Spatial analysis of murine lungs exposed to fibrogenic stress

Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. To better study immune cell heterogeneity during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. SP-CI73T mice received corn oil (controls) or tamoxifen (175 mg/kg, orally) to excise a neomycin cassette placed on the surfacant protein C gene locus, hence inducing the expression of the mutant transcript. We have previously shown that a complex inflammatory response arises by 14 days post tamoxifen administration. Paraffin sections of murine lungs were therefore collected in controls and 14 days post induction. Immunofluorescence for DNA, CD45, CD68 was performed to identify macrophages (CD45+CD68+) and non-immune ("other", DNA+CD45-) cells within healthy, peri-injured and injured regions of the lung. A total of 12 regions (680um x 700um in size) were selected and macrophages and "other" cells isolated. RNA sequencing was then performed. Principal component and pathway analysis revealed spatial heterogeneity with an hyperactive peri-injured compartment (extracellular matrix remodeling, innate and adaptive immunity, senescence). Cells isolated from injured areas of the lung were relatively quiescent. These results illustrate regional heterogeneity in immune and non-immune cells of the lung during fibrogenic stress.