The Impact of NO2 on Epithelial Barrier Integrity of a Primary Cell-Based Air-Liquid Interface Model of the Nasal Respiratory Epithelium

Abstract

Nitrogen dioxide (NO₂) is a pervasive gaseous air pollutant with well-documented hazardous effects on health, necessitating precise toxicological characterization. While prior research has primarily focused on lower airway structures, the upper airways, serving as the first line of defense against airborne substances, remain understudied. This study aimed to investigate the functional effects of NO₂ exposure alone or in combination with hypoxia as a secondary stimulus on nasal epithelium and elucidate its molecular mechanisms because hypoxia is considered a pathophysiological factor in the onset and persistence of chronic rhinosinusitis, a disease of the upper airways. Air-liquid interface cell cultures derived from primary nasal mucosa cells were utilized as an in vitro model, offering a high in vitro-in vivo correlation. Our findings demonstrate that NO₂ exposure induces malfunction of the epithelial barrier, as evidenced by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate (FITC)-dextran permeability. mRNA expression analysis revealed a significant increase in IL-6 and IL-8 expressions following NO₂. Reduced mRNA expression of the tight junction component occludin was identified as a structural correlate of the damaged epithelial barrier. Notably, hypoxic conditions alone did not alter epithelial barrier integrity. These findings provide information on the harmful effects of NO₂ exposure on the human nasal epithelium, including compromised barrier integrity and induction of inflammatory responses. Overall, this study contributes to our understanding of pathophysiological mechanisms underlying also upper airway respiratory diseases associated with air pollution exposure and emphasizes the importance of mitigating NO₂ emissions to safeguard respiratory health.

Keywords: NO2; hypoxia; nasal epithelial barrier; tight junctions; upper airway.

FIGURE 1

FD4 (A) and FD40 (B) passage was measured 1, 4, 7, and 14 days after the transfer of NEC to ALI culture conditions. Permeability decreased at each of the time points, reaching a significant difference compared to Day 1 after 7 days for both tested dextran sizes. The permeability was calculated by dividing the measured fluorescence of the sample and the fluorescence of the apically applied FITC‐medium mixture, minus the fluorescence value of the pure medium. Asterisks indicate p ≤ 0.05 (Dunn's multiple comparison test). Data are presented with box plots, margins of them illustrate the 25th and 75th percentiles. Whiskers indicate minimal and maximal values. n = 10.

FIGURE 2

NEC were exposed to 0.1 ppm of NO₂ for 1 h. LPS (for FITC‐dextran and RT‐PCR) and tBHP (for TEER measurement) served as positive controls. FD4 permeability measured by FITC‐dextran assay (Figure 2A) increased and TEER (Figure 2B) significantly decreased in treated cells compared to the untreated control. Figure 2C shows the result of the quantitative Real‐Time PCR of occludin. Data are presented as 2^‐ΔΔCT, so that a change of 1 theoretically corresponds to a doubling of the number of mRNA. Untreated cells were set to 1. Asterisks indicate p ≤ 0.05 (as data were normally distributed, multiway ANOVA followed by Tukey's multiple comparison test was performed). The square in the boxplot in Figure 2C indicates a statistical outlier (outside 1.5 standard deviations from median); n = 10.

FIGURE 3

Results of FD4 permeability (A) and TEER measurement (B). NEC were exposed to 0.1 ppm of NO₂ alone, hypoxia alone, or in combination (NO₂ + hypoxia). Untreated cells served as the negative control, while cells treated with LPS for FITC‐dextran assay and tBHP for TEER analysis served as the positive control. There was a significant increase in FD4 passage after NO₂ + hypoxia and decrease in TEER in the NEC exposed to NO₂ alone and NO₂ + hypoxia. Results were not significant for the hypoxia single exposure group compared to untreated cells. Asterisks indicate p ≤ 0.05 (as data were normally distributed, multiway ANOVA followed by Tukey's multiple comparison test was performed). Squares indicate statistical outliers (outside 1.5 standard deviations from median); n = 10.

FIGURE 4

IL‐6 (A) and IL‐8 (B) mRNA abundance determined by qRT‐PCR. NEC were exposed to 0.1 ppm of NO₂ alone, hypoxia alone, or in combination (NO₂ + hypoxia). LPS was used as positive control. Data are presented as 2^‐ΔΔCT, with untreated cells set to 1. IL‐6 was significantly elevated after NO₂ exposure alone and in combination with hypoxia. Hypoxia alone did not induce significant changes. IL‐8 levels were significantly altered in all subgroups compared to the negative control. Asterisks indicate p ≤ 0.05 (as data were normally distributed, multiway ANOVA followed by Tukey's multiple comparison test was performed). Squares indicate statistical outliers (outside 1.5 standard deviations from median); n = 10.