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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

Development of human hematopoietic cells in the bone marrow of NSG mice 8 weeks after transplantation of human CD34+ cells. Representative dot plots show flow cytometry analysis of bone marrow cells with specific anti-human monoclonal antibodies. a Diagram showing mean frequency of human leukocyte subsets in the bone marrow of humanized mice. b Staining of bone marrow from non-humanized NSG mouse with anti-human CD45. c Morphology of bone marrow cells from hu-NSG. d Expression of pan-hematopoietic CD45 antigen in cells gated in R1. e Analysis of monocytes. f Analysis of B cells. g Analysis of T cells. h Analysis of myeloid cells. i Analysis of hematopoietic stem and progenitor cells (gated from R1). j Histogram comparing expression of TLR4 receptor on HSCs and progenitor cells (gated from i). k Expression of TLR4 is present on both undifferentiated Lineage− cells and mature Lineage+ cells. The values on graphs present percentages of a given population from a maternal gate. HSC hematopoietic stem cell, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ, NSG NOD.Cg-Prkdc/scidIL2rγ, TLR Toll-like receptor
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Fig1: Development of human hematopoietic cells in the bone marrow of NSG mice 8 weeks after transplantation of human CD34+ cells. Representative dot plots show flow cytometry analysis of bone marrow cells with specific anti-human monoclonal antibodies. a Diagram showing mean frequency of human leukocyte subsets in the bone marrow of humanized mice. b Staining of bone marrow from non-humanized NSG mouse with anti-human CD45. c Morphology of bone marrow cells from hu-NSG. d Expression of pan-hematopoietic CD45 antigen in cells gated in R1. e Analysis of monocytes. f Analysis of B cells. g Analysis of T cells. h Analysis of myeloid cells. i Analysis of hematopoietic stem and progenitor cells (gated from R1). j Histogram comparing expression of TLR4 receptor on HSCs and progenitor cells (gated from i). k Expression of TLR4 is present on both undifferentiated Lineage− cells and mature Lineage+ cells. The values on graphs present percentages of a given population from a maternal gate. HSC hematopoietic stem cell, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ, NSG NOD.Cg-Prkdc/scidIL2rγ, TLR Toll-like receptor

Mentions: Among all human hematopoietic cells, we identified HSPCs as well as differentiated myeloid and lymphoid cells (Fig. 1). Although human erythrocytes were not detected in the peripheral blood, the BM contained cells from the erythroid lineage as determined by the expression of hu-glycophorin A (CD235a). In the peripheral blood, the human CD45+ leukocytes were present at frequencies of 1–29 %, mostly being CD19+ or CD33+ (data not shown). Within the splenocytes, hu-CD45+ cells were present at the average of 17 ± 9 %. Human hematopoietic cells residing in the murine BM expressed TLR4: the antigen was present on both differentiated (Lineage+) and the Lineage− undifferentiated cells (Fig. 1j). Both CD34+ CD38− HSCs and CD34+ CD38+ progenitors (Fig. 1h) expressed TLR4; the TLR4 surface density was higher on the latter cells (Fig. 1i).Fig. 1


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)

Development of human hematopoietic cells in the bone marrow of NSG mice 8 weeks after transplantation of human CD34+ cells. Representative dot plots show flow cytometry analysis of bone marrow cells with specific anti-human monoclonal antibodies. a Diagram showing mean frequency of human leukocyte subsets in the bone marrow of humanized mice. b Staining of bone marrow from non-humanized NSG mouse with anti-human CD45. c Morphology of bone marrow cells from hu-NSG. d Expression of pan-hematopoietic CD45 antigen in cells gated in R1. e Analysis of monocytes. f Analysis of B cells. g Analysis of T cells. h Analysis of myeloid cells. i Analysis of hematopoietic stem and progenitor cells (gated from R1). j Histogram comparing expression of TLR4 receptor on HSCs and progenitor cells (gated from i). k Expression of TLR4 is present on both undifferentiated Lineage− cells and mature Lineage+ cells. The values on graphs present percentages of a given population from a maternal gate. HSC hematopoietic stem cell, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ, NSG NOD.Cg-Prkdc/scidIL2rγ, TLR Toll-like receptor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Development of human hematopoietic cells in the bone marrow of NSG mice 8 weeks after transplantation of human CD34+ cells. Representative dot plots show flow cytometry analysis of bone marrow cells with specific anti-human monoclonal antibodies. a Diagram showing mean frequency of human leukocyte subsets in the bone marrow of humanized mice. b Staining of bone marrow from non-humanized NSG mouse with anti-human CD45. c Morphology of bone marrow cells from hu-NSG. d Expression of pan-hematopoietic CD45 antigen in cells gated in R1. e Analysis of monocytes. f Analysis of B cells. g Analysis of T cells. h Analysis of myeloid cells. i Analysis of hematopoietic stem and progenitor cells (gated from R1). j Histogram comparing expression of TLR4 receptor on HSCs and progenitor cells (gated from i). k Expression of TLR4 is present on both undifferentiated Lineage− cells and mature Lineage+ cells. The values on graphs present percentages of a given population from a maternal gate. HSC hematopoietic stem cell, hu-NSG humanized NOD.Cg-Prkdc/scidIL2rγ, NSG NOD.Cg-Prkdc/scidIL2rγ, TLR Toll-like receptor
Mentions: Among all human hematopoietic cells, we identified HSPCs as well as differentiated myeloid and lymphoid cells (Fig. 1). Although human erythrocytes were not detected in the peripheral blood, the BM contained cells from the erythroid lineage as determined by the expression of hu-glycophorin A (CD235a). In the peripheral blood, the human CD45+ leukocytes were present at frequencies of 1–29 %, mostly being CD19+ or CD33+ (data not shown). Within the splenocytes, hu-CD45+ cells were present at the average of 17 ± 9 %. Human hematopoietic cells residing in the murine BM expressed TLR4: the antigen was present on both differentiated (Lineage+) and the Lineage− undifferentiated cells (Fig. 1j). Both CD34+ CD38− HSCs and CD34+ CD38+ progenitors (Fig. 1h) expressed TLR4; the TLR4 surface density was higher on the latter cells (Fig. 1i).Fig. 1

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