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 (O3). The mechanism(s) underlying these associations and potential links among diabetes, O3, 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 ppm O3, 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 O3 exposure, higher bronchoalveolar lavage fluid inflammatory cells were observed in all mice (KKAy>KK>C57BL/6), with a notable influx of neutrophils and eosinophils in KK and KKAy mice. Although the lungs of O3-exposed C57BL/6J and KK mice had minimal centriacinar histological changes without fibrosis, the lungs of O3-exposed KKAy mice contained marked epithelial hyperplasia in proximal alveolar ducts and adjacent alveoli with associated centriacinar fibrosis. Fibrosis in O3-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


Table of Contents
: Body Weight (g). Table S3. Data for Figure S1B: Change in Body Weight (g). Table S4. Data for Figure 1A: Blood Glucose (mg/dL). Table S5. Data for Figure 1B: Plasma Insulin (µU/ml). Table S6. Data for Figure 1C: HOMA-IR. Table S7. Data for Figure S2A: Plasma Leptin (ng/ml). Table S8. Data for Figure S2B: Plasma Adiponectin (ng/ml). Table S9. Data for Figure 1D: Blood Glucose (mg/dL). Table S10. Data for Figure 1E: Blood Glucose (AUC). Table S11. Data for Figure 2A: Total Cells (cells /ml x 10 5 ). Table S12. Data for Figure 2B: Macrophages (cells /ml x 10 5 ). Table S13. Data for Figure 2C: Eosinophils (cells /ml x 10 4 ). Table S14. Data for Figure 2D: Neutrophils (cells /ml x 10 4 ). Table S15. Data for Figure 2E: Lymphocytes (cells /ml x 10 4 ). Table S16. Data for Figure S3A: Neutrophils in Lung Tissue (%). Table S17. Data for Figure S3B: Eosinophils in Lung Tissue (%). Table S18. Data for Figure S3C: Macrophages in Lung Tissue (%). Table S19. Data for Figure S3D: YM 1/2 Macrophages (% total macrophages). Table S20. Data for Figure 3A: BALF IL-5 (pg/ml). Table S21. Data for Figure 3B: BALF IL-13 (fg/ml). Table S22. Data for Figure 3C: BALF IL-6 (pg/ml). Table S23. Data for Figure 3D: BALF KC (pg/ml). Table S24. Data for Figure 3E: BALF IL-17 (fg/ml). Table S25. Data for Figure 3F: BALF IL-1β (fg/ml). Table S26. Data for Figure 5G: Tissue Collagen (% lung parenchyma). Table S27. Data for Figure 5I: Lung Hydroxyproline (µg/ml). Table S28. Data for Figure 5H: Lung Hydroxyproline (µg/ml). Table S29. Data for Figure 6A: Eotaxin mRNA. Table S30. Data for Figure 6B: IL-13 mRNA. Table S31. Data for Figure 6C: Mcp2 mRNA. Table S32. Data for Figure 6D: Arg1 mRNA. Table S33. Data for Figure 6E: Mmp12 mRNA. Table S34. Data for Figure 6F: CCSP mRNA. Table S35. Data for Figure 6G: Chia1 mRNA. Table S36. Data for Figure 6H: Saa3 mRNA. Table S37. Data for Figure S5G CCSP in Airway Epithelium (%). Figure S1. Body weights after air or ozone exposure for 13 days. Final body weights of C57BL/6, KK and KKAy mice at the time of necropsy (A), and their change in body weight since the beginning of exposures to the time of necropsy 14 days later (B). C57BL/6J (open box), KK (gray box) and KKAy (black box). Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. Summary data for panels A, and B can be found in Tables S2, and S3, respectively. . Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. Summary data for panels A, and B can be found in Tables S7, and S8, respectively. . (E) Representative image of lung tissue from KKAy mouse that was stained with major basic protein to identify eosinophils (arrows) in the centriacinar lesions (identified with asterisk). tb = terminal bronchial, ad = alveolar duct, and a = alveoli. Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. ND = not detected. Summary data for panels A, B, C, and D can be found in Tables S16, S17, S18, S19, respectively. Tissue sections from air-exposed (A) and repetitive O 3 -exposed (B) KKAy mice underwent staining for α-smooth muscle actin (SMA) to identify myofibroblasts. α-SMA staining in air-exposed animals is noted in the subendothelial space (dashed arrow), while in the O 3 -exposed mice α-SMA staining is noted in the centriacinar regions (solid arrow). Images are representative images. a = alveoli, ad = alveolar duct, e = endothelium. Figure S5. Immunofluorescense staining of SFTPC, CCSP and HA in KK and KKAy mice. Increased sized images of KK and KKAy strains for visualization of the differences in staining and morphology in the air-and O 3 -exposed KK and KKAy strains.

Figure S6. CCSP immunohistology staining and morphometry in C57BL/6J, KK and KKAy mice.
Light photomicrographs of a centriacinar region in the lungs of C57BL/6 mice (A, B), KK mice (C, D) and KKAy mice (E, F) exposed to air (A, C, E) or ozone (B, D, F). Tissues were immunohistochemically stained for Club Cell Secretory Protein (CCSP; solid arrow; red chromagen) in epithelial cells (e) lining the terminal bronchiole (TB). Area of alveolitis (marked with stippled arrow), which included alveolar septal thickening, type two alveolar epithelial hyperplasia and macrophage accumulation in alveolar airspaces observed in the proximal alveolar duct (AD) and adjacent alveolar parenchyma (a). (G) Morphometry quantification of the airway was performed for CCSP in air-and O 3 -exposed C57BL/6, KK and KKAy strains. Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, p < 0.05. Summary data for panel G can be found in Table S37.

Table S1 -Primers used for real-time PCR.
Notes: List of all primers for gene expression analysis using the TaqMan gene expression assay from Applied Biosystems.

Gene Symbol
Assay ID Description (AB gene Name)

Tabular Data of Figures:
All data are expressed as Mean + SEM. Statistical differences are indicated where p < 0.05. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air. For gene expression analysis, *= significantly different from respective strain exposed to filtered air.  . Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. Summary data for panels A, and B can be found in Tables S2, and S3, respectively and KKAy (black box). Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. Summary data for panels A, and B can be found in Tables S7, and S8, respectively KKAy mouse that was stained with major basic protein to identify eosinophils (arrows) in the centriacinar lesions (identified with asterisk). tb = terminal bronchial, ad = alveolar duct, and a = alveoli. Data are expressed as mean ± SEM (n = 8/group). Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, c= significantly different from respective strain exposed to filtered air, p < 0.05. ND = not detected. Summary data for panels A, B, C, and D can be found in Tables S16, S17, S18, S19, respectively Figure S4 -α-Smooth Muscle Actin Staining in KKAy mice. Tissue sections from airexposed (A) and repetitive O 3 -exposed (B) KKAy mice underwent staining for α-smooth muscle actin (SMA) to identify myofibroblasts. α-SMA staining in air-exposed animals is noted in the subendothelial space (dashed arrow), while in the O 3 -exposed mice α-SMA staining is noted in the centriacinar regions (solid arrow). Images are representative images. a = alveoli, ad = alveolar duct, e = endothelium. Figure S5 -Immunofluorescense staining of SFTPC, CCSP and HA in KK and KKAy mice. Increased sized images of KK and KKAy strains for visualization of the differences in staining and morphology in the air-and O 3 -exposed KK and KKAy strains.

Figure S6
-CCSP immunohistology staining and morphometry in C57BL/6J, KK and KKAy mice. Light photomicrographs of a centriacinar region in the lungs of C57BL/6 mice (A, B), KK mice (C, D) and KKAy mice (E, F) exposed to air (A, C, E) or ozone (B, D, F). Tissues were immunohistochemically stained for Club Cell Secretory Protein (CCSP; solid arrow; red chromagen) in epithelial cells (e) lining the terminal bronchiole (TB). Area of alveolitis (marked with stippled arrow), which included alveolar septal thickening, type two alveolar epithelial hyperplasia and macrophage accumulation in alveolar airspaces observed in the proximal alveolar duct (AD) and adjacent alveolar parenchyma (a). (G) Morphometry quantification of the airway was performed for CCSP in air-and O 3 -exposed C57BL/6, KK and KKAy strains. Data were analyzed using a completely randomized analysis of variance with factors of mouse strain and exposure, and comparisons of group means made with the Student-Newman-Keuls post hoc test. a=significantly different from similarly exposed C57BL/6 mice, b= significantly different from similarly exposed KK mice, p < 0.05. Summary data for panel G can be found in Table S37