Figures & data
Figure 1. Experimental procedure of the smoke exposure.
Table 1. Network models and subnetworks applied for the computational analysis of network perturbation amplitudes (NPAs) and biological impact factor (BIF).
Figure 2. Tissue viability and epithelial barrier function assessment. LDH activity was measured in the culture medium immediately after exposure (0 h), and at 4, 24 and 48 h PE of CS of the buccal (A) and gingival (B) tissue cultures. TEER was measured at 48 h PE to CS in buccal (C) and gingival (D) tissue cultures. The charts on the right show the positive control tests using Triton X-100 treatment. Means ± SEM are shown (N = 3 inserts following a single exposure run). Asterisk indicates significant p < 0.05 compared with the air-exposed control within each of the post-exposure time-point, Dunnett adjusted for multiple comparison. Abbreviations: CS, cigarette smoke; LDH, lactate dehydrogenase; PE, post-exposure; RFU, raw fluorescence unit; TEER, transepithelial electrical resistance; SEM, standard error of the mean.
Figure 3. Histological examination of tissue structure and immunohistochemical identification of cell types and proliferation. H&E staining (40×) of the buccal (upper panels) and gingival tissues (lower panels) are shown in (A) at 48 h PE to CS at 19.7 and 40.7% compared with the air-exposed control. Insets show a 400× magnification. Quantification of immunostaining in the buccal (B) and gingival (C) tissues for various markers was performed. Basal cells (p63), proliferating basal cells (Ki-67) and a surface marker expressed in antigen presenting cells (HLA-DR, only in B) are shown as the ratios of immunostained cells per total cell count (number of DAPI-positive cells). Medians ± standard error of the medians are shown (N = 3 inserts following a single exposure run). Asterisk indicates significant p < 0.05 compared with the air-exposed control. Abbreviations: CS, cigarette smoke; DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin & eosin; HLA-DR, human leukocyte antigen DR; PE, post-exposure.
Figure 4. Enriched canonical pathways in the in vitro datasets. Significant canonical pathways (in different colors) were extracted from the differentially expressed genes in the buccal and gingival organotypic tissue cultures at various time-points of PE to CS (19.7 and 40.7%) compared with the air-exposed control using DAVID annotation tools (FDR < 0.05, with at least two gene count in the annotation). NA indicates no significant enrichment was identified using the before-mentioned threshold. All annotations and associated genes within each of the canonical pathways are listed in Supplemental Table S2. Abbreviations: CS, cigarette smoke; FDR, false discovery rate; NA, not available; PE, post-exposure.
Figure 5. Transcriptomic analysis using a network-based biological impact factor analysis. The overall biological impact calculated as biological impact factor (BIF) from all aggregated biological network models is shown in (A) for the various PE time-points following exposure to 40.7% CS in the gingival (GI) and buccal (BU) tissues. (B)–(E) show spider plots that display the normalized NPA values illustrating the quantification of the impacted biological networks for each of the post-exposure time-points. Gray areas in the center of the spider plots indicate statistically non-significant perturbation of the different networks according to the Specificity statistics, refer the “Materials and methods” section. Abbreviations: CS, cigarette smoke; PE, post-exposure.
Figure 6. Perturbation of various biological networks and subnetworks upon 40.7% CS exposure in the gingival (GI) and buccal (BU) tissues. Illustration of the decomposition of Stress network (A), Pulmonary Inflammation network (C) and Necroptosis network (E) into their subnetworks. Gray areas in the illustration indicate the subnetworks that were not significantly perturbed. Normalized NPA values indicated the levels of impact on the biological processes designated as Stress, Pulmonary Inflammation and Necroptosis networks and their subnetworks are shown in B, D and F, respectively. Bar charts above the gray area, those that were statistically significantly impacted (described in the “Materials and methods” section). Abbreviations: NPA, network perturbation amplitude, TNFR1, tumor necrosis factor receptor 1; RIPK-ROS, receptor-interacting serine/threonine-protein kinase-reactive oxygen species.
Figure 7. Comparability of the gene expression changes in vivo to the in vitro buccal samples upon exposure to CS. The comparison of the responses to CS exposure in vivo derived from buccal epithelial cells of smokers (GSE17913; Boyle et al., Citation2010) and that of in vitro buccal epithelium exposed to CS at various time-points of post-exposure illustrated in spider plots using the network-based approach (A–D, with the same conventions as described for ). Gray areas in the center of the spider plots indicate non-significant perturbation of the different networks. E shows the annotated canonical pathways (DAVID) extracted from the leading genes derived from the comparative enrichment analysis with FDR < 0.05, with at least two gene count in the annotation (Comparative enrichment scores are shown in Supplemental Figure S2). Red and blue bars indicate the enrichment scores of the upregulated and downregulated genes, respectively, derived from the in vivo data that were enriched with the in vitro datasets. All annotations and associated genes within each of the canonical pathways from DAVID are listed in Supplemental Table S3. N/A indicates no significant enrichment was identified using these thresholds. Abbreviations: CS, cigarette smoke; NA, not available; PE, post-exposure.
Figure 8. CYP1A1/1B1 enzyme activity and cytokine secretion in buccal and gingival tissues. Activity levels of CYP1A1/1B1 were measured at 24 h post-exposure to CS (17.9 and 40.7%) in the buccal (A) and gingival (B) tissues (N = 3 inserts following a single exposure run). Positive control tests are shown (right side of each panel) using TCDD as an inducer of CYP1A1/1B1 activity. Inflammatory mediators (C) and the corresponding gene expression (D) were measured at 24 h post-exposure of CS compared with the air-exposed tissues (N = 3 inserts following a single exposure run). Columns are representing different tissues as indicated in the label under the heatmap. Fold changes were obtained by taking the log2 ratio of the cytokine abundance (C) or of the gene expression (D) between the CS group and air-exposed exposure group for every tissue. Welch’s t-test was performed to test the null hypothesis that cytokine abundance (C) or log2-based gene expression (D) in the CS (19.7 and 40.7%) groups and air-exposed group was equivalent. Fold change was set to be zero for the p > 0.05. Blue and red colors indicate negative or positive fold-changes in the CS-exposed tissues as compared to the air-exposed tissues, respectively. Abbreviations: CCL, CC chemokine ligand; CS, cigarette smoke; CYP, cytochrome; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; IP-10, interferon gamma inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; SEM, standard error of the mean; RANTES, regulated on activation, normal T cell expressed and secreted; RLU, raw luminescence unit; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEGF, vascular endothelial growth factor; PE, post-exposure.
