Sub-chronic e-cig exposure altered exercise capacity and blood-gas saturation
After 30 days e-cig exposure, WT (floxed) mice showed no difference in exercise capacity among the groups (Additional File 1: Figure S2). However, nAChR α7 deficient (KO) mice showed less capacity to run when exposed to PG with nicotine compared to air control and PG alone groups. Similar results were observed in nAChR α7 lung epithelial cell-specific conditional KO mice exposed to PG with nicotine show reduced exercise capacity compared to air control and PG alone groups. E-cig aerosol with nicotine exposure in nAChR α7 KO show lowered exercise capacity indicating the possible role of nAChR α7 in the lung epithelium. 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 between WT and nAChR α7 KO mice exposed to PG with or without nicotine (Additional File 2: Table S1).
Sub-chronic e-cig exposure induces inflammatory cellular influx in the mouse lung
The total cell counts were increased in WT mice exposed to PG with or without nicotine relative to respective air group, and total cell counts in WT mice exposed to PG with nicotine increased significantly compared to air and PG alone groups (Figure 1A). The nAChR α7 KO mice showed no change in total cell counts when exposed to PG with nicotine (Figure 1A). However, the total cell counts were increased but not significant compared to respective control group (Figure 1A). Macrophage counts followed a similar trend as observed in total cell counts (Figure 1B). 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 loss of nAChR α7 prevents increase in T-lymphocytes (Figure 1D-E). PG-exposed nAChR α7 KO mice showed higher levels of neutrophils and CD4a+/CD8a+ T-lymphocytes than PG-exposed WT mice (Figure 1C-E).
Sub-chronic e-cig exposure augments influx of pro-inflammatory cytokines in BALF
We next determined the level of pro-inflammatory cytokines in BALF after sub-chronic e-cig exposure. Most of the pro-inflammatory cytokines in BALF were significantly increased in WT mice exposed to PG with nicotine compared to PG alone and air control (Figure 2 and Additional File 2: Table S2). We have noticed that macrophage mediated pro-inflammatory cytokines: IL-1α, MCP-1, and GM-CSF were significantly increased in WT mice exposed to PG with nicotine, and nAChR α7 KO blocks the release of those mediators (Figure 2). Similarly, lymphocytes-related cytokines: IL-2, IL-9, IFNγ, and RANTES showed nAChR α7-dependent reduction in cytokine levels (Figure 2). Additionally, TNFα, MIP-1β, IL-1β and IL-5 were increased in PG with nicotine in WT mice, but not in nAChR α7 KO mice exposed to PG with nicotine. The remaining cytokines IL-3, IL-4, IL-10, IL-12p40, IL-17A, G-CSF, and MIP-1α were not affected among any of the exposure groups in WT and nAChR α7 KO mice compared to respective air control (Additional File 2: Table S2).
Sub-chronic e-cig exposure alters mRNA transcript levels of inflammation and dysregulated repair response genes in WT and nAChR α7 KO mice
To further determine whether sub-chronic e-cig exposure mediated inflammatory responses and dysregulated repair/ECM remodeling was caused by altered expression of myeloid innate immune response target genes (~734 genes), we performed gene expression analysis using nCounter Mouse Myeloid Innate Immunity Panel (Nanostring Technologies, Inc.). The pairwise comparison between PG alone exposed WT and nAChR α7 KO mice, revealed 21 genes were differentially expressed compared to respective air control (Figure 7B). Additionally, pairwise comparison analysis among PG with nicotine exposed WT and nAChR α7 KO mice revealed 89 genes differentially expressed when compared to respective air control (Figure 7B). Furthermore, pairwise comparison between PG with nicotine and PG alone groups in WT and nAChR α7 KO mice revealed 57 genes differentially expressed among them (Figure 7B).
WT mice showed significantly higher RNA counts indicative of increased mRNA transcript levels for multiple targets in PG with or without nicotine exposed groups, compared to air control (Additional File 1: Figure S3). When we compared WT mice exposed to PG alone, air control or PG with nicotine showed altered gene expression of inflammatory and ECM remodeling targets such as matrix metalloproteases (MMP9, and MMP8), S100A8, and collagens (COL17A1 and COL14A1) (Additional File 1: Figure S3A). When WT mice exposed to PG with nicotine were compared with air control, we found significant difference in transcript levels of inflammatory targets such as ARG1 and LPL (Additional File 1: Figure S3A). PG with nicotine exposure caused significant alterations in the expression of several target genes such as SMAD7, KLF4, CDH1, COL4A2, ICAM1, LDLR, IL1B, TLR5, NFKBIA, and CXCL2 among WT and nAChR α7 KO mice that belong to the broad categories inflammation and dysregulated repaired/ECM remodeling (Additional File 1: Figure S3B). Based on the above-mentioned gene expression analysis by Nanostring, we choose specific target genes for further analysis (Figure. 3).
Based on the Nanostring nCounter analysis, transcript levels of target genes that showed significant differences between WT and nAChR α7 KO mice following PG with nicotine exposure were selectively represented in Figure 3A. The gene expression levels of SKIL (Ski-like protein) and LDLR (Low-density lipoprotein receptor) were significantly decreased in WT mice exposed to PG with nicotine, but not nAChR α7 KO mice. However, the gene expression levels of 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) were significantly increased only in nAChR α7 KO mice exposed to PG with nicotine compared to WT mice (Figure 3A). Finally, NECTIN1 gene expression in WT and nAChR α7 KO mice showed a decreased trend, but only WT mice exposed to PG with nicotine showed a significant reduction in the expression of NECTIN1 transcript compared to air control (Figure 3A).
Sub-chronic e-cig exposure with nicotine in nAChR α7 deficient mice prevents dysregulation of p50/p105
In order to determine the role of target molecules that contribute to e-cig exposure induced inflammatory response at the protein level, we measured the protein abundance of a NF-κB subunit (nuclear factor kappa-light-chain-enhancer; p50/p105). We have observed that the protein levels of both p50 and p105 were upregulated in WT female mice exposed to PG with nicotine, while nAChR α7 KO showed attenuation of p50 and p105 expression in the lungs (Figure 4A). However, there was no significant difference in male mice exposed to PG with or without nicotine compared to air control in both WT and nAChR α7 KO mice (Figure 4A). WT or nAChR α7 KO female mice exposed to PG alone 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 with nicotine exposure induced up-regulation of Angiotensin-converting enzyme (ACE2) in mouse lung
Recently, it has been shown that ACE2 (Covid-19 receptor) is a key point in migration of fibroblast to injured locations in lungs, and ACE2 is involved in lung ECM remodeling. We determined the protein abundance of ACE2 (Figure 4B). We have noticed that PG with nicotine has increased the protein abundance, whereas nAChR α7 deletion attenuated the dysregulation in female. While in male, there was no difference between PG with nicotine exposure and air control, PG exposure decreased the ACE2 protein level, and nAChR α7 knockdown lowered the baseline abundance of ACE2 in lungs.
Sub-chronic e-cig exposure with or without nicotine induces dysregulated repair/ECM remodeling in a nAChR α7-independent manner
In our previous study, we have identified that acute e-cig exposure causes dysregulated repair/ECM remodeling in mouse lungs [8]. In this study, we were interested to investigate the role of dysregulated repair/ECM remodeling in sub-chronic e-cig exposed WT and nAChR α7 KO mice. We were specifically interested in selected MMPs and ECM remodeling markers to determine their effects following sub-chronic e-cig exposure both at the gene expression level (Figures 3B), and protein abundance (Figure 5-6).
PG exposed WT and nAChR α7 KO mice showed decreased expression of MMP8 and MMP9 compared to air control or PG with nicotine group (Figure 3B). WT and nAChR α7 KO mice when exposed to PG with nicotine showed significant increase in TIMP3, which is an inhibitor of MMPs (Figure 3B). ECM target genes fibronectin (FN1) or Plasminogen activator inhibitor-1 (PAI-1, gene code: SERPINE1) remain unaffected, while genes that modulate collagen expression (COL1A2 and COL4A1) was significant decreases in PG with or without nicotine exposed WT and nAChR α7 KO mice.
Based on the gene expression changes in dysregulated repair markers, we measured the protein abundances of the same in e-cig exposed WT and nAChR α7 KO mice. Results from immunoblot analysis correlated with gene expression of MMP9 and MMP8 in both female and male mice (Figure 5A-B). Decreased MMP8 and MMP9 were also observed in PG-exposed WT and nAChR α7 KO mice compared to PG with nicotine and air control (Figure 5A-B). MMP2 protein abundance was increased in WT male and female mice exposed to PG alone, compared to air control and PG with nicotine-exposed mice. Moreover, nAChR α7 KO female mice show increased MMP2 protein abundance at baseline, while no change was observed in male mice (Figure 5A-B). The protein levels of MMP9, MMP2, and MMP12 were increased in WT male mice exposed to PG with nicotine, while nAChR α7 KO inhibits protein abundance of MMPs (Figure 5B). MMP8 protein levels were comparable in female and male mice and nAChR α7 KO showed decreased protein abundance of MMP8 at baseline (Figure 5A-B).
ECM proteins/regulator such as collagens, fibronectin, and PAI-1, at the protein levels were measured in WT and nAChR α7 KO mouse lungs (Figure 6). The protein abundance of PAI-1 showed no difference among air, PG alone and PG with nicotine exposed WT and nAChR α7 KO female mice (Figure 6A). Whereas, PAI-1 was increased in WT male mice exposed to PG with nicotine, but was reduced in nAChR α7 KO male mice (Figure 6B). Furthermore, two different subunits of type 1 collagens (COL1A1 and COL1A2) were differentially expressed at the protein level among the experimental groups. WT and nAChR α7 KO female and male mice showed increased expression of COL1A2 when exposed to PG alone (Figure 6). However, COL1A1 protein abundance was reduced in WT male mice exposed to PG with or without nicotine, and nAChR α7 deficiency further decreased the baseline of COL1A1 levels in male mice. There is no change in the protein levels of COL1A1 among PG alone and PG with nicotine exposure in WT and nAChR α7 KO mice. PG with or without nicotine exposure reduced the protein abundance of fibronectin in WT female and nAChR α7 KO mice compared to air control (Figure 6A). However, decreased fibronectin protein level was observed in the PG-exposed WT male mice, and there was no alteration in nAChR α7 KO male mice among all 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 exposed nAChR α7 lung epithelial cell-specific KO mice to e-cig with or without nicotine to determine the role of nAChR α7 in the lung epithelium. Surprisingly, we did not see any significant changes in pro-inflammatory cytokines following PG with nicotine exposure in nAChR α7 epithelial cell-specific KO mice. However, IL-5, MCP-1, KC, Eotaxin, GM-CSF, and G-CSF levels were significantly increased in PG alone group when compared to air control, and these cytokines were inhibited in nAChR α7 epithelial cell-specific KO mice (Additional File 1: Figure S4A). The rest of the cytokines from BALF showed no changes among all the different exposure groups in nAChR α7 epithelial cell-specific KO mice (Additional File 1: Figure S4B-C).