Altered cholesterol immunometabolism activates the macrophage NLRP3-inflammasome in lung fibrosis

Previous research has highlighted dysregulation in lipid metabolism during lung fibrosis. However, the impact of cholesterol immunometabolism during lung fibrosis progression remains unclear but has been related to the NLRP3-inflammasome activation in cardiovascular diseases. The main objective of this work was to investigate the link between altered cholesterol metabolism and NLRP3 inflammasome activation in fibrotic lungs. Different pulmonary fibrosis patient cohorts (from 2 centers and a publicly available dataset) and a murine model of lung fibrosis (aged SP-C-/-) were included. Expression of cholesterol metabolism proteins and cholesterol content were determined in lungs from patients and bronchoalveolar lavage fluid (BALF) cells of aging SP-C-/- mice. Metabolomic and lipidomic analyses were conducted in BALF and BALF cells of SP-C-/- versus wild-type (WT) mice. NLRP3 inflammasome components were assessed by immunoblotting, ELISA, and immunofluorescence. Lung samples from fibrosis patients showed higher cholesterol content, altered cholesterol metabolism, and higher IL-18 levels compared to controls. Moreover, key genes related to inflammasome activation and cholesterol metabolism were differentially expressed in alveolar macrophages from IPF patients with idiopathic pulmonary fibrosis. Accordingly, BALF cells of SP-C-/- mice showed alteration of their cholesterol metabolism and inflammasome activation with age and fibrosis development. Lipidomic analysis pointed at cholesterol esters as potential activating agent. The molecular mechanism linking cholesterol esters to NLRP3 inflammasome and fibrosis markers was confirmed in vitro in a human macrophage model. In conclusion, altered cholesterol esterification activates the NLRP3 inflammasome in alveolar macrophages during pulmonary fibrosis in a murine model and patients with fibrosis.