Polyphenols Modulate Quiescence/Mobilization of Hematopoietic Immature Cells through APC/EPCR/PAR-1 Axis

Coordinated arrangement of bone marrow (BM) microenvironment is essential for the maintenance of hematopoietic stem cells and their progenitors (HSPC), and for the continuous supply of peripheral blood (PB) cells. Quiescence and/or mobilization of these BM cells into the circulation are tightly regulated however non-physiological or stress conditions, such as infections, can accelerate them. Our previous findings have shown that polyphenols from green tea modulate the myeloid population (Gr-1⁺Mac-1⁺) of BM, spleen and PB of mice challenged by lipopolysaccharide (LPS). We have also observed a reduction in the circulating levels of thrombin-antithrombin complex (TAT) and soluble endothelial protein C receptor (sEPCR) of these mice. It has been reported that thrombin induces the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). In this context, our goal was to investigate the effects of polyphenols on the Lin^-Sca-1⁺c-Kit⁺ (LSK) primitive stem cell population and mature populations including: T and B lymphocytes, monocytes, granulocytes and erythrocytes (Lin⁺). The expression of PAR-1 and EPCR in these cells, and the levels of sEPCR in the BM fluid were also evaluated. The 250 mg/kg dose of polyphenol from green tea extract was given once every 7 days orally (gavage) to mice (n=6/group) challenged with i.p. injection of 100µg LPS. Control group received vehicle only. After 24h, mice were sacrificed; BM and PB were collected for the assays. LPS injection induced an increase in circulating LSK, without affecting the pool of these cells in BM. Treatment with polyphenols prevented recruitment of LSK into the PB, and did not alter the percentage of BM LSK. Corroborating these findings, polyphenols also reduced the number of immature progenitors (CFU-C) in the PB, evaluated by the clonal culture assay. In addition, polyphenols did not affect the increase in the number of Lin⁺ cells and circulating leukocytes (total WBC) induced by LPS injection. We also observed that LPS induced a rapid increase in the number of PB LSK and Lin+ cells expressing PAR-1, while reducing BM LSK expressing EPCR. Polyphenols did not affect PAR-1 expression in both populations, but partially recovered expression of EPCR in BM LSK. Polyphenols also reduced the increased levels of sEPCR induced by LPS in the BM niche. Taken together, our results demonstrate that polyphenols promote an effect on the quiescence/mobilization of both the most primitive hematopoietic cells and their progenitors, but does not affect mature populations. The reduction in vascular permeability observed in the BM of mice using Evans blue reinforces this action of polyphenols. It appears that the effect of polyphenols is thrombin dependent; although polyphenols did not affect the expression of PAR-1, they modulated EPCR, mainly in the medullary microenvironment with maintenance of barrier integrity and consequent cellular quiescence. In addition, polyphenols appear to have an anti-inflammatory effect, possibly triggered by their effect on the EPCR.