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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

Impact of LPS on the differentiation and proliferation of purified CD34+ human BM cells in vitro. a Effect of LPS on the frequency of the CD34+ CD38− subpopulation after 9 days of culture. b Effect of LPS on the proliferative index of CD34+ CD38+ subpopulation. c Proliferation of CD34+ CD38+ cells in the presence of LPS. Impact of LPS on the frequency of CD34+ CD38− cells (d) and CD34+ CD38+ cells (e) within first, second, and third generations of daughter cells (fewer than four generations). All results obtained after 9 days of culture in atmosphere of 1 % O2. n = 6, *P < 0.05, **P < 0.001. BM bone marrow, LPS lipopolysaccharide
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Fig5: Impact of LPS on the differentiation and proliferation of purified CD34+ human BM cells in vitro. a Effect of LPS on the frequency of the CD34+ CD38− subpopulation after 9 days of culture. b Effect of LPS on the proliferative index of CD34+ CD38+ subpopulation. c Proliferation of CD34+ CD38+ cells in the presence of LPS. Impact of LPS on the frequency of CD34+ CD38− cells (d) and CD34+ CD38+ cells (e) within first, second, and third generations of daughter cells (fewer than four generations). All results obtained after 9 days of culture in atmosphere of 1 % O2. n = 6, *P < 0.05, **P < 0.001. BM bone marrow, LPS lipopolysaccharide

Mentions: The modulation of hu-HSCs in in vivo models is the sum of complex signaling stimuli triggered by sepsis. Thus, we investigated how LPS-induced TLR4 stimulation influences proliferation of purified HSPCs. After 9 days of culture, the presence of LPS reduced the frequency of CD34+ CD38− HSCs by 44 % compared with control (Fig. 5a). As the cells were stained with a fluorescent dye (CellTrace Violet), we were able to trace the proliferation history of each cell. When cultured in the medium alone, CD34+ CD38+ cells displayed higher proliferative activity (defined by proliferative index [23]) in comparison with CD34+ CD38− cells (14.3 ± 5.4 versus 7 ± 2.1, P < 0.05) (Fig. 5b). The addition of LPS did not influence the proliferative index of CD34+ CD38− HSCs, but the index strongly increased in CD34+ CD38+ cells (P < 0.001) (Fig. 5c). We also calculated frequency of the daughter cells in the first, second, and third generations. LPS did not change the frequency of CD34+ CD38− cells within the sum of fewer than four generations, but it significantly decreased frequency of the CD34+ CD38+ cells in fewer than four generations (by nine-fold) (Fig. 5d, e). These observations suggest that LPS accelerated the proliferation of the more differentiated CD34+ CD38+ cell population.Fig. 5


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)

Impact of LPS on the differentiation and proliferation of purified CD34+ human BM cells in vitro. a Effect of LPS on the frequency of the CD34+ CD38− subpopulation after 9 days of culture. b Effect of LPS on the proliferative index of CD34+ CD38+ subpopulation. c Proliferation of CD34+ CD38+ cells in the presence of LPS. Impact of LPS on the frequency of CD34+ CD38− cells (d) and CD34+ CD38+ cells (e) within first, second, and third generations of daughter cells (fewer than four generations). All results obtained after 9 days of culture in atmosphere of 1 % O2. n = 6, *P < 0.05, **P < 0.001. BM bone marrow, LPS lipopolysaccharide
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Impact of LPS on the differentiation and proliferation of purified CD34+ human BM cells in vitro. a Effect of LPS on the frequency of the CD34+ CD38− subpopulation after 9 days of culture. b Effect of LPS on the proliferative index of CD34+ CD38+ subpopulation. c Proliferation of CD34+ CD38+ cells in the presence of LPS. Impact of LPS on the frequency of CD34+ CD38− cells (d) and CD34+ CD38+ cells (e) within first, second, and third generations of daughter cells (fewer than four generations). All results obtained after 9 days of culture in atmosphere of 1 % O2. n = 6, *P < 0.05, **P < 0.001. BM bone marrow, LPS lipopolysaccharide
Mentions: The modulation of hu-HSCs in in vivo models is the sum of complex signaling stimuli triggered by sepsis. Thus, we investigated how LPS-induced TLR4 stimulation influences proliferation of purified HSPCs. After 9 days of culture, the presence of LPS reduced the frequency of CD34+ CD38− HSCs by 44 % compared with control (Fig. 5a). As the cells were stained with a fluorescent dye (CellTrace Violet), we were able to trace the proliferation history of each cell. When cultured in the medium alone, CD34+ CD38+ cells displayed higher proliferative activity (defined by proliferative index [23]) in comparison with CD34+ CD38− cells (14.3 ± 5.4 versus 7 ± 2.1, P < 0.05) (Fig. 5b). The addition of LPS did not influence the proliferative index of CD34+ CD38− HSCs, but the index strongly increased in CD34+ CD38+ cells (P < 0.001) (Fig. 5c). We also calculated frequency of the daughter cells in the first, second, and third generations. LPS did not change the frequency of CD34+ CD38− cells within the sum of fewer than four generations, but it significantly decreased frequency of the CD34+ CD38+ cells in fewer than four generations (by nine-fold) (Fig. 5d, e). These observations suggest that LPS accelerated the proliferation of the more differentiated CD34+ CD38+ cell population.Fig. 5

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