Bone Marrow Derived Mesenchymal Cells Secrete Granulopoietic Cytokines upon Danger Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4115-4115
Author(s):  
Stefan Wirths ◽  
Stefanie Bugl ◽  
Markus P. Radsak ◽  
Melanie Märklin ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Feedback Loop ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Caspase 1 ◽  
Stem And Progenitor Cells ◽  
Myd88 Signaling

Abstract Granulopoietic homeostasis is regulated at steady-state to supply sufficient numbers of pooled and circulating neutrophils to maintain barrier function against commensal flora. In addition, upon pathogenic microbial challenge, an increased formation of neutrophils is induced, termed ‘emergency granulopoiesis’. Antibody-mediated reduction of neutrophil numbers in steady-state induces a feedback loop leading to an increase of bone marrow granulopoiesis with expansion of hematopoetic stem and progenitor cells. This feedback loop was demonstrated to depend on TLR4 and TRIF, but not MyD88 signaling (Bugl et al. Blood 2013). In contrast, emergency granulopoiesis was shown to be dependent on MyD88 signaling in endothelial cells (Boettcher et al. Blood 2014). Bone marrow mesenchymal stromal cells (MSC) are niche-forming cells, harboring and regulating hematopoiesis. Upon steady-state neutropenia an increase of niche size was observed. Here we investigated, whether niche-forming MSC act as sensors of pathogen-associated molecular patterns (PAMPs) and induce granulopoietic cytokines to stimulate expansion of adjacent hematopoietic stem and progenitor cells. MSC of C57BL/6 and TLR4-KO mice were cultured in vitro and treated with LPS for 24 hours. Cells were harvested and qRT-PCR for G-CSF, TLR4, MyD88, TRIF, GM-CSF, IL-1β, IL-18 and Casp-1 was performed After treatment with LPS, RNA of granulopoietic cytokines G-CSF and GM-CSF were massively up regulated in MSC of WT mice. Upstream regulating, inflammasome components IL-1ß and caspase-1 RNA levels increased as well, with little changes in IL-18, TLR4, MyD88 and TRIF. Unexpectedly, TLR4-KO MSC up regulated transcription of IL-1β and G-CSF upon LPS stimulation as well, and caspase-1 was found to be strongly up-regulated in unstimulated TLR4-KO compared to WT MSC. In summary, bone marrow stromal cells are found to be PAMP-sensing and secrete cytokines that regulate granulopoiesis. TLR4-independent sensing of LPS by MSC might correspond to the alternative noncanonical inflammasome pathway recently described (Kayagaki et al. Science 2013). Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1689-1689
Author(s):  
Jichun Chen ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Immune Mediated ◽  
Stem And Progenitor Cells

Abstract Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals. Figure Figure

scholarly journals Acute exercise mobilizes hematopoietic stem and progenitor cells and alters the mesenchymal stromal cell secretome

2016 ◽  
Vol 120 (6) ◽  
pp. 624-632 ◽  
Author(s):  
Russell Emmons ◽  
Grace M. Niemiro ◽  
Olatomide Owolabi ◽  
Michael De Lisio
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Acute Exercise ◽  
Marrow Cell ◽  
Exercise Bout ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Transplantation of hematopoietic stem and progenitor cells (HSPC), collected from peripheral blood, is the primary treatment for many hematological malignancies; however, variable collection efficacy with current protocols merits further examination into factors responsible for HSPC mobilization. HSPCs primarily reside within the bone marrow and are regulated by mesenchymal stromal cells (MSC). Exercise potently and transiently mobilizes HSPCs from the bone marrow into peripheral circulation. Thus the purpose of the present study was to evaluate potential factors in the bone marrow responsible for HSPC mobilization, investigate potential sites of HSPC homing, and assess changes in bone marrow cell populations following exercise. An acute exercise bout increased circulating HSPCs at 15 min (88%, P < 0.001) that returned to baseline at 60 min. Gene expression for HSPC homing factors (CXCL12, vascular endothelial growth factor-a, and angiopoietin-1) were increased at 15 min in skeletal muscle and HSPC content was increased in the spleen 48 h postexercise (45%, P < 0.01). Acute exercise did not alter HSPCs or MSCs quantity in the bone marrow; however, proliferation of HSPCs (40%, P < 0.001), multipotent progenitors (40%, P < 0.001), short-term hematopoietic stem cells (61%, P < 0.001), long-term hematopoietic stem cells (55%, P = 0.002), and MSCs (20%, P = 0.01) increased postexercise. Acute exercise increased the content of the mobilization agent granulocyte-colony stimulating factor, as well as stem cell factor, interleukin-3, and thrombopoeitin in conditioned media collected from bone marrow stromal cells 15 min postexercise. These findings suggest that the MSC secretome is responsible for HSPC mobilization and proliferation; concurrently, HSPCs are homing to extramedullary sites following exercise.

scholarly journals Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Bo O Zhou ◽  
Lei Ding ◽  
Sean J Morrison
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Leptin Receptor ◽  
Hematopoietic Cells ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Function ◽  
Angiopoietin 1

Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery.

scholarly journals EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 96-96
Author(s):  
Marta Derecka ◽  
Senthilkumar Ramamoorthy ◽  
Pierre Cauchy ◽  
Josip Herman ◽  
Dominic Grun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis. Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. Disclosures No relevant conflicts of interest to declare.

The Combination of AMD3100 + G-CSF Restores the Ineffective Hematopoietic Stem Cell Mobilization with G-CSF Alone in a Thalassemic Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3234-3234
Author(s):  
Evangelia Yannaki ◽  
Nikoleta Psatha ◽  
Maria Demertzi ◽  
Evangelia Athanasiou ◽  
Eleni Sgouramali ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Mouse Model ◽  
Progenitor Cells ◽  
State Condition ◽  
Hematopoietic Stem ◽  
Steady State Condition ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 3234 Poster Board III-171 Gene therapy has been recently postulated as a realistic therapeutic potential for thalassemia and the mobilized autologous hematopoietic stem cells (HSCs) may represent the preferable source of stem cells for genetic modification due to the higher yield of HSCs compared to conventional bone marrow (bm) harvest. We have previously shown (manuscript under revision) that G-CSF mobilization in the HBBth-3 thalassemic mouse model is less efficient compared to normal C57Bl6 strain, mainly due to increased trapping of hematopoietic stem (Lin-sca-1+ckit+–LSK) and progenitor cells (CFU-GM) in the enlarged thalassemic spleen. The novel mobilizer, AMD3100 (plerixafor, mozobil), has been shown to reversibly bind to CXCR4 and inhibit the interaction between SDF-1 and CXCR4 within the bm microenvironment, resulting in the egress of CD34+ cells into the circulation of healthy donors and cancer patients. The addition of AMD to G-CSF results in even greater increases in circulating CD34+cells. We explored in the current study whether AMD alone or in combination with G-CSF improves the mobilization efficiency of thalassemic mice. C57 and HBBth-3 mice received G-CSF-alone at 250microgr/kgX7 days, AMD-alone at 5mg/kgX3 days or the combination of two with AMD administered in the evening of days 5-7 of G-CSF administration. Hematopoietic tissues (blood, bm, spleen) were collected and the absolute LSK and CFU-GM numbers were calculated based on their frequency within tissues (by FCM and clonogenic assays) in relation to the individual cell count per tissue. AMD-alone didn't significantly affect the HSC yield as compared to G-CSF mobilization in thal mice (LSK/μl blood: 103±85 vs 69±26 p=ns), although it significantly increased the circulating Colony Forming Cells (CFU-GM/ml blood: 1205±533 vs 330±87, p=0,05). In contrast, the AMD+G-CSF combination significantly improved the mobilization efficiency of HBBth-3 mice over the G-CSF-treated group (LSK cells/μl blood: 224±104 vs 69±26 p=0,04, CFU-GM/ml blood: 1671±984 vs 330±87 p=0,05, respectively) at levels comparable to normal mice treated with G-CSF (LSK cells/ μl blood: 241±167, CFU-GM/ml blood: 1235±1140, respectively). AMD induced a “detachment” of stem cells from the bm because reduced numbers of bm LSK cells were counted in the AMD-alone group as compared to the untreated group (LSK/2 femurs×103: 692±429 vs 1687±1016, respectively, p=0,05). This was in contrast to the marrow hyperplasia caused by G-CSF over the steady-state condition (LSK/2 femurs×103: 2684±1743 vs 1687±1016 p=0,02 / CFU-GM/2femurs:111.841±15.391 vs 76.774±31.728 p=0,01). Consequently, the combination of AMD+G-CSF resulted in increased numbers of circulating stem and progenitor cells without inducing marrow hyperplasia as compared to steady-state condition (LSK/2femurs×103: 1681±862 vs 1686±1017, p=ns / CFU-GM/2femurs: 76.774±31.728 vs 82.905±26.277, p=ns). AMD, also in contrast to G-CSF, did not cause increased trapping of stem and progenitor cells in the spleen compared to the untreated condition (LSK cells/spleen×103: 4738±2970 vs 8303±4166 p=ns / CFU-GM/spleen:146.269±93.174 vs 98.518±25.549, p=ns). However, the combination of AMD+G-CSF still resulted in splenic sequestration of progenitor cells (CFU-GM/spleen: 412.176±157.417 vs 98.518±25.549, p=0,0003) but not of LSK cells (LSK cells/spleen×103: 10.200±7.260 vs 8.300±4.166 p=ns). Overall, the combination of AMD3100+G-CSF seems to restore the less efficient mobilization in a thalassemic mouse model. This combination may prove beneficial in a GT setting for obtaining the high numbers of HSCs needed for genetic correction. In addition, the combination of AMD3100+G-CSF, by avoiding the marrow hyperplasia induced by G-CSF alone, indicates a better safety profile because it will not further burden the hyperplastic –due to the increased erythroid demand and the intramarrow destruction of erythroblasts-thalassemic bone marrow. Disclosures No relevant conflicts of interest to declare.

Dendritic Cell Homeostasis Is Maintained by Non-Hematopoietic and T Cell-Produced Flt3-Ligand In Steady-State and During Immune Responses.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1547-1547
Author(s):  
Chandra Sekhar Boddupalli ◽  
Dior Baumjohann ◽  
Tim Sparwasser ◽  
Markus G Manz
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Progenitor Cells ◽  
T And B Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1547 Lymphoid tissue dendritic cells (DCs) have a short life-span of a few days and need to be continuously replenished from hematopoietic stem and progenitor cells. Flt3-Ligand (Flt3L) plays non-redundant role in development of DCs (McKenna. H.J. et al., Blood; 2000). Previously we found that Flk2 (fetal liver kinase-2), the cognate receptor for Flt3L is expressed on early dendritic cell progenitors and Flt3L-Flk2 signalling efficiently supports DC development from early progenitors to steady-state DCs in mice and men (Karsunky, H. et al., J Exp Med; 2003; Chicha L. et al. J Exp Med; 2004). Flk2 is also expressed on mature steady-state lymphoid organ DCs; however its function on mature cells remains to be determined. Flt3L is expressed in almost all the tissues in both mice and men (Hannum, C. et al., Nature; 1994) and this cytokine is critical in the maintenance of DC/T regulatory (Treg) cell homeostasis (Darrase-Jéze. G et al., J Exp Med; 2009; Swee LK et al., Blood; 2009; Manz MG, Blood 2009). However, the precise cellular source of Flt3L and the regulation of production in steady-state and immune responses in vivo is not well understood. Genetic ablation of the Flk2 receptor lead to 10-fold elevated Flt3L levels in the serum of mice. To evaluate if hematopoietic or non-hematopoietic cells are the main consumers of Flt3L in vivo, we generated bone marrow chimeras by transplanting wild type (WT) or Flt3L-/- c-Kit+ hematopoietic stem and progenitor cells into lethally irradiated Flk2-/- mice. This demonstrated that hematopietic progenitors and DCs expressing Flk2 receptor are the main consumers of Flt3L in vivo. Previously we showed that in vivo Flk2 tyrosine kinase inhibition and consecutive DC reduction lead to 10fold elevated levels of serum Flt3L (Tussiwand. R. et al., J Immunol; 2005). By using CD11c DTR mice (Zaft, T. et al., J Immunol; 2005) in which diphtheria toxin (DT) receptor is cloned under the CD11c promoter and treatment of mice with DT lead to selective depletion of DCs we here show that ablation Flk2 expressing DCs lead to immediate, about 4-fold elevated serum Flt3L levels in mice. However, we observed no change in mRNA expression of Flt3L, which strongly indicates that Flk2 expressed on DCs is acting as “scavenger” for Flt3L. We then studied sources of Flt3L in vivo. To this end we generated bone marrow chimeras by transplanting WT c-Kit+ hematopoietic stem and progenitor cells in to lethally irradiated Flt3L-/- hosts and vice versa (WT to Fllt3L-/-, Flt3L-/- to WT), and found that in vivo DC homeostasis can be achieved by non-hematopoietic and to lesser extend by hematopoietic cell produced Flt3L. Furhtermore, we found that compared to other hematopoietic cells Flt3L mRNA is highly expressed in lymphocytes (T and B cells) and in lymphoid tissues like thymus, spleen and lymph nodes. We thus used bone marrow c-Kit+ hematopoietic stem and progenitor cells from mice that lack T and B cells (Rag1-/-) or that lack T cells (CD3ε-/-) as donors to transplant lethally conditioned Flt3L-/- mice, and found that Flt3L produced by T and B cells is necessary to support DC development in non hematopoietic Flt3L deficient mice. Using BrdU incorporation we evaluated the functional relevance of Flt3L produced by T cells in an ongoing immune response. Experiments revealed that in lymph nodes with proliferating T cells producing Flt3L a higher percent of BrdU+ DCs, i.e. DCs derived from proliferating progenitors were detected. This indicates that Flt3L produced by T cells in an ongoing immune response helps in faster regeneration of DCs from DC committed progenitors. Earlier it has been shown that Treg ablation in Foxp3-DTR mice lead to expansion of DCs in lymph nodes and spleen through Flk2 mediated pathway (Liu, K. et al., Science; 2009); however, the source of Flt3L remained unknown. Here we provide evidence that Treg ablation leads to activation and proliferation of CD4+ T cells that in turn release Flt3L to enhance DC development. These key observations provide insight into the regulation of DC homeostasis and function via tailored adaptation of the Flt3L cytokine milieu by non-hematopoietic and T cells during steady state and during adaptive immune responses. Supported by the Swiss National Science Foundation (310000-116637) and the European Commission FP6 Network of Excellence initiative (LSHB-CT-2004-512074 DC-THERA) Disclosures: No relevant conflicts of interest to declare.

Direct Sensing of Toll-Like Receptor Agonists by Dendritic Cell Progenitors Leads to Their Homing to Inflamed Lymph Nodes Via Regulation of CXCR4 and CCR7

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3895-3895
Author(s):  
Michael A Schmid ◽  
Dior Baumjohann ◽  
Markus G Manz
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Lymph Nodes ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Tlr Agonists ◽  
Stem And Progenitor Cells

Abstract Abstract 3895 Dendritic cells (DCs), the key antigen-presenting cell population, continuously need to be regenerated from bone marrow (BM) hematopoietic stem and progenitor cells. Common dendritic progenitors (CDP) were previously shown to efficiently generate DCs in lymphoid and non-lymphoid tissues. How the dissemination of bone marrow (BM) DC-progenitors to peripheral tissues is regulated upon demand remains elusive to date. Acute microbial infections are sensed via Toll-like receptors (TLR). Recent studies showed that stem and progenitor cells express TLRs. We found that CDPs in the BM of mice express relative high levels of Tlr2, Tlr4 and Tlr9, and hypothesized that these might be involved in regulating CDP migration. CDPs in steady-state expressed high levels of Cxcr4, but no, or low Ccr7. Upon direct stimulation with the respective TLR-agonists in vitro, CDPs rapidly down-regulated Cxcr4 and up-regulated Ccr7 mRNA and protein. CDPs that were stimulated with TLR-agonists for only 2 h preferentially homed to the lymph nodes (LN) in expense of BM in steady-state recipients. When TLR-agonists were injected subcutaneously, CDPs gave rise to increased numbers of plasmacytoid DCs, classical DCs, and DCs with a skin-derived migratory phenotype in inflamed LNs on day 4. This was not due to increased proliferative activity. Injecting the CXCR4 antagonist AMD3100 demonstrated that the retention of CDPs in the BM depends on CXCR4. Furthermore, CCR7 was important for the engraftment of CDP-derived DCs into LNs in steady-state and during inflammation. In conclusion, DC progenitors in the bone marrow are capable to directly sense TLR-agonists via their cognate receptors in systemic infections. This results in differential expression of chemokine receptors and consecutive migration of DC-progenitors to inflamed LNs. This mechanism helps to restore DC subsets during ongoing immune responses and to return to DC homeostasis once the inflammation ceases. Disclosures: No relevant conflicts of interest to declare.

C/EBPβ Isoforms Distinctively and Collaboratively Regulate the Behavior of Hematopoietic Stem and Progenitor Cells in Regenerative Conditions

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3580-3580
Author(s):  
Atsushi Sato ◽  
Hideyo Hirai ◽  
Asumi Yokota ◽  
Akihiro Tamura ◽  
Tsukimi Shoji ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Cell Cycle Regulation ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells ◽  
Cycle Regulation ◽  
Cell Cycle Status

Abstract CCAAT Enhancer Binding Protein b (C/EBPb) is a leucine zipper type transcription factor. While C/EBPa plays a critical role in maintaining steady-state granulopoiesis, C/EBPb is required for stress-induced granulopoiesis (Hirai et al., 2006). We have been focusing on the functions of C/EBPb in the regulation of hematopoietic stem and progenitor cells (HSPCs) especially under stressed conditions. Last year in this meeting, we have shown that 1) C/EBPb was upregulated at protein level in HSPCs after hematopoietic stresses, 2) C/EBPb was required for initial expansion of HSPCs after transplantation, and 3) C/EBPb promoted exhaustion of HSPCs under repetitive hematopoietic stresses (56th ASH, abstract #67850). Here, we further investigated the significance of C/EBPb in cell cycle regulation of HSPCs and the distinct roles of C/EBPb isoforms in HSPCs during regenerative conditions. To clarify the involvement of C/EBPb in cell cycle regulation of HSPCs, we compared the cell cycle status of wild-type (WT) and Cebpb knockout (KO) HSPCs by intracellular Ki67 staining and short-term BrdU incorporation assay in combination with multi-color flow cytometric analysis. In order to exclude the difference in the bone marrow microenvironment, CD45.2+ WT or Cebpb KO bone marrow (BM) cells were transplanted into lethally irradiated CD45.1+ WT mice. At steady state (12 weeks after the BM transplantation), the cell cycle status of Cebpb KO HSPCs was identical to that of WT HSPCs. Then cell cycle status of HSPCs was assessed at various time points during regeneration after intraperitoneal administration of 5-fluorouracil (5-FU, 150mg/kg). We found that significantly more Cebpb KO HSPCs remained in the G0 phase than WT HSPCs (in LT-HSCs on days 3-10; in MPPs on days 6-12). Significantly less Cebpb KO HSPCs were BrdU+ and were in the S/G2/M phase on day 7. These findings suggest that C/EBPb, in a cell-intrinsic manner, facilitates cell cycle entry, progression and consequent earlier expansion of HSPCs in response to hematopoietic stresses. Next, we investigated the distinct roles of C/EBPb isoforms in regulation of HSPCs. C/EBPb is a unique single exon gene and utilization of three different initiating codons result in three distinct isoforms. Liver-enriched activating protein* (LAP*) and LAP are the longer isoforms containing transactivating domains, DNA binding and dimerization domains, and liver-enriched inhibitory protein (LIP) is the shortest isoform which lacks the transactivating domains. In order to examine the expression pattern of C/EBPb isoforms in vivo in scarce populations of regenerating HSPCs, we developed a novel flow cytometric method to distinguish the cells predominantly expressing shorter isoform (LIP) from the cells expressing both LIP and the longer isoforms (LAP* and LAP) by intracellular double staining. Using this method, we found that predominantly LIP-expressing cells transiently emerged within MPP fraction in the regenerating bone marrow (on days 5-6 after administration of 5-FU, Figure below), while overall C/EBPb expression levels were significantly upregulated in most cells. To examine the roles of respective C/EBPb isoforms in regulation of HSPCs, EML cells, a murine hematopoietic stem cell line, were retrovirally transduced with one of the C/EBPb isoforms and the transduced cells were subjected to further analysis (vectors are kind gifts from Dr Watanabe-Okouchi N and Dr Kurokawa M, Tokyo Univ). LIP-expressing EML cells were more proliferative and actively cycling than EML cells transduced with a control vector, whereas the proliferation of LAP*- or LAP-expressing cells were markedly suppressed. LIP-expressing cells remained undifferentiated status (c-kithigh CD11b-) for more than 2 weeks, while LAP*- or LAP-expressing cells rapidly differentiated into c-kitlow CD11b+ myeloid cells and eventually exhausted within a week. These results indicate LIP plays quite distinct roles from LAP* and LAP in regulation of HSPCs. Collectively, our data suggest that C/EBPb isoforms distinctively and collaboratively regulate HSPCs in regenerative conditions: early transient elevation of LIP contributes to cell cycle activation and rapid expansion of HSPC population, which is in turn converted into supply of mature myeloid cells by more abundant upregulation of LAP* and LAP. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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