Novel DNA methylation changes in mouse lungs associated with chronic smoking

Abstract

Smoking is a potent cause of asthma exacerbations, chronic obstructive pulmonary disease (COPD) and many other health defects, and changes in DNA methylation (DNAm) have been identified as a potential link between smoking and these health outcomes. However, most studies of smoking and DNAm have been done using blood and other easily accessible tissues in humans, while evidence from more directly affected tissues such as the lungs is lacking. Here, we identified DNAm patterns in the lungs that are altered by smoking. We used an established mouse model to measure the effects of chronic smoke exposure first on lung phenotype immediately after smoking and then after a period of smoking cessation. Next, we determined whether our mouse model recapitulates previous DNAm patterns observed in smoking humans, specifically measuring DNAm at a candidate gene responsive to cigarette smoke, Cyp1a1. Finally, we carried out epigenome-wide DNAm analyses using the newly released Illumina mouse methylation microarrays. Our results recapitulate some of the phenotypes and DNAm patterns observed in human studies but reveal 32 differentially methylated genes specific to the lungs which have not been previously associated with smoking. The affected genes are associated with nicotine dependency, tumorigenesis and metastasis, immune cell dysfunction, lung function decline, and COPD. This research emphasizes the need to study CS-mediated DNAm signatures in directly affected tissues like the lungs, to fully understand mechanisms underlying CS-mediated health outcomes.

Keywords: Chronic smoking; DNA methylation; epigenome-wide; lung function.

Figure 1.

Development of a mouse model to study the effects of heavy smoking. (a) Adult female mice (N = 16 control, N = 16 CS) were exposed to heavy doses of whole-body CS for 9 weeks, from a pre-pregnancy period of 3 weeks and ending 3 weeks after birth of offspring. 15 of these had their litters within one week of each other and were included in this study. Following lung function measurements, tissues were collected from dams 48 hours after the last day of 9 weeks of smoke exposure and then 15 weeks after smoking cessation. (b) Dam cotinine levels measured in plasma 48 hours and 15 weeks after smoking cessation. N = 5 per group. Differences in plasma cotinine were analysed using student t-tests.

Figure 2.

Smoke exposure causes transient changes to immune cell infiltration into dam lungs, but does not alter dam baseline lung function over time. (a) Total immune cells per mL lavage immediately after smoking and 15 weeks after smoking cessation. (b) Eosinophils per mL lavage in the CS-exposed dams (mean = 7528, SD = 3736) was over 4 times more elevated than control dams (mean = 1569, SD = 1029) immediately after smoking, but normalized to controls 15 weeks after smoking cessation. (c) Macrophages per mL lavage immediately after smoking and 15 weeks after smoking cessation. (d) Lymphocytes per mL lavage in the CS-exposed dams (mean = 5680, SD = 2357) was over 8 times elevated compared to control dams (mean = 649, SD = 256) immediately after smoking, but normalized to controls 15 weeks after smoking cessation. (e) Total lung resistance at baseline. (f) Airway resistance at baseline. (g) Tissue resistance at baseline. (h) Alveolar elastance at baseline. N = 4–6 per group. Differential cell counts were normalized to lavage volume. Lung function values were measured using 90^th percentile values after injection of saline into the lungs. Two-group comparisons were conducted using a student t-test and p < 0.05 was considered significant.

Figure 3.

Smoke exposure alters methacholine responsiveness in mouse lungs up to 15 weeks after smoking cessation. (a) Total lung resistance immediately after smoking. (b) Airway resistance immediately after smoking. (c) Tissue resistance immediately after smoking. (d) Alveolar elastance immediately after smoking. (e) Total lung resistance after 15 weeks of smoking cessation. (f) Airway resistance after 15 weeks of smoking cessation. (g) Tissue resistance after 15 weeks of smoking cessation. (h) Alveolar elastance after 15 weeks of smoking cessation. N = 5–6 per group. Lung function values were measured using 90^th percentile values upon administration of increasing doses of methacholine. Data was analyzed using one-way ANOVA, followed by multiple comparisons at each methacholine dose where significant. *p < 0.05 in control vs smoke-exposed group.

Figure 4.

Cyp1a1 and Ahrr DNAm and expression levels in dam blood and lungs immediately after smoking and 15 weeks after smoking cessation. (a) Cyp1a1 DNAm in dam blood 15 weeks after smoking cessation. (b) Cyp1a1 expression in dam lungs 15 weeks after smoking cessation. (c) Cyp1a1 DNAm in dam lungs immediately after 9 weeks of smoking (d) Cyp1a1 DNAm in dam lungs 15 weeks after smoking cessation. (e) Ahrr DNAm in dam blood after 15 weeks of smoking cessation. (f) Ahrr DNAm in dam lungs after 15 weeks of smoking cessation. (g) Ahrr expression in dam lungs after 15 weeks of smoking cessation. N = 2–5 per group. Differences in DNAm and expression were analysed using student t-tests.