DOI: 10.5061/dryad.4f4qrfjh1
title: Lipidomic analysis of bronchoalveolar lavage from mice exposed to ozone

description: 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, increased relative amounts of SP-B, and normalized pulmonary
 function in ozone-exposed mice. This was associated with increases in lung
 macrophage expression of CD36, a scavenger receptor important in lipid
 uptake and a transcriptional target of PPARγ. These findings highlight the
 role of alveolar lipids as regulators of surfactant activity and pulmonary
 function following ozone exposure and suggest that targeting lipid uptake
 by lung macrophages may be an efficacious approach for treating altered
 respiratory mechanics.
description: <strong>Bronchoalveolar lavage fluid collection and
 analysis</strong> 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). <strong>UHPLC-mass
 spectrometry lipidomics</strong> 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-performance liquid chromatography (UHPLC)-mass spectrometry at the
 Rutgers Cancer Institute of New Jersey Metabolomics Core following a
 chromatography protocol adapted from Chen et al. (Chen et al., 2020). Full
 scan mass spectrometry analysis was performed on a Thermo Q Exactive PLUS
 with a HESI source under positive mode. Relative quantitation of select
 lipids was performed by back-calculation based on the average ion
 intensities of deuterated-DPPC and normalizing to 32 µg
 spike-in.