To determine whether the effects of PGE2 on CXCR4 were due effects on HIF1 transcriptional activity, we employed two mouse hepatoma cell lines (ARNT- and ARNT+). These cells express detectable levels of CXCR4 as well as all four of the PGE2 G-protein coupled receptors (EP1-4). However the ARNT- cell line lacks the HIF nuclear translocator, and thus lacks HIF1 transcriptional activity. While ARNT+ cells showed the characteristic increase in CXCR4 cell surface protein (91.3 ± 28.7%) and mRNA (5.69-fold increase) after treatment with 1μM dmPGE2, ARNT- cells failed to show a similar up-regulation of CXCR4 protein (15 ± 6.1% increase) and mRNA (0.44-fold increase) after PGE2 treatment. These data suggest that HIF1 transcriptional activity is necessary for PGE2-mediated CXCR4 regulation. To determine whether HIF1α stabilization has similar effects on HSC function after transplant, in vivo homing studies were performed. Pulse-treatment of mouse SKL cells with 5μM DMOG ex vivo for 1 hour resulted in a 2.9-fold increase in homing (p<0.005), compared to a 2.5-fold increase for cells treated with dmPGE2. Treatment of donor cells with the CXCR4 antagonist AMD3100 resulted in a statistically significant reduction in SKL homing (74.8 ± 9%, p<0.05), indicating that DMOG's positive effects on homing are mainly due to CXCR4 up-regulation and are similar to effects seen after dmPGE2 treatment. These data suggest the effects of PGE2 on CXCR4 expression are at least partially due to the stabilization of HIF1α. Due to the importance of HIF1α and its involvement in HSC regulation, PGE2 treatment could also have effects on other pathways associated with the hypoxic response mediated through HIF1α that could be targeted to modulate HSC function. Further studies could potentially identify more specific targets to improve the efficacy of HSCs after transplant. Disclosures: Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Differential Maintenance of Primitive Human SCID-Repopulating Cells, Clonogenic Progenitors, and Long-Term Culture-Initiating Cells After Incubation on Human Bone Marrow Stromal Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 641-650 ◽  
Author(s):  
Olga I. Gan ◽  
Barbara Murdoch ◽  
Andre Larochelle ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Stromal Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Long Term Culture ◽  
Cell Engraftment ◽  
Ex Vivo Culture

Abstract Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

Impaired Engraftment of Lineage-Depleted Marrow Cultured for Ex Vivo Gene Transfer in Submyeloablated Murine Hosts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5545-5545
Author(s):  
Brandon K. Wyss ◽  
Justin L. Meyers ◽  
Anthony L. Sinn ◽  
Shanbao Cai ◽  
Karen E. Pollok ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Cell Function ◽  
Ex Vivo ◽  
Transduction Efficiency ◽  
Cell Recovery ◽  
Donor Chimerism ◽  
Ex Vivo Culture ◽  
Cells Cultured ◽  
Marrow Cells

Abstract We previously demonstrated that engraftment of murine whole bone marrow (WBM) transduced with an oncoretroviral vector using an optimized 5-fluorouracil (5-FU)-based transduction protocol is reduced ~3-fold compared to fresh WBM upon transplantation into sublethally irradiated hosts, although competitive repopulating ability in ablated hosts is not decreased (Goebel et al., Exp. Hematol. 30:1324, 2002). We therefore sought to determine whether marrow cells transduced using a clinically relevant, non-5-FU-containing protocol would engraft more efficiently. Li et al. (Exp. Hematol. 31:1206, 2003) showed that lineage-depleted (lin−) marrow cells from donor mice not treated with 5-FU were effectively transduced, and repopulated myeloablated hosts. We hypothesized that ex vivo culture for gene transfer in the absence of 5-FU would lead to improved donor marrow engraftment in submyeloablated hosts. Lin− cells, isolated from B6.SJL (Boy J; CD45.1+) WBM using a Miltenyi kit and VarioMACS apparatus, were prestimulated in StemSpan serum-free medium with SCF and IL-6 for 48 hours, followed by overnight transduction on RetroNectin-coated plates preloaded with ecotropic SF1-EGFP retroviral supernatant. Cell recovery from the MACS column (1.9 ± 1.4%), bulk transduction efficiency (77.3 ± 12%), and lin− cell purity (58 ± 17%; all from 7–13 experiments) was similar to that previously described. Transplantation of 106 lin− transduced cells into 300 cGy-conditioned congenic C57Bl6/J (B6; CD45.2+) hosts produced only 1.4 ± 0.5% donor chimerism 4–6 months post-transplant, significantly lower than that observed using 106 fresh lin− cells (29 + 18.8%; N = 8–9 hosts each from 2–3 experiments). The percentage of EGFP+ cells in the donor population, nevertheless, was 55.6 ± 18%, indicating that stem cells were marked but engrafted poorly. The repopulating ability of transduced lin− marrow was reduced ~10-fold compared to fresh lin− cells as determined in competitive repopulation assays in ablated hosts. Together, these data suggest that lin cells cultured ex vivo for gene transfer acquired an engraftment defect despite the absence of 5-FU. Increasing the conditioning radiation dose to 550 cGy, a dose used in prior canine and non-human primate gene transfer studies, markedly improved donor chimerism following transplantation of 106 fresh lin− cells (90 ± 1.3% at 4 months, N = 5) or 106 transduced lin− cells (38.5 ± 14% at 2 months, N = 10), suggesting that greater reduction in host stem cell function may be needed for engraftment of cells cultured ex vivo for gene transfer. Ongoing studies to investigate the mechanism responsible for this engraftment defect indicate that expression of adhesion molecules important for homing and engraftment (CD29, 44, 49d, 49e, 62L), CXCR4 expression, and the percentage of cells actively cycling are not significantly altered by the transduction process, although functional studies are underway. The percentage of Sca-1+lin−c-kit+ (SLK) cells in the transduced cell pool is similar to that of freshly isolated lin− cells; thus, transplantation of lin− cells cultured ex vivo for gene transfer results in significantly lower donor chimerism than fresh lin− cells despite the grafts containing similar numbers of SLK cells. Secondary transplants and limiting dilution studies to determine stem cell self-renewal and engraftment capacity before and after ex vivo culture for gene transfer are in progress.

scholarly journals Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1624-1632
Author(s):  
NP Christiansen ◽  
KM Skubitz
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Lectin Affinity Chromatography ◽  
Con A ◽  
Major Surface

Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1677-1677
Author(s):  
Zejin Sun ◽  
Yanzhu Yang ◽  
Yan Li ◽  
Daisy Zeng ◽  
Jingling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

Hif1α and Rac1 Are Necessary For Enhanced HSPC Migration and Homing In Response To Prostaglandin E2 Treatment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 889-889
Author(s):  
Jennifer M. Speth ◽  
Maegan L. Capitano ◽  
Jonathan Hoggatt ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Prostaglandin E2 ◽  
Gene Transcription ◽  
Ex Vivo ◽  
Cxcr4 Expression ◽  
Direct Result ◽  
Pulse Treatment ◽  
Hematopoietic Stem ◽  
Hypoxia Response ◽  
Transformed Cell Lines

Abstract Hematopoietic stem cell (HSC) transplant is a lifesaving therapy for a number of different hematologic disorders, and takes advantage of the HSC’s ability to engraft and reconstitute the entire blood system. A critical step in the engraftment process is the ability of HSC’s to travel to or “home” to the bone marrow after transplant. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution. Enhancing HSC homing is one strategy to improve HSC function and transplantation efficiency. We have previously shown that ex vivo treatment of mouse HSCs with 16-16 dimethyl Prostaglandin E2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting from upregulation of the homing receptor CXCR4. The exact mechanism behind this effect, however, has not been defined. The transcription factor Hypoxia Inducible Factor 1α (HIF1α) has been shown to transcriptionally regulate CXCR4 in hypoxia, and contains three hypoxia response elements within its promoter. Furthermore, PGE2 has been shown to stabilize HIF1α under normoxic conditions in a number of transformed cell lines. We hypothesized that the effects of PGE2 on enhancing HSC CXCR4 expression and homing might be mediated through stabilization of HIF1α. Here we show that pulse-treatment of mouse bone marrow cells with dmPGE2 stabilizes HIF1α protein and increases HIF1α-dependent gene transcription in hematopoietic stem and progenitor cells (HSPCs). In addition, we show that similar treatment of HSPCs with the hypoxia mimetic dimethyloxalylglycine (DMOG) produces analogous effects to dmPGE2 on enhanced CXCR4 expression, gene transcription and functional migration, and produces a 2-fold enhanced homing effect as a direct result of CXCR4 upregulation. Limiting-dilution competitive transplants demonstrated a significant increase in peripheral blood chimerism using DMOG-treated cells compared to vehicle, which resulted from a ∼2-fold increase in HSC frequency. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP) results in abrogation of dmPGE2-induced CXCR4 upregulation and enhanced migration, confirming the requirement of HIF1α for these effects. In addition, the requirement for HIF1α in dmPGE2-enhanced in vivo homing was confirmed using a mouse model of conditional HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb upstream from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after dmPGE2 treatment. Unexpectedly, we also observed a significant increase in the small GTPase Rac1 after dmPGE2 treatment. Rac1 is necessary for successful HSC engraftment, and has been shown to affect cell sensitivity to SDF-1 through colocalization with CXCR4. Rac1 is also required for HIF1 activity, suggesting that it may be involved in regulation of HIF1α and CXCR4 after dmPGE2 treatment. Conditional knockout of Rac1, but not Rac2 in HSPCs results in lower basal levels of HIF1α protein, and loss of dmPGE2-enhanced migration and CXCR4 expression, supporting the hypothesis that Rac1 is necessary for downstream effects on HIF1α and CXCR4. Using an ImageStream flow cytometer and Bright Detail Similarity analysis, we observed an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment, which resulted in an approximate 10-fold enhanced HSPC sensitivity to SDF-1. Taken together, these data define a novel mechanism whereby ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing as a result of HIF1α and Rac1 modulation. We also define a new ex vivo pharmacologic strategy to improve HSC engraftment and repopulation, and for the first time, provide evidence that PGE2 can enhance HSPC function through modulation of Rac1. Disclosures: Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.

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