RNA-seq analysis of sorted colon epithelial cells and colon lamia propria macrophages from mice where Tsc2 is deleted in Lyz2-expressing cells

The intestinal epithelium has a high turnover and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here we show that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, mTORC1 activation in macrophages protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism for optimizing proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as “commensals” that provide metabolic support to promote efficient self-renewal of the colon epithelium. Overall design: Colon lamina propria macrophages (Cd45+, Ly6G-, SiglecF-, Cd24-, Cd11c+/-, Cd103-, Cd11b+, Ly6C-, Cd64+, MHCII+) and Cd44+ epithelial cells (Cd45-, EpCAM+, Cd44+) from 3 Tsc2fl/fl and 4 Tsc2Lyz2-cre mice were isolated as per manufacturer's instructions (MACS Miltenyi Biotec, 130-097-410) and FACS sorted at the FACS Core Unit of the Children's Cancer Research Institute (CCRI) of Vienna. Freshly sorted cells were harvested in RLT-Buffer (RNeasy RNA isolation kits, QIAGEN) containing ß-mercaptoethanol (Sigma) and stored at -80 °C. RNA was isolated using an RNeasy Micro Kit (QIAGEN, Cat No.ID: 74004).