Analysis of transcriptional profile of lung resident macrophages during acute and resolution phase of LPS inhalation induced lung injury.

Analysis of transcriptional profile of lung resident macrophages during acute and resolution phase of LPS inhalation induced lung injury. Because macrophages coordinate both the induction and resolution of inflammatory lung injury, we examined the transcriptional signatures of resident lung macrophages isolated from LysM-GFP mice during baseline (0h), peak of injury (4h), and during the resolution phase (24h). Acute lung injury was induced in LysM-GFP mice through nebulization of a solution of LPS in saline (1mg/ml). At experimental timepoints, lungs were surgically removed, minced, and digested in a collogenase solution for 50min at 37 degrees, followed by mechanical separation by repeatedly passing tissue through a narrow gauge syringe. Single cell suspension was stained with florescently tagged antibodies for myeloid cell surface markers. Cells were isolated by FACS sorting: GFP+ cells were selected, followed by ly6g negative gating to exclude neutrophils, and CD64+ cells for mature macrophages. RNA from the sorted cells was collected in Trizol and isolated from cells via ethanol precipitation, then submitted for RNA sequencing.