Mechanism for HSPC expansion in the endothelial cuddling niche microenvironment through inflammatory lipid mediators receptor CysLTR1

Abstract Hematopoietic stem and progenitor cells (HSPCs) closely interact with the niche, allowing dynamic regulation of hematopoiesis. Despite fetal liver and bone marrow being considered general hematopoietic niches, HSPCs are reported to reside in confined niche microenvironments that orchestrate their proliferation, differentiation and self-renewal. HSPC arrival stimulates the remodeling of a subgroup of endothelial cells (ECs) to form a ‘pocket-like’ structure and HSPCs physically interact with these ECs, termed ‘endothelial cuddling’. A HSPC often undergoes a self-renewal division within the endothelial ‘pocket’, yet the mechanism is unknown. From a chemical screen to identify compounds that expand HSPCs using runx1+23:GFP zebrafish, I identified a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist that expanded HSPCs in the caudal hematopoietic niche by 1.7 fold (p<0.0001). Intriguingly, CysLTR1 antagonist specifically increased HSPC divisions within the endothelial ‘pocket’ using high-resolution time-lapse imaging (2.22 fold, p<0.01). Genetic knockout of CysLTR1 recapitulated the phenotypes of CysLTR1 antagonists in increasing HSPCs (1.83 fold increase, p<0.01). CysLTR1 is a G-coupled receptor of cysteinyl leukotrienes that are bioactive lipid mediators playing key roles in inflammation. High-sensitivity lipidomics detected cysteinyl leukotrienes in HSPC niches. Vascular administration of cysteinyl leukotriene LTD4 inhibited HSPC division (p<0.001). scRNA-seq analysis revealed that CysLTR1 is specifically expressed in a subgroup of sinusoidal endothelial cells (ECs). To identify the downstream targets of CysLTR1, I performed ultra-low-input RNA-seq on sinusoidal ECs upon CysLTR1 antagonist or LTD4 treatment. Sinusoidal ECs showed upregulation of genes in angiogenesis, vascular integrity and cytokine il11a upon CysLTR1 inhibition and corresponding downregulation with LTD4 treatment. Cytokine il11a overexpression abolished the inhibitory effect of LTD4 administration on HSPC increase (p<0.0001), suggesting that IL11 is the downstream target of CysLTR1 signaling. CysLTR1 KO fish have an increased number of smaller sinusoids in the marrow, indicating a role of CysLTR1 in niche vascular remodeling. Together, our studies revealed a novel mechanism of lipid mediator signaling through CysLTR1 that modulates the niche endothelial cells to regulate HSPC division in the niche microenvironment and an undiscovered role of CysLTR1 in niche vascular remodeling.