Sub-chronic e-cig exposure altered exercise capacity and blood-gas saturation
After sub-chronic 30 days of e-cig exposure, WT (floxed) mice showed no difference in exercise capacity among the groups (Additional File 1: Figure S2). However, mice lacking nAChR α7 showed less capacity to run after being exposed to PG with nicotine compared to the other two exposure conditions. Interestingly, mice with a nAChR α7 lung epithelial cell-specific conditional deletion showed the same results, which indicates that nicotine inhalation with a nAChR α7 KO lowered exercise capacity, and that nAChR α7 in the epithelium played an essential role in this change. Blood pH, O2 pressure, concentrations of HCO3 and CO2 were shown to be dysregulated when WT mice were exposed to PG with nicotine compared to PG alone, while no difference was observed in nAChR α7 deficient mice exposed to PG with or without nicotine (Additional File 1: Table S1).
Sub-chronic exposure of e-cig aerosols induces upregulation of inflammatory cells
in mouse lung
The total cell counts in WT mice exposed to PG with or without nicotine all increased relative to their respective air group, and total cell counts for mice exposed to PG with nicotine increased significantly (Figure 1A). Interestingly, nAChR α7 KO mice showed no change in total cell counts when exposed to PG with nicotine. However, exposure to PG alone insignificantly increased the total cell counts (Figure 1A). Macrophage counts followed a similar trend to total cell counts (Figure 1B). Neutrophil counts showed a significant increase in PG-exposed nAChR α7 KO mice compared to WT mice (Figure 1C). WT and nAChR α7 KO mice exposed to PG with nicotine showed no change in neutrophil counts in BALF (Figure 1C). Additionally, CD4a+ and CD8a+ T-lymphocyte counts were significantly increased when WT mice were exposed to PG with nicotine, and lacking nAChR α7 helped to prevent dysregulation of T-lymphocytes (Figure 1D-E). Interestingly, PG-exposed nAChR α7 KO mice showed higher levels of neutrophils and CD4a+/CD8a+ T-lymphocytes than PG-exposed WT mice.
Sub-chronic e-cig exposure augments influx of pro-inflammatory cytokines in BALF
We next determined the level of pro-inflammatory cytokines in BALF. The levels of IL-1α, MCP-1, TNFα, GM-CSF, MIP-1β, IL-2, IL-5, IL-9, IFNg, RANTES, IL-6, IL-12p70, IL-13, KC, IL-1β, and Eotaxin were significantly increased when exposed to PG with nicotine compared to PG alone and the air control group in WT mice (Figure 2 and Additional File 1: Table S2). Interestingly, IL-1α, GM-CSF, IL-2, IL-9, IFNg, RANTES, MCP-1, and Eotaxin were decreased significantly with nAChR α7 KO compared to WT mice exposed to PG with nicotine (Figure 2 and Additional File 1: Table S2), which is nAChR α7 dependent. Besides, IL-1β and IL-5 both increased in either nAChR α7 KO or WT mice when exposed to PG with nicotine, which might indicate that these two cytokines are nAChR α7 independent. Additionally, IL-3, IL-4, IL-10, IL-12p40, IL-17A, G-CSF, and MIP-1α all showed no significant difference among the different conditions (Additional File 1: Table S2).
Sub-chronic e-cig exposure alters mRNA expression of inflammation and dysregulated repair response genes in WT and nAChR α7 KO mouse lung
To further understand the inflammatory responses and dysregulated repair/ECM remodeling occurring in this study, a inflammatory-myeloid panel of 734 genes for gene expression analysis was used (Nanostring Inc). The gene datasets from nCounter Mouse Myeloid Innate Immunity Panel were analyzed via Nanostring nSolver and R programming language (Additional File 2: Table S3). The boxplots show the distribution of the normalized gene-level among the different experimental conditions (Additional File 1: Figure S3A). From the R analysis, 110 and 109 dysregulated genes were found when mice were exposed to PG alone in WT and nAChR α7 KO mice, respectively, and 21 genes were found in common between WT and nAChR α7 KO mice when exposed to PG alone, compared to air controls (Additional File 1: Figure S3B). Similarly, a total of 190 and 228 genes were found to be altered in WT and nAChR α7 KO mice, respectively, when exposed to PG with nicotine. Additionally, 89 genes were found in common when mice of different genotypes were exposed to PG with nicotine (Additional File 1: Figure S3B). In a comparison between PG alone and PG with nicotine, 146 genes were dysregulated in WT mice, 177 genes were changed nAChR α7 KO mice, and 57 genes were found to be common between WT and nAChR α7 KO mice when exposed to PG alone, compared to PG with nicotine (Additional File 1: Figure S3B).
In the WT mice, significantly higher RNA counts were found for multiple targets in both the PG with or without nicotine exposed groups, compared to the air control (Additional File 1: Figure S4). When WT mice were exposed to PG alone, compared to air control mice, and mice exposed to PG with nicotine, some inflammatory markers and ECM remodeling markers, including MMP9, MMP8, S100A8, and some collagens (COL17A1 and COL14A1), were dysregulated (Additional File 1: Figure S4A). Also in the WT mice, comparisons between the air control and PG with nicotine exposure groups showed differences in more inflammatory focused markers, such as ARG1 and LPL (Additional File 1: Figure S4A). As expected, nAChR α7 KO mice exposed to PG or air showed no difference in gene levels compared to WT mice. However, PG with nicotine exposure dysregulated multiple targets with significant differences between WT and nAChR α7 KO mice in levels of gene expression, including both inflammation and dysregulated repaired/ECM remodeling markers, such as SMAD7, KLF4, CDH1, COL4A2, ICAM1, LDLR, IL1B, TLR5, NFKBIA, and CXCL2 (Additional File 1: Figure S4B). We then selected specific gene targets and focused on inflammatory responses and dysregulated repair/ECM remodeling for further analysis (Figures 3).
Based on the Nanostring nCounter analysis, gene targets with significant differences between WT and nAChR α7 KO mice, when exposed to PG with nicotine, were selected and plotted (Figure 3A). Notably, SKIL (Ski-like protein) and LDLR (Low-density lipoprotein receptor) were significantly decreased in WT mice exposed to PG with nicotine, while nAChR α7 KO helped to blocked it. Interestingly, CCL9 (Chemokine ligand 9), KLF4 (Kruppel-like factor 4), DUSP1 (Dual Specificity Phosphatase 1), BTLA (B and T Lymphocyte Associated), and SMAD7 (SMAD Family Member 7) only showed dysregulated gene-level in nAChR α7 KO mice exposed to PG with nicotine compared to WT mice (Figure 3A). As for NECTIN1, both WT and nAChR α7 KO mice showed decreased trend, but only WT mice showed a significant difference between the air control and PG with nicotine exposure groups (Figure 3A).
Sub-chronic e-cig exposure with nicotine in nAChR α7 deficient mice prevents dysregulation of p50/p105 in a sex-dependent manner
In order to understand the inflammation responses in protein level, we measured the protein abundance of p50/p105, one of the subunits of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) via Western blot (Figure 4). We have observed that the protein levels of both p50 and p105 upregulated in female WT mice exposed to PG with nicotine, while attenuated by lacking nAChR α7 (Figure 4A). However, there was no significant difference in male mice exposed to PG with or without nicotine compared to air controls in either WT or nAChR α7 KO mice. Notably, in males only, decreased p50/p105 protein abundances were found in nAChR α7 KO mice compared to WT mice (Figure 4B). Female mice exposed to PG alone, whether WT or nAChR α7 KO, showed similar upregulation of p105, indicating that PG alone could induce pro-inflammatory responses in a nAChR α7 independent manner (Figure 4A).
Sub-chronic e-cig exposure with or without nicotine induced dysregulated repair/ECM remodeling in a sex-dependent but nAChR α7 independent manner
In recent studies, we have identified that acute e-cig exposure has induced dysregulated repair/ECM remodeling in mouse lungs . We are therefore interested in how ECM remodeling could be induced via sub-chronic exposure. We have selected MMPs and ECM markers to measure in both protein abundance (Figure 5-6) and gene expression level (Figures 3B). Mice exposed to PG alone showed prominent dysregulation in both protein and gene expression levels, and protein level was in a sex-dependent manner (Figures 5-6).
From the absolute RNA count, MMP8 and MMP9 were found to be lower in the PG groups than in air control or PG with nicotine exposed WT or nAChR α7 KO mice (Figure 3B). A slightly increased MMP8 level was observed in PG with nicotine-exposed mice compared to air controls, but this difference was not significant (Figure 3B). As an inhibitor of MMPs, TIMP3 was shown to be upregulated statistically in both WT and nAChR α7 KO mice when exposed to PG with nicotine (Figure 3B). For the ECM proteins, no change in gene expression was detected in fibronectin (FN1) or Plasminogen activator inhibitor-1 (PAI-1, gene code: SERPINE1), while significant decreases were detected in collagens (COL1A2 and COL4A1), when exposed to PG with or without nicotine in both WT and nAChR α7 KO mice.
Since we observed dysregulated gene levels, protein abundances were also measured. The protein levels of MMP2, MMP8, MMP9, and MMP12 were selected, and PAI-1, COL1A1, COL1A2, and fibronectin were selected as ECM markers. Both female and male mice were used here to clarify any sex-dependent differences. We noticed similar trends of MMP9 and MMP8 protein levels compared to RNA transcript levels in both female and male mice (Figure 5). Decreased MMP8 and MMP9 were observed in PG-exposed WT and nAChR α7 KO mice, while PG with nicotine-exposed mice showed a lesser decrease when compared to air controls in both females and males (Figure 5). Notably, in both females and males, upregulated MMP2 protein abundance was seen in both male and female WT mice when exposed to PG alone, compared to air controls and PG with nicotine-exposed mice. Moreover, there was an increased MMP2 baseline when female mice lack the nAChR α7 while no such change is seen in male mice (Figure 5). Interestingly, MMP9, MMP2, and MMP12 were found to be upregulated in male WT mice exposed to PG with nicotine, while the nAChR α7 deletion helped prevent the dysregulation of these MMPs (Figure 5B). For MMP8, female and male mice showed similar protein levels, and nAChR α7 deficiency induced downregulation of protein abundance (Figure 5).
ECM proteins/regulator, collagens, fibronectin, and PAI-1, were measured as well (Figure 6). The protein abundance of PAI-1 showed no difference among all conditions in females (Figure 6A), while it was increased in male WT mice exposed to PG with nicotine, and slightly decreased in nAChR α7 KO males (Figure 6B). Further, COL1A1 and COL1A2, two different subunits of type 1 collagens, showed different expression levels among the experimental groups. Interestingly, both female and male WT mice showed upregulated COL1A2 when exposed to PG alone in both WT and nAChR α7 KO mice (Figure 6). While, COL1A1 exhibited no changes in either WT or nAChR α7 KO female mice, decreased protein abundances were seen when male WT mice were exposed to PG with or without nicotine, and a lower baseline of COL1A1 was seen in mice lacking nAChR α7. Similarly, we found a lower protein expression level of fibronectin in female WT and nAChR α7 KO mice when exposed to PG with or without nicotine compared to air group (Figure 6A). However, decreased fibronectin was seen in the PG-exposed male WT mice, and there was no alteration in male nAChR α7 KO mice among the different exposure conditions (Figure 6B).
Sub-chronic e-cig exposure in lung epithelial cell-specific nAChR α7 deletion protects against inflammation
Considering the inflammatory responses seen in WT and nAChR α7 KO mice, we next determined the role of nAChR α7 in the lung epithelial cells when exposed to e-cig with or without nicotine. Surprisingly, we did not see any significant changes in cytokines in PG with nicotine-exposed in nAChR α7 epithelial cell-specific KO mice. However, we did find IL-5, MCP-1, KC, Eotaxin, GM-CSF, and G-CSF to be significantly up-regulated in the PG alone group when compared to air control mice, and inhibited in nAChR α7 epithelial cell-specific KO mice (Additional File 1: Figure S5A). The rest of the cytokines, IL-1α, TNFα, MIP-1β, IL-2, IL-9, IFNg, RANTES, IL-6, IL-12p70, IL-13, IL-1β, Eotaxin, IL-3, IL-4, IL-10, IL-12p40, IL-17A, and MIP-1α, showed no changes among the different groups (Additional File 1: Figure S5B-C).