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Early severe impairment of hematopoietic stem and progenitor cells from the bone marrow caused by CLP sepsis and endotoxemia in a humanized mice model.

Skirecki T, Kawiak J, Machaj E, Pojda Z, Wasilewska D, Czubak J, Hoser G - Stem Cell Res Ther (2015)

Bottom Line: Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM.In contrast, in vitro LPS stimulated differentiation of CD34(+) CD38(-) HSCs but did not induce proliferation of these cells in contrast to the CD34(+) CD38(+) progenitors.It is suggestive that the Notch pathway contributed to this effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Flow Cytometry, The Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland. tskirecki@gmail.com.

ABSTRACT

Introduction: An effective immune response to severe bacterial infections requires a robust production of the innate immunity cells from hematopoietic stem and progenitor cells (HSPCs) in a process called emergency myelopoiesis. In sepsis, an altered immune response that leads to a failure of bacterial clearance is often observed. In this study, we aimed to evaluate the impact of sepsis on human HSPCs in the bone marrow (BM) microenvironment of humanized mice subjected to acute endotoxemia and polymicrobial sepsis.

Methods: Humanized mice (hu-NSG) were generated by transplanting NOD.Cg-Prkdc/scidIL2rγ (NSG) mice with the human cord blood CD34(+) cells. Eight weeks after the transplantation, hu-NSG mice were subjected to sepsis induced by endotoxemia-Escherichia coli lipopolysaccharide (LPS)-or by cecal ligation and puncture (CLP). Twenty-four hours later, HSPCs from BM were analyzed by flow cytometry and colony-forming unit (CFU) assay. CLP after inhibition of Notch signaling was also performed. The effects of LPS on the in vitro proliferation of CD34(+) cells from human BM were tested by CellTrace Violet dye staining.

Results: The expression of Toll-like receptor 4 receptor was present among engrafted human HSPCs. Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM. In addition, in both models, accumulation of early CD34(+) CD38(-) HSCs was observed, but the number of CD34(+) CD38(+) progenitors decreased. After CLP, there was a 1.5-fold increase of proliferating CD34(+) CD38(-)Ki-67(+) cells. Moreover, CFU assay revealed a depressed (by 75 % after LPS and by 50 % after CLP) production of human hematopoietic colonies from the BM of septic mice. In contrast, in vitro LPS stimulated differentiation of CD34(+) CD38(-) HSCs but did not induce proliferation of these cells in contrast to the CD34(+) CD38(+) progenitors. CLP sepsis modulated the BM microenvironment by upregulation of Jagged-1 expression on non-hematopoietic cells, and the proliferation of HSCs was Notch-dependent.

Conclusions: CLP sepsis and endotoxemia induced a similar expansion and proliferation of early HSCs in the BM, while committed progenitors decreased. It is suggestive that the Notch pathway contributed to this effect. Targeting early hematopoiesis may be considered as a viable alternative in the existing arsenal of supportive therapies in sepsis.

No MeSH data available.


Related in: MedlinePlus

Analysis of the growth of human hematopoietic colonies from bone marrow cells isolated from humanized mice 24 h after induction of experimental sepsis. Twenty-four hours after induction of experimental sepsis, bone marrow cells (5 × 105) from hu-NSG were seeded on methylocellulose medium supplemented with human growth factors. After 20 days of culture, the colonies formed by human progenitor cells were counted. a Results from the model of endotoxemia. b Results from the CLP-induced sepsis model. n = 6, *P < 0.05, **P < 0.01. BMC bone marrow cell, CLP cecal ligation and puncture surgery, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ
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Fig3: Analysis of the growth of human hematopoietic colonies from bone marrow cells isolated from humanized mice 24 h after induction of experimental sepsis. Twenty-four hours after induction of experimental sepsis, bone marrow cells (5 × 105) from hu-NSG were seeded on methylocellulose medium supplemented with human growth factors. After 20 days of culture, the colonies formed by human progenitor cells were counted. a Results from the model of endotoxemia. b Results from the CLP-induced sepsis model. n = 6, *P < 0.05, **P < 0.01. BMC bone marrow cell, CLP cecal ligation and puncture surgery, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ

Mentions: In the next step, we aimed to test the impact of endotoxemia and CLP on the functionality and number of hu-hematopoietic progenitors. To do this, we used the CFU assay with medium supplemented with recombinant human cytokines which do not support growth of murine cells. The differences in baseline production of CFUs from control mice in both models (Fig. 3) are a result of different human chimerism after transplantation, but each time control mice were paired with experimental mice on the basis of human cell chimerism. Human hematopoietic cells isolated from the BM of mice injected with LPS formed four-fold fewer colonies compared with control mice. A similar effect occurred in cells from CLP mice: a two-fold reduction was observed.Fig. 3


Early severe impairment of hematopoietic stem and progenitor cells from the bone marrow caused by CLP sepsis and endotoxemia in a humanized mice model.

Skirecki T, Kawiak J, Machaj E, Pojda Z, Wasilewska D, Czubak J, Hoser G - Stem Cell Res Ther (2015)

Analysis of the growth of human hematopoietic colonies from bone marrow cells isolated from humanized mice 24 h after induction of experimental sepsis. Twenty-four hours after induction of experimental sepsis, bone marrow cells (5 × 105) from hu-NSG were seeded on methylocellulose medium supplemented with human growth factors. After 20 days of culture, the colonies formed by human progenitor cells were counted. a Results from the model of endotoxemia. b Results from the CLP-induced sepsis model. n = 6, *P < 0.05, **P < 0.01. BMC bone marrow cell, CLP cecal ligation and puncture surgery, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536694&req=5

Fig3: Analysis of the growth of human hematopoietic colonies from bone marrow cells isolated from humanized mice 24 h after induction of experimental sepsis. Twenty-four hours after induction of experimental sepsis, bone marrow cells (5 × 105) from hu-NSG were seeded on methylocellulose medium supplemented with human growth factors. After 20 days of culture, the colonies formed by human progenitor cells were counted. a Results from the model of endotoxemia. b Results from the CLP-induced sepsis model. n = 6, *P < 0.05, **P < 0.01. BMC bone marrow cell, CLP cecal ligation and puncture surgery, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ
Mentions: In the next step, we aimed to test the impact of endotoxemia and CLP on the functionality and number of hu-hematopoietic progenitors. To do this, we used the CFU assay with medium supplemented with recombinant human cytokines which do not support growth of murine cells. The differences in baseline production of CFUs from control mice in both models (Fig. 3) are a result of different human chimerism after transplantation, but each time control mice were paired with experimental mice on the basis of human cell chimerism. Human hematopoietic cells isolated from the BM of mice injected with LPS formed four-fold fewer colonies compared with control mice. A similar effect occurred in cells from CLP mice: a two-fold reduction was observed.Fig. 3

Bottom Line: Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM.In contrast, in vitro LPS stimulated differentiation of CD34(+) CD38(-) HSCs but did not induce proliferation of these cells in contrast to the CD34(+) CD38(+) progenitors.It is suggestive that the Notch pathway contributed to this effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Flow Cytometry, The Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland. tskirecki@gmail.com.

ABSTRACT

Introduction: An effective immune response to severe bacterial infections requires a robust production of the innate immunity cells from hematopoietic stem and progenitor cells (HSPCs) in a process called emergency myelopoiesis. In sepsis, an altered immune response that leads to a failure of bacterial clearance is often observed. In this study, we aimed to evaluate the impact of sepsis on human HSPCs in the bone marrow (BM) microenvironment of humanized mice subjected to acute endotoxemia and polymicrobial sepsis.

Methods: Humanized mice (hu-NSG) were generated by transplanting NOD.Cg-Prkdc/scidIL2rγ (NSG) mice with the human cord blood CD34(+) cells. Eight weeks after the transplantation, hu-NSG mice were subjected to sepsis induced by endotoxemia-Escherichia coli lipopolysaccharide (LPS)-or by cecal ligation and puncture (CLP). Twenty-four hours later, HSPCs from BM were analyzed by flow cytometry and colony-forming unit (CFU) assay. CLP after inhibition of Notch signaling was also performed. The effects of LPS on the in vitro proliferation of CD34(+) cells from human BM were tested by CellTrace Violet dye staining.

Results: The expression of Toll-like receptor 4 receptor was present among engrafted human HSPCs. Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM. In addition, in both models, accumulation of early CD34(+) CD38(-) HSCs was observed, but the number of CD34(+) CD38(+) progenitors decreased. After CLP, there was a 1.5-fold increase of proliferating CD34(+) CD38(-)Ki-67(+) cells. Moreover, CFU assay revealed a depressed (by 75 % after LPS and by 50 % after CLP) production of human hematopoietic colonies from the BM of septic mice. In contrast, in vitro LPS stimulated differentiation of CD34(+) CD38(-) HSCs but did not induce proliferation of these cells in contrast to the CD34(+) CD38(+) progenitors. CLP sepsis modulated the BM microenvironment by upregulation of Jagged-1 expression on non-hematopoietic cells, and the proliferation of HSCs was Notch-dependent.

Conclusions: CLP sepsis and endotoxemia induced a similar expansion and proliferation of early HSCs in the BM, while committed progenitors decreased. It is suggestive that the Notch pathway contributed to this effect. Targeting early hematopoiesis may be considered as a viable alternative in the existing arsenal of supportive therapies in sepsis.

No MeSH data available.


Related in: MedlinePlus