The impact of electronic cigarettes on airway epithelial barrier integrity in preclinical mouse model

Abstract

The increasing use of electronic cigarettes (e-cigs) among adolescents poses significant public health risks. This study investigates the impact of e-cigs on the airway epithelial barrier, focusing on apical junctional complexes (AJCs), including tight junctions (TJs) and adherens junctions (AJs). We hypothesized that e-cigs disrupt AJCs in a mouse model, leading to increased airway barrier permeability. C57BL/6 mice were exposed to 36 mg/mL e-cig aerosols (3 puffs/min) for 1 h daily over 4 days. Bronchoalveolar lavage (BAL) fluid analysis, lung inflammation assessment, immunohistochemistry (IHC) staining, Western blotting (WB), and permeability assays were performed to evaluate the structure and function of the airway barrier. E-cig-exposed mice showed weight loss and elevated serum cotinine levels. BAL fluid analysis revealed elevated white blood cells. Histological analysis confirmed lung inflammation, whereas IHC and WB showed significant AJC disruption. Notably, claudin-2 levels were elevated in e-cig-exposed mice compared with controls. Claudin-2, known for its role in promoting permeability in "leaky" epithelia, increased alongside decreases in other TJ components, signifying structural barrier impairment. After e-cig exposure, instilling fluorescein isothiocyanate (FITC)-dextran into the airway increased serum FITC-dextran levels, indicating enhanced barrier permeability. E-cig aerosol exposure disrupts airway epithelial barrier structure and function, primarily through the disassembly of TJs and AJs. These findings suggest potential pathways for further clinical investigation into the health risks of e-cig use.NEW & NOTEWORTHY The rising use of e-cigs among youth has become a significant public health concern. This study, using a mouse model, demonstrates that exposure to e-cig aerosol leads to airway inflammation, structural damage to the airway epithelial barrier, and increased epithelial barrier permeability.

Keywords: FITC—fluorescein isothiocyanate; airway epithelial barrier; electronic cigarette; immunohistochemistry; vaping.

Fig. 1:. Acute exposure of mice to e-cig aerosol induces weight loss.

C57BL/6 mice were exposed to e-cig aerosol at 36 mg/mL using the SCIREQ inExpose system, delivering 3 puffs/min with a puff volume of 70 mL for 60 minutes per session, once a day for 4 days. (A) Weight reduction was noted on day 4 in mice exposed to e-cig aerosol in comparison to controls. Data are shown as mean ± SD, 35 mice per group, ****p = 0.000001 (B) Elevated cotinine levels were observed in the blood serum of mice exposed to e-cig aerosol in comparison to control mice. Data are shown as mean ± SD, 6 mice per group, ****p < 0.0001, as determined by using unpaired t tests with two-stage step-up for grouped analyses (Benjamini, Krieger, and Yekutieli) followed by Shapiro-Wilk test.

Fig. 2:. E-cig aerosol exposure increases airway and lung inflammation.

C57BL/6 mice were exposed to e-cig aerosol at a concentration of 36 mg/mL using the SCIREQ inExpose system. The exposure regimen consisted of 3 puffs/min, each with a volume of 70 mL, administered for 60 minutes per session, once daily for 4 consecutive days. (A) BAL fluid analysis demonstrated a significant increase in white blood cell (WBC) counts in mice exposed to e-cig aerosols compared to control mice. Data are shown as mean ± SD, 7–8 mice per group, **p = 0.0078 (B) WBC counts showed no notable differences in differentiation between control and e-cig mice. (C) Histological examination with H&E staining revealed lung inflammation in e-cig-exposed mice, characterized by enhanced peribronchial infiltration of immune cells. (D) Inflammation levels quantified through histopathology scoring, were notably higher in e-cig-exposed mice compared to HEPA-filtered air-exposed mice. Data are shown as mean ± SD, 17–20 mice per group, ****p < 0.0001. Scale bar of 200 μm. Statistical analyses were completed using unpaired t tests with two-stage step-up for grouped analyses (Benjamini, Krieger, and Yekutieli) followed by Shapiro-Wilk test.

Fig. 3:. Impact of e-cig aerosol exposure on apical junctional complexes structure.

(A) Lung tissue sections collected on day 4, post-exposure, were examined for the localization of key AJC proteins including occludin, E-cadherin, β-catenin, and claudin-2. In control mice, a well-defined AJC structure was observed in the bronchiolar epithelium, while exposure to e-cig aerosol led to a noticeable disruption of epithelial TJ and AJ integrity. Scale bar of 50 μm. (B) In lung homogenates, were subjected to Western blot analysis. (C-F) Densitometry analysis confirmed the significant reduction in occludin, no significant changes in E-cadherin or β-catenin, and a significant increase in claudin-2 expression in e-cig-exposed mice. The data is represented as mean ± SD, 3 mice per group, not significant (ns), *p =0.0204, **p = 0.0031, as determined by paired t tests followed by the Shapiro-Wilk test.

Fig. 4:. Exposure of mice to e-cig aerosol induces leaky airway.

Mice were exposed to e-cig aerosol at a concentration of 36 mg/mL, 3 puffs/min, each with a volume of 70 mL, administered for 60 minutes per session. (A) BAL fluid was collected, and protein levels were measured. Data are shown as mean ± SD, 9–10 mice per group, **p = 0.0083 (B) Mice were intranasally inoculated with 5 μg/g FITC-dextran in PBS or vehicle on day 4, and blood was collected 1 hour later. The serum level of FITC-dextran was significantly higher in e-cig aerosol-exposed animals compared to controls. A significant increase in protein concentrations was observed in e-cig-exposed mice. Data are normalized to the average of control group. Data are shown as mean ± SD., with 7 mice per group, **p = 0.009. Statistical analyses were completed using unpaired t tests followed by Shapiro-Wilk test.