PS1225 NOVEL PRO‐INFLAMMATORY MACROPHAGES ARE INVOLVED IN HEMATOPOIETIC STEM CELL DEVELOPMENT
Authors: E. Dzierzak, S. Mariani, Z. Li, S. Rice, C. Krieg, M. Fragkogianni, C.S. Vink, J. Pollard
Journal: HemaSphere (2019)

Background: Knowledge of the developmental cells and factors that affect the generation of Hemaotpoietic Stem Cells (HSC) in the embryo will inform strategies on ex vivo HSC generation for therapeutic application. HSC are generated from specialized endothelial cells of the embryonic aorta. In the rapidly changing microenvironment surrounding the aorta, the so‐called Aorta‐Gonad‐Mesonephros (AGM) region provides inducting factors for generation of the potent HSC that are responsible for the life‐long production of the blood system. Previously, inflammatory factors have been implicated in regulating HSC development in the mouse embryo, but it is unclear what cells in the AGM microenvironment are the source. Whereas it is known that macrophages play both pro‐ and anti‐inflammatory roles and influence HSCs in the adult, it is as yet unknown whether the macrophages found in the embryo prior to HSC generation are involved in the AGM HSC‐generative microenvironment. Aims: To investigate whether innate immune cell types and macrophages, which are generated prior to HSCs in the mouse embryo, play a role the AGM microenvironment and to characterize those cells phenotypically and functionally to understand whether and how these cells influence the development of the potent adult‐type HSCs. Methods: We examined the cell types in the midgestation mouse AGM by mass cytometry (CyTOF) with 23 hematopoietic markers. Immuno‐histology of embryos was performed to localize macrophages and vital time‐lapse imaging to study cell motility. Quantitative (FACS, developmental time courses) and qualitative analyses (functional hematopoietic progenitor and in vivo hematopoietic cell transplantation assays) were performed to characterize AGM macrophages. Co‐cultures of aortic hemogenic endothelial cells and AGM macrophages on OP9‐DL1 stromal cells tested the induction abilities of macrophages. Mouse genetic models and specific macrophage‐depletion methods were used to eliminate macrophages or prevent mobilization. RNA sequencing and qRT‐PCR identified and validated the transcriptome of AGM macrophages. Results: Our CyTOF results on mouse embryonic day 10 AGM regions indicate two abundant myeloid cell types – mannose‐receptor positive AGM‐associated macrophages (AGM‐aM) and mannose‐receptor negative progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1/CX3CL1 – in the absence of this chemokine signaling pathway, macrophages are not mobilized to the AGM and instead are retained in the yolk sac. Depletion of AGM macrophages by two complementary methods resulted in reduced hematopoietic progenitor numbers and an almost complete absence of HSCs in the AGM. Importantly, AGM‐aM express a pro‐inflammatory signature, localize to the embryonic aorta and dynamically interact with nascent and emerging intra‐aortic hematopoietic cells (IAHC). Summary/Conclusion: Macrophages that are produced prior the generation of HSCs dynamically interact with nascent/emerging hematopoietic cells in the midgestation aorta to affect the generation of HSCs. Mannose receptor positive macrophages specifically localize to intra‐aortic cluster cells. The timely presence of macrophages in the AGM enhances the generation of fully functional hematopoietic progenitor cells and HSCs. Together, our results identify a novel pro‐inflammatory macrophage subset that regulates the embryonic development of adult‐type HSCs.