Fig 1.
Ozone-induced Neutrophilia and Eosinophilia in Bronchoalveolar Lavage.
The changes in bronchoalveolar lavage (BAL) neutrophils and eosinophils from 0 to 200 ppb ozone exposure were not significantly different between the subjects with or without asthma or atopy. The horizontal dashed lines represent the 50% increase in count of neutrophils or eosinophils from 0 to 200 ppb ozone exposure. The shaded gray areas represent the subjects categorized as those without an ozone-induced neutrophilic or eosinophilic response (non-responders).
Table 1.
Bronchoscopy Subjects Characteristics.
Table 2.
Ozone-induced cellular inflammation in airways.
Fig 2.
Heatmap of BAL Cell Gene Expression After Ozone Exposure.
A. Ozone exposure had a consistent effect on the gene expression of human BAL cells with up-regulation of a group of genes in a dose-dependent manner from 0 to 100 to 200 ppb. The subjects are presented on the horizontal axis in the same order in each level of exposure. Covariates of atopy, lung function response, neutrophilic response, asthma, and gender are marked above the heatmap, with color indicating presence of covariates (for gender, blue = male; pink = female; for lung function and neutrophilic response, green and blue = no response; see Methods for details). B & C. Cluster analysis identified a relatively distinct classification of subjects with and without asthma or those with and without lung function response to 200 ppb ozone.
Table 3.
Differentially Expressed Genes with Increasing Levels of Ozone Exposure.
Fig 3.
Molecular Function of Processes Associated with the Differentially Expressed Genes (DEGs) After Ozone Exposure.
Analysis using DAVID showed 20 enriched gene ontologies to be associated with the DEGs based on linear regression and two-group comparison analyses with FDR threshold <25%. The gene ontologies are categorized into groups by similar molecular function.
Table 4.
Enriched Gene Ontologies From Differentially Expressed Genes Across Ozone Exposures.
Table 5.
Enriched Gene Ontologies From Differentially Expressed Genes Across 0 and 200 ppb Ozone Exposure.
Table 6.
Gene Set Analysis (GSA) Using Ingenuity Pathway Analysis (IPA).
Fig 4.
Stratification of BAL Cells Gene Expression After Ozone Exposure by Covariates.
Rate of increase in expression (parameter estimate ±SEM in fold change per 100 ppb increase in level of ozone exposure) of DEGs involved in some of the identified processes from DAVID with stratification for (A) asthma status or (B) lung function response to ozone. O3: ozone.
Fig 5.
Expression of Osteopontin RNA and Protein in BAL After Ozone Exposure.
A. Osteopontin gene (SPP1) RNA expression increased with increasing ozone exposure (q-value of 9.1x10-7 in linear regression analysis). B. Immunoblot of BAL samples from four representative subjects after exposure to 0, 100, and 200 ppb ozone probed with anti-OPN antibody. C. Osteopontin total protein (monomeric and polymeric forms) signal (from immunoblot densitometry analysis) increased with increasing ozone exposure (linear regression p-value = 0.03). D & E. The ratio of polymeric to monomeric forms of osteopontin decreased with increasing ozone exposure in those subjects with asthma (linear regression p-value = 0.01). Bars represent Mean ± SEM.
Fig 6.
Effect of Osteopontin on Epithelial Wound Closure.
16HBE14o- cells were grown to confluence on trans-well membrane plates and used in a scratch assay as a model of wound closure. A. Open wound area was measured 14 hours after scratch and treatment with saline (PBS), monomeric osteopontin (mOPN), transglutaminase 2 (TG2), polymeric osteopontin (pOPN), with and without anti-osteopontin antibody (anti-OPN) or anti-α9 integrin antibody (anti-α9). The scale bar is 50 μm. B. Average width of open wound area was calculated by adjusting wound area for length of scratch. The graph shows mean (middle horizontal bar) and standard deviation (whiskers).