N-3 Polyunsaturated Fatty Acid (N-3 PUFA) Derived Lipid Mediators Increase Hematopoiesis during Zebrafish Embryogenesis

Abstract

Polyunsaturated fatty acids (PUFAs), which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. The richest source of PUFAs is fish oil. N-3 PUFAs have been used in the prevention and treatment of coronary artery disease, diabetes, hypertension, arthritis, cancer, and other inflammatory and autoimmune disorders in humans. Lipid mediators derived from n-3 PUFAs are implicated in mediating the resolution of inflammation. However, their effect on hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation and on hematopoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-derived lipid mediators. We exposed wild-type zebrafish embryos to different n-3 PUFA-derived lipid mediators. After fixation, we performed whole mount in situ hybridization (WISH) using antisense RNA probes such as c-myb (hematopoietic stem cell marker), gata1 (erythroid progenitor cell marker), hbbe1 (erythrocyte marker), l-plastin (macrophage and neutrophil cell marker ), mpx (neutrophil cell marker), lyz (neutrophil cell marker), and rag1 (immature lymphocyte marker); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and fluorescence-activated cell sorting (FACS) to understand the effect of lipid mediator exposure on the proliferation of HSPCs, erythroid progenitor cells, erythrocytes, granulocytic cells, neutrophils, macrophages, and T lymphocytes in different development stages during vertebrate hematopoiesis. In addition, we also used o-dianisidine staining to study red blood cell (RBC) formation and transgenic zebrafish to observe different blood lineages including HSPCs, neutrophils, macrophages, erythroid progenitors, and erythrocytes with fluorescent microscopy. We identified two novel lipid mediators which promote HSPC proliferation, enhance erythroid progenitor cell and erythrocyte production, and increase neutrophil cell number. These two molecules could be applied as novel therapeutics for blood disorders and blood cancers such as anemia and leukemia in humans. These two compounds can be intensely investigated to become candidate supplements to enhance HSPC amplification before HSPC transplantation for leukemia patients.