Roles of DPA derived specialized pre-resolving mediators during vertebrate hematopoiesis

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Blood disease, including anemia, blood clotting, and blood cancers, affects millions of people worldwide each year, causing tremendous economic and social burdens. The American Cancer Society estimated 178,520 blood cancer cases in 2020 in America and approximately 5,600 patients died of anemia in 2019 in the US. Our country faces at least a 45 billion dollars economic burden annually due to blood related diseases. However, the current treatments of blood-related diseases only palliate the symptom and temporarily prevent the disease progression. The goal of this project is to investigate the roles played by specialized pro-resolving mediators (SPMs) derived from polyunsaturated fatty acid (PUFA) n-3 docosapentaenoic acid (DPAn-3) during vertebrate hematopoiesis and evaluate in vivo their therapeutic potentials for blood disorders. I performed chemical screen in zebrafish embryos using several ecosanoid derived SPMs and whole-mount in situ hybridization (WISH) to identify compounds that are able to increase Hematopoietic stem cell (HSC) and red blood cell (RBC). This screen identified two promising SMP compound hits: 7,17 dihydroxy DPA (7,17 diHDPAn-3) and 19,20 epoxy DPA (19,20 EpDPA). In this proposal I will evaluate their effects on the proliferation of downstream hematopoietic lineages, including myeloid and lymphoid lineages by exposing wild-type zebrafish embryos to 1.5 µM 7,17 diHDPAn-3 and 2.5 µM 19,20 EpDPA from 11-36 hours post fertilization (hpf). After fixation, I will perform WISH and real-time quantitative PCR (RT-qPCR) using c-myb, gata1, l-plastin, lyz, mpeg, rag1 and CD79 mRNA probes and primers to descriptively and quantitatively evaluate the resulting effects of the two SPMs exposure on the proliferation of hematopoietic stem cells, progenitor erythroid, granulocytic cells, neutrophils, and macrophages, T and B cells respectively. Our preliminary data show that both 7,17 diHDPAn-3 and 19,20 EpDPA promote HSC proliferation, increase RBC progenitors and RBC number, and increase neutrophil cell number. Therefore, I hypothesize that 7,17 diHDPAn-3 and 19,20 EpDPA exposure enhances HSC proliferation, increasing downstream mature blood cell types in vertebrates. To test our central hypothesis, I developed the following two specific aims: Aim 1. To test the hypothesis that the two selected SPMs increase HSC number, leading to increases in myeloid-derived cell and lymphoid-derived cell numbers and Aim 2. To test the hypothesis that the two selected SPMs can be used as therapeutic interventions in disease models such as bacteria- mediated infection, hematopoiesis genetic diseases, and post-irradiated adult zebrafish. This work will characterize the roles of these two SPMs during vertebrate hematopoiesis and will test their therapeutic potential in vivo. The outcome of this proposal will discover an unknown role for these two SPMs in regulating hematopoiesis, opening a new road to developing a novel therapeutic therapy for blood-related diseases.