RNA-seq gene expression profiles of primary murine peritoneal mesothelial cells.

Local factors produced in the tissue microenvironment play essential roles in promoting the ontogeny and phenotype of tissue resident macrophages (TRM). In the peritoneal cavity, large peritoneal macrophages (LPM) are the dominant TRMs that functionally mediate type 2 immunity, facilitate tissue repair of the mesothelium, and protect against peritoneal fibrosis. It is established that retinoic acid derived from the omentum induces transcription factor Gata6 expression in LPMs, which in turn regulates gene expression of factors that define peritoneal macrophages. It is still unclear whether retinoic acid is the sole local factor that regulates Gata6 expression in LPMs. Mesothelial cells line the entire peritoneal cavity and produce a protective, non-adhesive barrier against injury, at least in part by recruiting immune cells with secreted cytokines, such as M-CSF. We hypothesized that secreted factors from peritoneal mesothelial cells are also responsible for regulating LPM development including both ontogeny and function. Due to their immediate proximity to the peritoneal cavity, we propose that mesothelial cells can produce and secrete proteins into the peritoneum to maintain Gata6 expression by LPMs. To identify secreted factors that are highly and specifically expressed in mesothelial cells, we harvested primary mesothelial cells from 10-week-old C57BL/6 mice using FACS selection (CD45- PDPN+ GPM6a+). Total RNA was isolated from these cells and subjected to RNA-seq analysis after depletion of ribosomal RNA. To measure mesothelial gene expression at homeostasis, 10-week-old C57BL/6 mice were sacrificed (n=4 animals). Total peritoneal cells were isolated by trypsin digestion of the peritoneal cavity. Primary mesothelial cells were sorted from the cell suspension on a FACS Aria II Flow cytometer, using CD45- Pdpn+ GPM6a+ gating. Cells were sorted into DMEM + 10% FBS and stored on ice. Total RNA was extracted from the sorted mesothelial cells using the NucleoSpin RNA kit (Macherey-Nagel). rRNA was depleted using Ribo-Zero kit. RNA libraries were prepared for sequencing using standard Illumina protocols at the Washington University Genome Technology Access Center (GTAC).