Prostaglandin E2 Is Rapidly Produced In Response to Bone Marrow Injury and Improves Survival of Primitive Hematopoietic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 407-407
Author(s):  
Rebecca L Porter ◽  
Laura M Calvi
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Prostaglandin E2 ◽  
Radiation Injury ◽  
Conflicts Of Interest ◽  
Lethal Dose ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic System ◽  
Post Radiation

Abstract Abstract 407 Since the hematopoietic system is exquisitely sensitive to environmental and iatrogenic injury, the bone marrow microenvironment likely provides protective mechanisms during times of injury or stress. We have previously demonstrated that prostaglandin E2 (PGE2), which can be produced by many cell types in the bone marrow, targets both the bone marrow microarchitecture and primitive hematopoietic cells when administered systemically to mice (Porter, Frisch et. al., Blood, 2009). Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. To test this hypothesis, we injured mice with a sub-lethal dose of gamma radiation, 6.5 Gy TBI, and sacrificed mice at varying time points from 1 hour to 6 days post-radiation. Bone marrow supernatant was collected and used for quantification of local PGE2 levels by ELISA. We found that, compared to non-irradiated mice, the PGE2 levels were increased greater than two-fold by 4 hours after irradiation (p=0.0030; n=3–6 mice/group), and these levels remain elevated until at least 6 days after injury (p<0.0001 by ANOVA). These data clearly demonstrate that PGE2 production is rapidly upregulated following bone marrow injury. To determine if HSPCs could be responding to this increase in local PGE2, we sorted Lin− c-Kit+ Sca1+ (LSK) cells from murine bone marrow and assayed the expression of the four PGE2 receptors, EP1-EP4. RT-PCR analysis demonstrated that all four receptors are expressed on LSK cells, suggesting that PGE2 could be acting on these primitive hematopoietic cells during times of injury. We next tested whether supplying additional PGE2 to mice could protect hematopoietic cells after injury. Mice were subjected to 6.5 Gy TBI and were treated with 0.5 mg/kg 16,16-dimethyl-PGE2 (dmPGE2) immediately after radiation and once daily thereafter until time of sacrifice. At 24 hours after radiation injury, mice that were treated with dmPGE2 had greater than 8-fold more surviving LSK cells, a population which still retains HSC repopulating activity in competitive transplantation studies, in their bone marrow compared with vehicle treated mice (n=4/group, p=0.046). Similarly, at 72 hr post-radiation, the dmPGE2 treated mice continued to have almost 2-fold greater numbers of LSK cells remaining viable in their bone marrow compared with vehicle treated mice (n=2–3/group). These data suggest that dmPGE2 treatment after bone marrow injury may provide protection, at least in the days immediately following injury, to primitive hematopoietic cells that remain capable of regenerating the hematopoietic system. To further support this idea, we also pretreated uninjured bone marrow cells in vitro with PGE2 (1 μ M) for 90 minutes and then exposed them to the chemotherapeutic agent cytarabine (Ara-C, 10 μ M for 4 hours). Pretreatment with PGE2 results in lower levels of apoptotic LSK cells compared with vehicle pre-treated LSK cells (30.26% vs. 39.02%; n=9/group; 3 independent experiments; p=0.0012). This result correlates with our in vivo radiation injury data and suggests that PGE2 may target primitive hematopoietic cells and render them more resistant to cell death from injury. Taken together, these results suggest that PGE2, which is released in the bone marrow after radiation exposure, may be an important microenvironmental regulator of HSPC response to injury, by preventing cell death, and/or increasing their recovery. Amplification of this physiological signal by treatment with exogenous PGE2 could provide a beneficial means of protecting hematopoietic cells in clinical situations of hematopoietic system injury and bone marrow transplantation, allowing patients to tolerate bone marrow suppressive treatments or to recover more easily. Further, these results also bring forth a potential concern about the safety of blocking prostaglandin synthesis by using anti-inflammatory medications during times of bone marrow injury. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Zebra "Fishing" the Role of Granulin in Hematopoiesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1194-1194
Author(s):  
Raquel Espin Palazon ◽  
Xiaoyi Cheng ◽  
Clyde A Campbell ◽  
Liangdao Li ◽  
Bettina Schmid ◽  
...  
Keyword(s):  
Animal Model ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Myeloid Differentiation ◽  
Hematopoietic System ◽  
High Conservation

Granulin (GRN) is a multifunctional protein with anti-inflammatory properties and involved in neurological diseases and tumorigenesis. It contains several cysteine-rich motifs that are unique to this protein, which are conserved from sponges to humans indicating their ancient evolutionary origin. Despite being highly expressed by certain hematopoietic cell lineages, the role that GRN plays in hematopoiesis has reminded elusive. The multifunctional nature of this protein, together with its wide expression in all mammalian cell types has challenged the characterization of its functional role in hematopoiesis due to its effects on other tissues. Therefore, we took advantage of the whole genomic duplication of the zebrafish (Danio rerio) and the high conservation of the cysteine-rich motifs among the zebrafish and human granulins to address this knowledge gap and explore their role in hematopoiesis in vivo. The whole genome duplication that separated teleost fish from mammals resulted in two copies of the granulin gene in the zebrafish (Granulin a and Granulin b, Grna and Grnb respectively). This has allowed us an unprecedented view into the function of this protein in hematopoiesis. We show that like mammals, grnb transcripts are found in all cell types, including hematopoietic cells. In contrast, grna is restricted to hematopoietic cells, including myeloid populations. The distinct cell expression of grna and grnb suggests that, in the zebrafish, grna evolved to specifically function in hematopoiesis, while grnb may have taken on the rest of the biological roles assigned to the mammalian granulin. The zebrafish is an animal model with unique advantages for in vivo studies. Its external development allows us to circumvent the challenges of in utero experimentation required using mammals, permitting the use of non-invasive imaging techniques to study developmental hematopoiesis. In addition, more than 70% of genes identified in the zebrafish are conserved in humans. These, together with its high conservation with the human hematopoietic system has led to a greater understanding and prevention of human hematologic diseases by using this elegant animal model. These unique advantages of the zebrafish, in addition to its genetic amenability allowed us to generate Grna and Grnb single mutants and identify their impact in the hematopoietic system in vivo. While the absence of Grnb did not affect the development of the hematopoietic system, lack of Grna led to decreased differentiation of myeloid precursors into neutrophils and macrophages. Therefore, Grna knockout allowed us to disrupt the hematopoietic function of granulin while keeping unaltered its function in the brain and other non-hematopoietic tissues. Although viable, adult Grna mutants developed kidney marrow (the fish analogous to the mammalian bone marrow) failure, with increased progenitors and decreased mature myeloid cells. Mechanistically, we found that pu.1, the main transcription factor that leads to myeloid differentiation, directly bound grna enhancers, upregulating its expression. We have demonstrated that Grna enhanced myeloid gene expression, and decreased gata1 expression thereby facilitating myeloid differentiation and inhibiting the erythroid genetic program. Finally, we show that these findings in the zebrafish are also conserved in humans. Altogether, we have identified the hematopoietic role of granulin without disturbing its biological functions in other tissues. We have unveiled a powerful and novel master regulator for myeloid differentiation that could potentially be utilized for the treatment of hematological disorders such as neutropenia and leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals CXCR4 is required for the quiescence of primitive hematopoietic cells

2008 ◽  
Vol 205 (4) ◽  
pp. 777-783 ◽  
Author(s):  
Yuchun Nie ◽  
Yoon-Chi Han ◽  
Yong-Rui Zou
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Regulatory Factor ◽  
Cell Compartment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Stem Cell Compartment

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

scholarly journals Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1165-1172 ◽  
Author(s):  
Russell S. Taichman ◽  
Marcelle J. Reilly ◽  
Rama S. Verma ◽  
Stephen G. Emerson
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Transforming Growth Factor ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Colony Stimulating Factor ◽  
Cd34 Cells ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.

Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 73-73
Author(s):  
Rebecca L Porter ◽  
Benjamin J Frisch ◽  
Regis J O’Keefe ◽  
Laura M Calvi
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Prostaglandin E2 ◽  
Flow Cytometric Analysis ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Fate Decisions ◽  
Flow Cytometric ◽  
Incorporated Brdu

Abstract HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

F-18 Fluorothymidine (FLT) Imaging of the Bone Marrow Compartment in Rats Following Whole Body Irradiation, Stem Cell Transplantation and Spontaneous Recovery.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3529-3529
Author(s):  
Jennifer L. Holter ◽  
Vibhudutta Awasthi ◽  
Kristin Thorp ◽  
Anderson Stacy ◽  
Sandra Bryant ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Lethal Dose ◽  
Whole Body ◽  
Hematopoietic Tissue ◽  
Post Transplantation ◽  
Early Recovery ◽  
Noninvasive Technique ◽  
Imaging Results ◽  
Group 2

Abstract Abstract 3529 Poster Board III-466 Pet imaging using F-18 glucose (FDG) is increasingly being used for evaluation and staging of malignancy. However, staging in hematopoietic tissue using this agent has been hampered by poor specificity. F-18 flourothymidine (FLT) is currently being evaluated clinically as an imaging technique for tumor detection and staging. Secondary to its inclusion in DNA during the S phase, FLT is much more specific to proliferative tissue and less hampered by inflammatory background. As FLT uptake occurs in proliferating cell populations, we attempted to determine if imaging could provide useful information for evaluating global hematopoietic injury and recovery following radiation and transplantation. Three major groups of Wistar-Furth rats were studied. Group 1 consisted of rats receiving 950cGy of Whole Body Irradiation (TBI). Group 2 consisted of rats transplanted with syngeneic bone marrow 24-48 hrs following irradiation. Group 3 consisted of 6 rats exposed to a potentially sub-lethal dose of 500cGy and not transplanted. FLT imaging was performed before irradiation (n=4), 24-48 hrs. following irradiation, and on day 4-5 post transplantation. Subsequent imaging was carried out in 4 transplanted rats on days 8 and 14. Comparative FDG studies were also performed in selected animals. Table 1 summarizes the imaging studies performed in various subsets of rats. Table 1 Imaging Subsets of Experimental Animals and Histologic Correlations Experimental rat subsets FLT # studies performed FDG # studies performed Histologic correlation Normal or baseline rat studies n=10 n=6 Normal cellular marrow 24-48hrs post 950 cGy TBI n=6 n=4 Marrow damage hypocellularity Day 7 post 950 cGy TBI n=4 not done Aplastic marrow Day 6-7 post 950cGy TBI (4-5 days post transplantation) n=4 n=4 Focal areas of cellular regeneration Day 10 post 950 cGy TBI (and transplantation) n=4 n=2 Cellular marrow Day 6-7 post 500cGy TBI (No transplantation) n=6 not done Moderate hypocellularity FLT imaging results were correlated with marrow histology and clinical survival in treated and control groups. Six of 6 irradiated control rats died with marrow aplasia during the second week following 950 cGy. Sub-lethally irradiated and transplanted rats animals showed clear evidence of definitive recovery as early as 6 days post irradiation or 4 days post transplantation respectively. FLT activity of all major marrow sites was easily identified and was superior to FDG images. Findings correlated with histologic evidence of early marrow repopulation and survival. Figure 1 illustrates FLT and FDG imaging performed in normal, post radiation and post transplanted rats. We conclude that FLT imaging represents a practical noninvasive technique to evaluate marrow injury and early recovery following radiation and hematopoietic transplantation. Disclosures: No relevant conflicts of interest to declare.

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2504-2504
Author(s):  
Russell Garrett ◽  
Gerd Bungartz ◽  
Alevtina Domashenko ◽  
Stephen G. Emerson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Hematopoietic Stem ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Latexin Inhibition Mitigates Irradiation Induced Hematopoietic Injury

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2562-2562
Author(s):  
Cuiping Zhang ◽  
Xiaojing Cui ◽  
Ying Liang
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Progenitor Cells ◽  
Knockout Mice ◽  
Lethal Dose ◽  
Negative Regulator ◽  
Cell Counts ◽  
Survival Pathway ◽  
Radiation Induced

Abstract Radiation-associated bone marrow (BM) injury is one of the most serious limiting factors of radiotherapy. Radiation-induced hematopoietic injury, no matter how transient or long lasting, can ultimately impair HSC function and decrease the HSC reserve, leading to increased risk for the development of BM failure or cancer. However, molecular mechanisms underlying radiation-induced HSC functional decline are largely unknown. We previously identified a stem cell regulatory gene, latexin (Lxn), as a novel negative regulator of HSCs in mice. HSCs in Lxn knockout mice (Lxn-/-) had increased self-renewal and survival. In our new findings, we surprisingly found that Lxn-/- mice had the significant survival advantages under lethal dose of total body irradiation (TBI). We further found that HSCs and hematopoietic progenitor cells (HPCs), measured by immunophenotypes and colony assay, recovered much faster in Lxn-/- mice than wild-type mice (WT) within one month after sub-lethal dose of TBI. The better preserved HSC/HPC pool was due to the decreased apoptosis in which the percentage of Annexin V + PI- apoptotic HSCs/HPCs cells was significantly lower in Lxn-/- mice than WT mice. These data suggest that Lxn inactivation protects HSCs and HPCs from radiation-induced cell death, thus mitigating acute hematopoietic suppression and conferring a survival advantage. To determine the long-term effect of TBI on Lxn-/- HSCs, we performed limiting dilution competitive repopulation unit assay (CRU), and found that Lxn-/- CRU was significantly higher than WT CRU. Moreover, we performed serial transplantation experiment, and found that Lxn-/- HSC continuously regenerated blood and bone marrow cells even at the 4th round of transplantation whereas WT HSCs were exhausted. These data provide robust evidence that Lxn inactivation protects functional long-term HSCs from radiation-induced injury. Radiation can increase the risk of hematological malignancy later in the life. We thus maintained a group of mice that were subject to either a single dose of 6.5Gy TBI or split low doses of TBI (2 Gy daily for 6 days), and monitored their gross condition and blood cell counts for 20 months. At 20 month post-radiation, we performed bone marrow analysis and histopathology analysis. We found that Lxn-/- mice did not spontaneously develop hematopoietic malignancies, their bone marrow HSCs/HPCs had normal population size, and bone marrow had normal histopathology. These data suggest that Lxn inactivation mitigates radiation-induced short-term myelosuppression and long-term HSC functional impairment without induction of hematologic malignancy. At the molecular level, we previously reported that Lxn sensitized leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway, and Rps3 was a binding protein of Lxn. Rps3 has been shown to be involved in the NFkB pathway. We found that Rps3 bound Lxn in primary hematopoietic stem and progenitor cells (HSPCs) using Co-IP assay. Lxn-/- HSPCs had the increased expression of Rps3 and NFkB p65 before or post-irradiation. Knockdown of Rps3 in Lxn-/- HSPCs decreased NFkB p65 and increased radiation-induced apoptosis. Moreover, when Lxn-/- HSPCs were treated with NFkB p65 specific inhibitor, the similar phenotypes were also shown, suggesting that Lxn functions through Rps3-NFkB-mediated pro-survival pathway in primary HSPCs. We are currently proving this molecular pathway using the in vivo model by crossing p65 knockout mice with Lxn-/- mice. In conclusion, latexin inhibition mitigates irradiation induced hematopoietic injury via Rps3-NFkB-mediated pro-survival pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone Marrow Granulocytes Drive Vascular and Hematopoietic Regeneration

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 427-427
Author(s):  
Emily Bowers ◽  
Slaughter Anastasiya ◽  
Daniel Lucas-Alcaraz
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Erythroid Cell ◽  
Vascular Regeneration ◽  
Hematopoietic Recovery

Abstract In addition to eliminating host hematopoietic cells myeloablation also disrupts the blood vessels that sustain hematopoiesis. Regeneration of the bone marrow (BM) vasculature is necessary for hematopoietic recovery and survival after transplantation (Cell Stem Cell. 2009 Mar 6;4(3):263-74) but the mechanisms that drive vascular regeneration are not clear. We found that, fourteen days after lethal irradiation and transplantation, mice transplanted with 20x106 bone marrow nucleated cells (BMNC) had ~6-fold more CD45-Ter119-CD31+CD105+ endothelial cells (6.9x103 vs 0.96x103 EC/femur, p<0.001), 2-fold more blood vessels (195 vs 87 blood vessels/sternum, p<0.05) and ~2-fold less vascular leakage (4.8 vs 9.3 ng of Evans Blue/ml of BM extracellular fluid, p<0.001) than mice transplanted with 105 BMNC. Transplant experiments using GFP+donor BMNC revealed that all endothelial cells after transplantation were host derived. Because hematopoietic progenitors inhibit vascular regeneration via angiopoietin 1 (Elife2015 Mar 30;4:e05521) we hypothesized that mature hematopoietic cells mediated vascular recovery. To test this we adoptively transferred, B and T cells, monocytes and macrophages (MO), granulocytes and erythroid cells into lethally irradiated recipients previously transplanted with 105 donor BMNC. Only CD115-Gr1+ granulocytes promoted endothelial cell regeneration (2.5x103 for granulocyte treated mice vs 0.9x103 for PBS, 0.3x103 for B- and T-cell, 0.7 1x103 for MO and 0.3x103 EC/femur for erythroid cell-treated mice; p<0.01). Granulocyte transfer also promoted survival (granulocytes=100%, PBS=50% p<0.05), probably due to faster host platelets and red blood cells recovery (granulocytes= 4.5x107, PBS=2.1x107 platelets/ml of blood, p<0.001; granulocytes=4x109, PBS=6.2x109 RBC/ml of blood, p<0.01). Importantly, competitive BM transplants showed that granulocytes did not exhaust donor HSC. These demonstrate that granulocyte transfer is sufficient to promote survival and drive vascular and hematopoietic recovery after transplantation. We then generated Mrp8-cre:iDTR mice which allowed us to specifically ablate BM granulocytes via diphtheria toxin (DT) injection. We transplanted lethally irradiated WT recipients with 106 BMNC purified from C67BL/6 WT or Mrp8-cre:iDTR mice followed by DT treatment for 7 days. This led to granulocyte depletion (1.6x106 vs 0.4x106 p<0.001) and impaired endothelial cell recovery (5.7x103 vs 2.4.x103 p<0.05) in mice transplanted with Mrp8-cre:iDTR BMNC. These results demonstrate that donor granulocytes are necessary for vascular regeneration. We found that granulocytes produced high levels of the angiogenic cytokine TNFα. This cytokine signals via Tnfrsf1aand Tnfrsf1b. Tnfrsf1a was upregulated specifically in BM endothelial cells. After myeloablation with 5-fluorouracil Tnfa-/-mice have reduced survival (Tnfa-/-= 13% vs WT= 93%; p<0.001) and reduced endothelial cell numbers (WT=9x103, Tnfa-/-=4.1x103 EC/femur; p<0.05) indicating that TNFα is necessary for survival and vascular regeneration after myeloablation. To test whether granulocytes promoted vascular regeneration via TNFα we lethally irradiated and transplanted C57BL/6 recipients followed by treatment with PBS or adoptive transfer of 106 WT or Tnfa-/- granulocytes. Only WT granulocytes induced vascular recovery as demonstrated by quantification of endothelial cells (PBS=0.9 x103, WT granulocytes=5.24x103 and Tnfa-/- granulocytes=3.0x103 cells/femur, p<0.05) and blood vessel numbers (PBS=126, WT granulocytes=186 and Tnfa-/- granulocytes=84 vessels per sternum BM; p<0.05). Further, adoptive transfer of WT granulocytes promoted survival and vascular regeneration (WT+PBS=1.4x103 vs WT+granulocytes=2.6x103, p<0.05; Tnfrsf1a-/-:Tnfrsf1b-/- +PBS=0.8x103 vs Tnfrsf1a-/-:Tnfrsf1b-/-+granulocytes=0.7x103 EC/femur p=0.83) in WT but not Tnfrsf1a-/-:Tnfrsf1b-/-recipients after transplantation. These experiments demonstrate that granulocytes crosstalk directly with stromal cells (likely endothelial cells) via TNFα to drive vascular regeneration. We have identified a new type of cellular crosstalk in the microenvironment that drives regeneration. Our research also provides proof of principle for studies targeting BM granulocytes to enhance vascular recovery and survival after transplantation in patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals TGF-Beta Signaling in Mesenchymal Stromal Cells Contributes to Myelofibrosis but Not Hematopoietic Phenotypes in Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3053-3053
Author(s):  
Juo-Chin Yao ◽  
Grazia Abou Ezzi ◽  
Joseph R. Krambs ◽  
Eric J. Duncavage ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Cells ◽  
Dismal Prognosis ◽  
Collagen Iii

Abstract The development of myelofibrosis in patients with myeloproliferative neoplasms (MPNs) is associated with a dismal prognosis. The mechanisms responsible for the progression to myelofibrosis are unclear, limiting the development of therapies to treat or prevent it. The cell of origin responsible for the increased collagen deposition is controversial, with recent studies implicating Gli1+ or leptin receptor+ mesenchymal stromal cells, monocytes, or even endothelial cells. Moreover, the signals generated by malignant hematopoietic cells in MPN that induce increased collagen expression are uncertain. There is some evidence that elevated expression of cytokines/chemokines in the bone marrow microenvironment of patients with MPN may contribute. In particular, recent studies have implicated transforming growth factor-β (TGF-β), platelet-derived growth factor and CXCL4 in the development of myelofibrosis. Here, we test the specific hypothesis that TGF-β signaling in mesenchymal stromal cells is required for the development of myelofibrosis. Moreover, we hypothesize that TGF-β signaling, by altering the expression of key niche factors by mesenchymal stromal cells, contributes to the myeloid expansion in MPN. To test this hypothesis, we abrogated TGF-β signaling in mesenchymal stem/progenitor cells (MSPCs) by deleting Tgfbr2 using a doxycycline-repressible Sp7 (osterix)-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells, osteoblasts, adipocytes, or arteriolar pericytes. We previously showed that TGF-β signaling plays a key role in the lineage specification of MSPCs during development (2017 ASH abstract #2438). In contrast, we show that post-natal deletion of Tgfbr2, by removing doxycycline at birth, is not associated with significant changes in mesenchymal stromal cells in the bone marrow. Moreover, expression of key niche factors, including Cxcl12 and stem cell factor, and basal hematopoiesis were normal in these mice. Thus, we used the post-natal Osx-Cre; Tgfbr2-deleted mice as recipients to assess the role of TGF-β signaling in mesenchymal stromal cells on the hematopoietic and myelofibrosis phenotype in Jak2V617For MPLW515Lmodels of MPN. Specifically, we transplanted hematopoietic cells from Mx1-Cre; Jak2V617Fmice (4 weeks after pIpC treatment) or hematopoietic cells transduced with MPLW515Lretrovirus into irradiated wildtype or post-natal Osx-Cre; Tgfbr2-deleted mice. Both MPN models have elevated Tgfb1 expression in the bone marrow. As reported previously, transplantation of MPLW515Ltransduced hematopoietic cells into wildtype recipients produced a rapidly fatal MPN characterized by neutrophilia, erythrocytosis, thrombocytosis, splenomegaly, and reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype was observed in Osx-Cre; Tgfbr2fl/flrecipients. However, a trend to decreased reticulin fibrosis was observed in Osx-Cre; Tgfbr2fl/flcompared to wildtype recipients (reticulin histology score: 0.5 versus 1.1, respectively, n=5, p=0.23). Likewise, the degree of neutrophilia, erythrocytosis, thrombocytosis, and splenomegaly in wildtype and Osx-Cre; Tgfbr2fl/flrecipients of Jak2V617Fcells was similar. As reported previously, we did not observe overt myelofibrosis in this model (as measured by reticulin staining). However, we were able to detect increased collagen III deposition using immunofluorescence staining in 4 of 5 wildtype recipients compared to 1 of 4 Osx-Cre Tgfbr2fl/flrecipients of Jak2V617Fcells (p=0.21). In conclusion, our data suggest that TGF-β signaling in mesenchymal stromal cells contributes, but is not absolutely required, for the development of myelofibrosis. Alterations in mesenchymal stromal cells induced by increased TGF-β signaling do not appear to be a major driver of the myeloid expansion in MPN. The contribution of increased TGF-β signaling in hematopoietic cells or other bone marrow stromal cell populations to the MPN phenotype is under investigation. Disclosures No relevant conflicts of interest to declare.

scholarly journals The PAF1c Subunit Cdc73 Is Essential for Hematopoiesis and Displays Differential Gene Regulation in MLL-AF9 Driven Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1280-1280
Author(s):  
Nirmalya SAHA ◽  
James Ropa ◽  
Lili Chen ◽  
Hsiang-Yu Hu ◽  
Maria Mysliwski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Flow Cytometry Analysis ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
A Cell

Abstract The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is a multi-protein complex composed of Paf1, Cdc73, Leo1, Ctr9, Rtf1 and WDR61, which have all been shown to play a role in disease progression and different types of cancer. Previous reports demonstrated that the PAF1c is required for MLL-fusion driven acute myeloid leukemia. This is due, in part, to a direct interaction between the PAF1c and wild type MLL or MLL fusion proteins. Importantly, targeted disruption of the PAF1c-MLL interaction impairs the growth of MLL-fusion leukemic cells but is tolerated by normal hematopoietic stem cells. These data point to differential functions for the PAF1c in normal and malignant hematopoietic cells that may be exploited for therapeutic purposes. However, a detailed exploration of the PAF1c in normal hematopoiesis is currently lacking. Here, we utilize a mouse genetic model to interrogate the role of the PAF1c subunit, Cdc73, in the development and sustenance of normal hematopoiesis. Using hematopoietic-specific constitutive and conditional drivers to express Cre recombinase, we efficiently excise floxed alleles of Cdc73 in hematopoietic cells. VavCre mediated excision of Cdc73 results in embryonic lethality due to hematopoietic failure. Characterization of the hematopoietic system demonstrated that cKit+ hematopoietic stem and progenitor cells (HSPC) are depleted due to Cdc73 knockout. We next investigated the role of Cdc73 in adult hematopoiesis using Mx1Cre mediated excision. Conditional knockout of Cdc73 in the adult hematopoietic system leads to lethality within 15 days of Cdc73 excision while no phenotype was observed in heterozygous Cdc73fl/wt controls. Pathological examination of bones in these mice showed extensive bone marrow failure. Flow cytometry analysis revealed that cKit+ HSPCs in adult mice are ablated following loss of Cdc73. Bone marrow transplantation assays demonstrated a cell autonomous requirement of Cdc73 for HSC function in vivo. To perform cellular characterization of HSPCs upon Cdc73 KO, we optimized excision conditions to capture cKit+ HSPCs with excised Cdc73 but before their exhaustion. Flow cytometry analysis demonstrated that Cdc73 KO leads to a cell cycle defect. Cdc73 excision leads to a 2.5 fold increase in the accumulation of HSPCs in the G0 phase of cell cycle with a reduction in the proliferative phases. This is accompanied with an increase in cellular death as indicated by Annexin V staining. Together, these data indicate that Cdc73 is required for cell cycle progression and HSPC survival. To understand the molecular function of Cdc73, we performed RNAseq analysis to identify genes regulated by Cdc73 in HSPCs. We observed 390 genes are upregulated and 433 genes are downregulated upon loss of Cdc73. Specifically, Cdc73 excision results in upregulation of cell cycle inhibitor genes such as p21 and p57, consistent with the cell cycle defect observed following Cdc73 excision. Further, when comparing our results to leukemic cells, we uncovered key differences in Cdc73 gene program regulation between ckit+ hematopoietic cells and MLL-AF9 AML cells. Loss of Cdc73 in leukemic cells leads to downregulation of genes associated with early hematopoietic progenitors and upregulation of myeloid differentiation genes consistent with previous studies. Interestingly, we observed a more even distribution of expression changes (non-directional) within these gene programs following Cdc73 inactivation in HSPCs. Most importantly, while loss of Cdc73 in MLL-AF9 AML cells leads to a profound downregulation of the Hoxa9/Meis1 gene program, excision of Cdc73 in HSPCs results in a modest non-directional change in expression of the Hoxa9/Meis1 gene program. This was attributed to no change in Hoxa9 and Meis1 expression in HSPCs following excision of Cdc73, in contrast to MLL-AF9 cells where these pro leukemic targets are significantly downregulated. Together, these data indicate an essential role for the PAF1c subunit Cdc73 in normal hematopoiesis but differential roles and context specific functions in normal and malignant hematopoiesis, which may be of therapeutic value for patients with AMLs expressing Hoxa9/Meis1 gene programs. Disclosures No relevant conflicts of interest to declare.

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