Early severe impairment of hematopoietic stem and progenitor cells from the bone marrow caused by CLP sepsis and endotoxemia in a humanized mice model.
BACKGROUND
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.
Department of Flow Cytometry, The Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland. tskirecki@gmail.com.|Department of Anesthesiology and Intensive Care Medicine, The Center of Postgraduate Medical Education, Czerniakowska 231, 00-416, Warsaw, Poland. tskirecki@gmail.com.
Tomasz
T
Skirecki
Department of Flow Cytometry, The Center of Postgraduate Medical Education, Marymoncka 99/103, 01-813, Warsaw, Poland. jkawiak@ibib.waw.pl.
Jerzy
J
Kawiak
Department of Cellular Engineering, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, W. Roentgena 5, 02-781, Warsaw, Poland. ekmachaj@coi.waw.pl.
Eugeniusz
E
Machaj
Department of Cellular Engineering, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, W. Roentgena 5, 02-781, Warsaw, Poland. zpojda@coi.waw.pl.
Zygmunt
Z
Pojda
Department of Clinical Cytology, The Center of Postgraduate Medical Education, Marymocnka 99/103, 01-813, Warsaw, Poland. wasilewska-danuta@tlen.pl.
Danuta
D
Wasilewska
Department of Pediatric Orthopedics, The Center of Postgraduate Medical Education, Konarskiego 13, 05-400, Otwock, Poland. wicedyrdn@cmkp.edu.pl.
Jarosław
J
Czubak
Department of Clinical Cytology, The Center of Postgraduate Medical Education, Marymocnka 99/103, 01-813, Warsaw, Poland. graho@cmkp.edu.pl.
Grażyna
G
Hoser
D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D001854:Bone Marrow Cells / Q000473:pathology*; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D019446:Endotoxemia / Q000503:physiopathology*; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D018805:Sepsis / Q000503:physiopathology*; D013234:Stem Cells / Q000473:pathology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4