Repetitive Ozone Exposures and Evaluation of Pulmonary Inflammation and Remodeling in Diabetic Mouse Strains

Background: Epidemiological studies support the hypothesis that diabetes alters pulmonary responses to air pollutants like ozone ($O_3$). The mechanism(s) underlying these associations and potential links among diabetes, $O_3$, and lung inflammation and remodeling are currently unknown.

Objectives: The goal was to determine whether pulmonary responses to repetitive ozone exposures are exacerbated in murine strains that are hyperglycemic and insulin resistant.

Methods: Normoglycemic and insulin-sensitive C57BL/6J mice; hyperglycemic, but mildly insulin-resistant, KK mice; and hyperglycemic and markedly insulin-resistant KKAy mice were used for ozone exposure studies. All animals were exposed to filtered air (FA) or repetitive ozone ($0.5\text{ppm}$ $O_3$, 4 h/d, for 13 consecutive weekdays). Tissue analysis was performed 24 h following the final exposure. This analysis included bronchoalveolar lavage (BAL) for cell and fluid analysis, and tissue for pathology, immunohistology, mRNA, and hydroxyproline.

Results: Following repetitive $O_3$ exposure, higher bronchoalveolar lavage fluid inflammatory cells were observed in all mice ($\text{KKAy}>\text{KK}>C57\text{BL}/6$), with a notable influx of neutrophils and eosinophils in KK and KKAy mice. Although the lungs of $O_3$-exposed C57BL/6J and KK mice had minimal centriacinar histological changes without fibrosis, the lungs of $O_3$-exposed KKAy mice contained marked epithelial hyperplasia in proximal alveolar ducts and adjacent alveoli with associated centriacinar fibrosis. Fibrosis in $O_3$-exposed KKAy lungs was confirmed with immunohistochemistry, tissue hydroxyproline content, and tissue mRNA expression of fibrosis-associated genes (Ccl11, Il13, and Mmp12). Immunofluorescence staining and confocal microscopy revealed alterations in the structure and composition of the airway and alveolar epithelium in regions of fibrosis.

Discussion: Our results demonstrate that in diabetic animal strains repetitive ambient ozone exposure led to early and exaggerated pulmonary inflammation and remodeling. Changes in distal and interstitial airspaces and the activation of Th2 inflammatory and profibrotic pathways in experimental animals provide a preliminary, mechanistic framework to support the emerging epidemiological associations among air pollution, diabetes, and lung disease. https://doi.org/10.1289/EHP7255.