Bulk RNA of IL-4 and squalene treated MDM

We performed bulk RNA-seq on squalene/M-CSF-treated macrophages to compute the fold change of genes in the squalene/M-CSF compared to M-CSF alone treated macrophages. We then looked at the distribution of the log fold changes in the skin-derived TREM2 (607 genes) and M2-like (709 genes) macrophage scRNA-seq signatures. We found that the TREM2 gene signature was more highly induced by squalene/M-CSF than the M2-like macrophage signature. In these squalene-induced macrophages, we found upregulation of several of the lipid metabolism and pro-inflammatory genes present in the skin-derived TREM2 macrophage gene signature. The study was performed in accordance with protocols approved by the institutional review board at University of California, Los Angeles. All patients provided written informed consent. Healthy donors were recruited from the University of California, Los Angeles. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from peripheral blood using a Ficoll-Paque (GE Healthcare) density gradient. Monocytes were isolated from PBMCs via positive selection with CD14 microbead (Miltenyi Biotec, cat# 130-050-201). Monocytes were seeded at 500,000 cells/well in a 24-well plate and differentiated in M-CSF (50 ng/ml) (R&D Systems, cat. # 216MC025/CF) for 5 day in RPMI with 10% FCS at 37°C. Macrophages were then stimulated with M-CSF (50 ng/ml) (R&D Systems, cat. # 216MC025/CF), IL-4 (20ng/mL) (Peprotech, cat. # 200-04), and squalene (1 mM) (Thermofisher Scientific, cat.# S3626-100ML) for 1 days. RNA were extracted using Trizol RNA extraction protocol and Qiagen RNeasy Micro Kit . We quantified the RNA concentration and quality using TapeStation.