Secondary bile acids function through the vitamin D receptor in myeloid progenitor cells to promote myelopoiesis

Metabolic products of the microbiota can alter hematopoiesis. However, the contribution and site of action of bile acids is poorly understood. Here we demonstrate that the secondary bile acids, deoxycholic acid (DCA), and lithocholic acid (LCA) increase bone marrow myelopoiesis. Treatment of bone marrow cells with DCA and LCA preferentially expanded immunophenotypic and functional (CFU-GM) granulocyte-monocyte progenitors (GMPs). DCA treatment of sorted hematopoietic stem/progenitor cells (HSPCs) increased CFU-GMs, indicating that direct exposure of HSPCs to DCA sufficed to expand GMPs. We determined that the vitamin D receptor (VDR) was required for the DCA-induced increase in CFU-GMs and GMPs. Finally, single-cell RNA sequencing revealed that DCA significantly upregulated genes associated with myeloid differentiation and proliferation in GMPs. The action of DCA on HSPCs to expand GMPs in a VDR-dependent manner suggests a mechanism for how microbiome-host interactions may directly impact bone marrow hematopoiesis and the severity of infectious and inflammatory disease. Overall design: Pre-irradiated murine embryonic liver fibroblasts (AFT024; ATCC- SCRC 1007.1) were seeded to confluency in 24 well plates in DMEM + 10%FBS + 0.05mM 2-mercaptoethanol in a 33°c, 5% CO2 incubator. Long-term culture media was made by the addition of 0.22uM filter sterilized Alpha-MEM with nucleosides (StemCell Inc) containing 1mM hydrocortisone (StemCell Inc) to Myelocult M5300 (StemCell Inc) to a final concentration of 1uM hydrocortisone. AFT024 media was then removed from the wells and two hundred thousand whole bone marrow cells in one mL of complete media was added to each well. Cells were cultured for 1 week either in a media control or in the presence of deoxycholic acid (35uM) or lithocholic acid (18uM). After 1 week, media was removed and placed into a collection tube, cells were then washed with HBSS (StemCell Inc), and this wash was placed into the same collection tube. Cells were then treated with Trypsin-EDTA (0.05%, Fisher Scientific) briefly to detach any cells still adhering to the culture plate. Trypsinization was halted by washing cells with heat-inactivated FBS, followed by IMDM+2%FBS. This wash was taken and added to the same collection tube used prior. Cells in the collection tubes were then centrifuged to pellet them and the media was decanted via aspiration. Cells were resuspended in FACS buffer (PBS+1%FBS), and subsequently stained for spectral flow cytometry to assay the wells for the growth of myeloid populations. All data is shown and is derived from 2 experimental replicates. contributor: Bioinformatics Core Uva ([e-mail])