RNAseq for peritoneal macrophages phagocytosing E.coli in the absence or presence of retinoic acid

Peritoneal macrophages comprise two distinct subpopulations: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs), each with unique phenotypic and functional characteristics. This study investigates the role of retinoic acid (RA) in modulating the response of peritoneal macrophages to bacterial infections. Using a murine peritonitis model, we demonstrate that RA enhances the phagocytic capacity of macrophages, delays the decline in phagocytic function, and alters macrophage distribution in the peritoneal cavity. RA treatment induced notable transcriptional changes in both LPMs and SPMs. Overall design: The E. coli ER2272 strain utilized in the experimental procedure incorporates the fluorescent construct pUC19-tdTomato, in which the pUC19 plasmid is capable of constitutive expression of the fluorescent protein tdTomato. Prior to injection, the E. coli bacteria were cultured overnight in LB medium at 37°C and then mixed with 0.2 ml PBS to create a suspension of 1 × 107 CFU/mouse, which was subsequently injected into the peritoneal cavity. To compared the effect of 24 hours treatment of retinoic acid (RA) on the SPMs and LPMs, we separated the mice into two groups: control and RA treatment. In some cases, RA was injected 24 hours before the E. coli injection.Upon the injection of bacteria containing pUC19-tdTomato, both SPMs and LPMs in the the peritoneal cavity can be detected based on the antibody staining of F4/80 and the phagoytosis of E.coli. SPMs and LPMs from the respective group were isolated from the peritoneal cavity using flow cytometry sorting. Total RNA was extracted from cell pellets using TRIzol reagent according to the manufacturer's protocol. High-throughput RNA sequencing was performed using Illumina NovaSeq Xplus platform.