Fig 1.
Mean static lung compliance (Cst).
Cst measured by the flexiVent™ instrument for each experimental group at each time point. C57Bl/6 mice were exposed to ambient air (AC, grey bars) or chronic cigarette smoke (CS, black bars) for the indicated duration of time measured in days (d) or months (mo). In one group of mice that underwent smoking cessation (SS; stop smoking, dark grey bar), mice were allowed to recover at ambient air (AC) for 3 months following a 6 months CS exposure. N = 10 mice /group. Horizontal bars with asterisk indicate significant differences in mean static lung compliance (p<0.05). Bars represent the standard error of the mean.
Table 1.
Number of significantly differentially expressed genes, perturbed pathways and differentially used exons identified between CS- and AC-exposed mice, and between pairwise comparisons with SS (cessation group, for stop smoking) mice (FDR≤0.10, |FC| ≥ 1.5).
Fig 2.
Heatmap of the relative change in gene expression.
Log2 FC in CS-exposed mice compared to AC mice of the 9 genes found to statistically significantly differentially expressed at four of five time points (FDR≤0.10). Positive fold change (pink) indicates upregulation in CS-exposed mice; negative fold change (blue) indicates downregulation in CS-exposed mice.
Fig 3.
Behavior of influential genes in relevant metabolic pathways.
Expression was summarized as base-2 fold change (log2 FC). Influential genes determined by GAGE using coreGeneSets, and are those member genes that substantially contribute to the significance of the entire pathway [14]. (A) Pyrimidine metabolism was initially enriched for downregulated genes, but later enriched for upregulated genes at 9 months of CS exposure. A gradual transition from downregulation to upregulation is apparent, especially for the gene Dpys. (B) Glutathione metabolism was the most consistently enriched KEGG pathway for upregulated genes. While 1 day of CS exposure did not elicit upregulation in many genes, at 7 days of CS most of the influential genes were upregulated. (C) Phosphatidylinositol (PI) signaling system was initially enriched for upregulated genes, but later enriched for downregulated genes at 9 months of CS exposure. Grey bars indicated 95% confidence intervals.
Fig 4.
KEGG pathways displaying unique enrichment patterns at 9 months of CS (FDR≤ 0.10).
The pathways were either exclusively differentially enriched or exhibited an opposite direction of enrichment after 9 months CS exposure compared to other time points. *Denotes pathways enriched for upregulated genes after 9 months of CS exposure, but was not significantly enriched at any other time point. †Denotes pathways enriched for upregulated genes after 9 months of CS exposure, but initially enriched for downregulated genes after 7 days; ‡ denotes pathways enriched for downregulated genes after 9 months of CS exposure, but initially enriched for upregulated genes after 7 days of CS exposure. No pathways were significantly enriched for downregulated genes after 9 months that were not significantly enriched at any other time point.
Table 2.
Abundance of genes found significantly differentially expressed using edgeR (FDR≤0.10) between CS- and AC-exposed mice and enriched pathways using GAGE (FDR≤0.10) exhibiting patterns detailed in S1 Fig.
Normalization, filtering, and modeling was performed using data from three groups (AC, CS, and smoking cessation).
Fig 5.
Genes showing a previously unsuspected significant time trend with CS exposure.
(A) Fam162b shows a significant downward trend for linear time and significant effect of group. (B) Grm4 shows significant upward trend for linear time and significant effect of group. (C) Lox was bi-directionally regulated in individual time point analysis. (D) Slc22a12 displayed significant linear time by treatment interaction, with no change in expression in the AC group.
Table 3.
Pathways overlapping between transcriptomic and metabolomic data.
Checkmarks signify significant pathway perturbation in metabolomic data (p<0.05), pink shading denotes enrichment for upregulated genes in that pathway in CS-exposed mice; blue shading indicates enrichment for downregulation in CS-exposed mice (FDR≤0.10). Perturbations indicated by shaded fields under the “Cessation” column persisted following cessation when compared to AC mice.