Resolving multi-image spatial lipidomic responses to inhaled toxicants by machine learning.

Regional responses to inhaled toxicants are essential to understand the pathogenesis of lung disease under exposure to air pollution. We evaluated the effect of combined allergen sensitization and ozone exposure on eliciting spatial differences in lipid distribution in the mouse lung that may contribute to ozone-induced exacerbations in asthma. Lung lobes from male and female BALB/c mice were cryosectioned and acquired by high resolution mass spectrometry imaging (MSI). Processed MSI peak annotations were validated by LC-MS/MS data from scraped tissue slides and microdissected lung tissue. Images were normalized and segmented into clusters. Interestingly, segmented clusters overlapped with stained serial tissue sections, enabling statistical analysis across biological replicates for morphologically relevant lung regions. Spatially distinct lipids had higher overall degree of unsaturated fatty acids in distal lung regions compared to proximal regions. Furthermore, the airway and alveolar epithelium exhibited significantly decreased sphingolipid and glycerophospholipid abundance in females, but not in males. We demonstrate the potential role of lipid saturation in healthy lung function and highlight sex differences in regional lung lipid distribution following ozone exposure. Our study provides a framework for future MSI experiments capable of relative quantification across biological replicates and expansion to multiple sample types, including human tissue.

Alteration of glycosphingolipid metabolism by ozone is associated with exacerbation of allergic asthma characteristics in mice.

Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma, which may be attributable to altered sphingolipid metabolism. This study tested the hypothesis that acute ozone exposure alters sphingolipid metabolism within the lung, which contributes to exacerbations in characteristics of asthma in allergen-sensitized mice. Adult male and female BALB/c mice were sensitized intranasally to house dust mite (HDM) allergen on days 1, 3, and 5 and challenged on days 12-14. Mice were exposed to ozone following each HDM challenge for 6 h/day. Bronchoalveolar lavage, lung lobes, and microdissected lung airways were collected for metabolomics analysis (N = 8/sex/group). Another subset of mice underwent methacholine challenge using a forced oscillation technique to measure airway resistance (N = 6/sex/group). Combined HDM and ozone exposure in male mice synergistically increased airway hyperreactivity that was not observed in females and was accompanied by increased airway inflammation and eosinophilia relative to control mice. Importantly, glycosphingolipids were significantly increased following combined HDM and ozone exposure relative to controls in both male and female airways, which was also associated with both airway resistance and eosinophilia. However, 15 glycosphingolipid species were increased in females compared with only 6 in males, which was concomitant with significant associations between glycosphingolipids and airway resistance that ranged from R2 = 0.33-0.51 for females and R2 = 0.20-0.34 in male mice. These observed sex differences demonstrate that glycosphingolipids potentially serve to mitigate exacerbations in characteristics of allergic asthma.