Lipidomic analysis of bronchoalveolar lavage from mice exposed to ozone

Exposure to ozone causes decrements in pulmonary function, a response associated with alterations in lung lipids. Pulmonary lipid homeostasis is dependent on the activity of peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that regulates lipid uptake and catabolism by alveolar macrophages (AMs). Herein, we assessed the role of PPARγ in ozone-induced dyslipidemia and aberrant lung function in mice. Exposure of mice to ozone (0.8 ppm, 3 hr) resulted in a significant reduction in lung hysteresivity at 72 hr post-exposure; this correlated with increases in levels of total phospholipids, specifically cholesteryl esters, ceramides, phosphatidylcholines, phosphorylethanolamines, sphingomyelins, and di- and triacylglycerols in lung lining fluid. This was accompanied by a reduction in relative surfactant protein-B (SP-B) content, consistent with surfactant dysfunction. Administration of the PPARγ agonist, rosiglitazone (5 mg/kg/day, i.p.) reduced total lung lipids, in..., Bronchoalveolar lavage fluid collection and analysis
Lung cells and cell-free lung lining fluid were collected by bronchoalveolar lavage as previously described (Francis et al., 2020). For preparation of large aggregate fractions for phospholipid quantitation, lung lining fluid was centrifuged at 20,000 x g for 1 hr at 4 °C. Pellets containing lipid-rich large aggregates were resuspended in 25 µL of saline. Inorganic phosphates were then extracted and measured as previously described (Bligh and Dyer, 1959; Massa et al., 2014).
UHPLC-mass spectrometry lipidomics
Deuterated 1-palmitoyl-2-palmitoyl-d31-sn-glycero-3-phosphocholine (DPPC, 32 µg; Avanti Polar Lipids, Alabaster, AL) was added to total large aggregate BAL fractions for absolute quantitation of lipid species. Lipids were extracted following the lipid tert-butyl methyl ether (MTBE) extraction method (Matyash et al., 2008). The upper layer was aliquoted (80 µL), dried and resuspended in 2 mL of 1:1 MeOH:IPA for analysis by high-pe...,