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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

Effects of Notch pathway inhibition in human HSPCs 24 h after induction of CLP sepsis in hu-mice. Pretreatment with DAPT (60 mg/kg) 2 h before CLP surgery inhibited activation of Notch-1 assessed by fluorescence intensity of NICD (a). Impact of the inhibition of Notch signaling on the CLP-induced expansion of CD34+ CD38− HSCs (b, c) and effect on CD34+ CD38+ progenitors (d, e). Influence of DAPT treatment on the frequency of proliferating HSCs (f), and the rate of apoptosis of CD34+ CD38− cells (g). Effect of DAPT on the expression of G-CSFR on HSPCs after CLP (h), and the phosphorylation of STAT3 (i). n = 3–5. *P < 0.05, **P < 0.001, ***P < 0.001. CLP cecum ligation and puncture, G-CSFR granulocyte colony-stimulating factor receptor, GMF geometric mean fluorescence, HSC hematopoietic stem cell, HSPC hematopoietic stem and progenitor cell, NICD Notch-1 intracellular domain
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Fig7: Effects of Notch pathway inhibition in human HSPCs 24 h after induction of CLP sepsis in hu-mice. Pretreatment with DAPT (60 mg/kg) 2 h before CLP surgery inhibited activation of Notch-1 assessed by fluorescence intensity of NICD (a). Impact of the inhibition of Notch signaling on the CLP-induced expansion of CD34+ CD38− HSCs (b, c) and effect on CD34+ CD38+ progenitors (d, e). Influence of DAPT treatment on the frequency of proliferating HSCs (f), and the rate of apoptosis of CD34+ CD38− cells (g). Effect of DAPT on the expression of G-CSFR on HSPCs after CLP (h), and the phosphorylation of STAT3 (i). n = 3–5. *P < 0.05, **P < 0.001, ***P < 0.001. CLP cecum ligation and puncture, G-CSFR granulocyte colony-stimulating factor receptor, GMF geometric mean fluorescence, HSC hematopoietic stem cell, HSPC hematopoietic stem and progenitor cell, NICD Notch-1 intracellular domain

Mentions: CLP led to a significant increase in the fluorescence of NICD in CD34+ CD38− cells which was abrogated by DAPT pretreatment (Fig. 7a). This effect was similar in CD34+ CD38+ cells, although the differences did not reach significance (Fig. 7a). The Notch blockade resulted in a seven-fold reduction of the expansion of primitive CD34+ CD38− (Fig. 7b, c) but had no effect on the more mature CD34+ CD38+ population (Fig. 7d, e). In addition, the percentage of actively proliferating CD34+ Lin− HSCs decreased in the presence of DAPT from 58 % to 37 % (P < 0.001, Fig. 7f).Fig. 7


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)

Effects of Notch pathway inhibition in human HSPCs 24 h after induction of CLP sepsis in hu-mice. Pretreatment with DAPT (60 mg/kg) 2 h before CLP surgery inhibited activation of Notch-1 assessed by fluorescence intensity of NICD (a). Impact of the inhibition of Notch signaling on the CLP-induced expansion of CD34+ CD38− HSCs (b, c) and effect on CD34+ CD38+ progenitors (d, e). Influence of DAPT treatment on the frequency of proliferating HSCs (f), and the rate of apoptosis of CD34+ CD38− cells (g). Effect of DAPT on the expression of G-CSFR on HSPCs after CLP (h), and the phosphorylation of STAT3 (i). n = 3–5. *P < 0.05, **P < 0.001, ***P < 0.001. CLP cecum ligation and puncture, G-CSFR granulocyte colony-stimulating factor receptor, GMF geometric mean fluorescence, HSC hematopoietic stem cell, HSPC hematopoietic stem and progenitor cell, NICD Notch-1 intracellular domain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Effects of Notch pathway inhibition in human HSPCs 24 h after induction of CLP sepsis in hu-mice. Pretreatment with DAPT (60 mg/kg) 2 h before CLP surgery inhibited activation of Notch-1 assessed by fluorescence intensity of NICD (a). Impact of the inhibition of Notch signaling on the CLP-induced expansion of CD34+ CD38− HSCs (b, c) and effect on CD34+ CD38+ progenitors (d, e). Influence of DAPT treatment on the frequency of proliferating HSCs (f), and the rate of apoptosis of CD34+ CD38− cells (g). Effect of DAPT on the expression of G-CSFR on HSPCs after CLP (h), and the phosphorylation of STAT3 (i). n = 3–5. *P < 0.05, **P < 0.001, ***P < 0.001. CLP cecum ligation and puncture, G-CSFR granulocyte colony-stimulating factor receptor, GMF geometric mean fluorescence, HSC hematopoietic stem cell, HSPC hematopoietic stem and progenitor cell, NICD Notch-1 intracellular domain
Mentions: CLP led to a significant increase in the fluorescence of NICD in CD34+ CD38− cells which was abrogated by DAPT pretreatment (Fig. 7a). This effect was similar in CD34+ CD38+ cells, although the differences did not reach significance (Fig. 7a). The Notch blockade resulted in a seven-fold reduction of the expansion of primitive CD34+ CD38− (Fig. 7b, c) but had no effect on the more mature CD34+ CD38+ population (Fig. 7d, e). In addition, the percentage of actively proliferating CD34+ Lin− HSCs decreased in the presence of DAPT from 58 % to 37 % (P < 0.001, Fig. 7f).Fig. 7

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