Resolution of Inflammation Increases Hematopoietic Stem Cell Number and Erythropoiesis

Hematopoietic stem cell (HSC) homeostasis is tightly control from early embryogenesis through adult life. To identify new modulators of HSC formation, we performed a targeted screen by selecting only bio-active Polyunsaturated fatty acids (PUFAs) metabolites. PUFAs, which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. Some lipid mediators derived from n-3 PUFAs named specialized pro-resolving mediators (SPMs) are implicated in mediating the resolution of inflammation. However, their effect on HSCs proliferation and erythropoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-SPMs. We exposed wild-type zebrafish embryos to several n-3 PUFA-derived SPMs and identified a natural occurring docosapentaenoic acid (DPA, 22:5n3) derived SPM as being able to increase c-myb (an HSC marker) positive cells by whole mount in situ hybridization and qPCR during zebrafish primitive hematopoiesis. We found that our selected SPM also increase the expression of scl/tal-1 (a primitive HSC marker) and runx1 another HSC marker. SPM exposure also increases the expression of the erythroid progenitor gata1 by 40% during zebrafish primitive erythropoiesis. Red blood cells (RBCs) staining in SPM exposed zebrafish embryos revealed a strong increase in RBCs while the expression of the erythrocyte markers hemoglobin beta embryonic 1 and hemoglobin alpha embryonic 3 are increased by 40% increase. As RBC and platelet share the same progenitor cells, we took advantage of the CD41-GFP transgenic zebrafish that expresses GFP in platelet cells and measure the expression of the platelet maker CD41 revealing that the number of platelet cells is not affected by SPM exposure. We confirm that the increase in RBC numbers during primitive hematopoiesis holds true for definitive hematopoiesis by exposing zebrafish larvae after the primitive wave wears off in the organism. we investigated the quality of these excess of RBCs under SPM exposure. To this end we used the gata1-DsRed transgenic zebrafish embryos in which a red fluorescence will be solely present in immature and mature erythrocytes. We exposed the gata1-DSred embryos to SPM and collected the red fluoresce cells by cell sorting. Metabolomics analysis indicated that SPM exposure does not modify the metabolomic content of the selected cells as only three out of eighty-two metabolite investigated (nicotinamide, citrate, and myristoyl-L-carnitine (AC(14:0)), showed a significant decreased. Additionally, RNA-sequencing analysis revealed that SPM exposure increases the expression of the xpo1b and hsp90 genes involved in erythrocyte maturation and heme synthesis respectively. SPM are specialized molecules involved in the resolution of inflammation. To understand the link between inflammation resolution and HSC formation, we exposed zebrafish embryos to 75 µM of dexamethasone and detected a strong increase in runx1 and c-myb positive cells and expression. Co-treatment of zebrafish embryos will sub-phenotypic doses of dexamethasone and our selected SPM showed an increase in c-myb and runx1 expression demonstrated additive affects between steroid and SPM action during HSC formation. Our results demonstrated that SPM exposure can increase HSC formation and erythropoiesis without affecting platelet number. Moreover, our results identified a novel role for the resolution of inflammation during HSC formation.