Allergy Asthma Immunol Res.  2020 Jan;12(1):56-71. 10.4168/aair.2020.12.1.56.

Particulate Matter 2.5 Causes Deficiency in Barrier Integrity in Human Nasal Epithelial Cells

• Xian M
• Ma S
• Wang K
• Lou H
• Wang Y
• Zhang L
• Wang C
• Akdis CA

• KMID: 2462572
• DOI: http://doi.org/10.4168/aair.2020.12.1.56

Abstract

PURPOSE
The effect of air pollution-related particulate matter (PM) on epithelial barrier function and tight junction (TJ) expression in human nasal mucosa has not been studied to date. This study therefore aimed to assess the direct impact of PM with an aerodynamic diameter less than 2.5 μm (PM2.5) on the barrier function and TJ molecular expression of human nasal epithelial cells.
METHODS
Air-liquid interface cultures were established with epithelial cells derived from noninflammatory nasal mucosal tissue collected from patients undergoing paranasal sinus surgery. Confluent cultures were exposed to 50 or 100 µg/mL PM2.5 for up to 72 hours, and assessed for 1) epithelial barrier integrity as measured by transepithelial resistance (TER) and permeability of fluorescein isothiocyanate (FITC) 4 kDa; 2) expression of TJs using real-time quantitative polymerase chain reaction and immunofluorescence staining, and 3) proinflammatory cytokines by luminometric bead array or enzyme-linked immunosorbent assay.
RESULTS
Compared to control medium, 50 and/or 100 µg/mL PM2.5-treatment 1) significantly decreased TER and increased FITC permeability, which could not be restored by budesonide pretreatment; 2) significantly decreased the expression of claudin-1 messenger RNA, claudin-1, occludin and ZO-1 protein; and 3) significantly increased production of the cytokines interleukin-8, TIMP metallopeptidase inhibitor 1 and thymic stromal lymphopoietin.
CONCLUSIONS
Exposure to PM2.5 may lead to loss of barrier function in human nasal epithelium through decreased expression of TJ proteins and increased release of proinflammatory cytokines. These results suggest an important mechanism of susceptibility to rhinitis and rhinosinusitis in highly PM2.5-polluted areas.

Keyword

Epithelial cells; particulate matters; nasal mucosa; rhinitis; asthma; cytokines

MeSH Terms

Asthma
Budesonide
Claudin-1
Cytokines
Enzyme-Linked Immunosorbent Assay
Epithelial Cells*
Fluorescein
Fluorescein-5-isothiocyanate
Fluorescent Antibody Technique
Humans*
Interleukin-8
Mucous Membrane
Nasal Mucosa
Occludin
Particulate Matter*
Permeability
Polymerase Chain Reaction
Rhinitis
RNA, Messenger
Tight Junctions
Budesonide
Claudin-1
Cytokines
Fluorescein
Fluorescein-5-isothiocyanate
Interleukin-8
Occludin
Particulate Matter
RNA, Messenger

Figure

• 

  Fig. 1 (A) Representative images of immunofluorescent-stained claudin-1, ZO-1 and occludin in noninflammatory nasal mucosa collected during different seasons. The immunofluorescent staining for ZO-1 (red) and occludin (green) was mainly restricted to the apical compartment of epithelium, whereas the distribution of claudin-1 (green) was more extensive in the epithelium. A relatively weak staining was seen for claudin-1, occludin and ZO-1 in biopsy specimens collected during the winter compared to specimens collected during the summer. Bar = 20 μm. (B) Fluorescence intensities were quantified using ImageJ software (winter vs. summer; n = 7 and 5, respectively). *Statistical significance at a level of P < 0.05.

• 

  Fig. 2 Effect of PM2.5 on the barrier function of ALI cultures established from NECs. (A) TER at different time points following exposure to medium alone (control), 50 μg/mL PM2.5 or 100 μg/mL PM2.5 intermittently. Due to some inherent variability in baseline TER measurements across samples, all measurements were normalized to a value of 1.0 at the beginning of the measurement (0 hour). (B) Dose-dependent increase in FITC-dextran paracellular flux in ALI cultures treated with different doses of PM2.5. Barrier integrity was determined by paracellular flux in response to PM2.5 treatment after 72 hours. (C) TER negatively correlates with FITC-dextran permeability in ALI cultures (exposed to 100 and 50 μg/mL PM2.5 or medium alone). Data are expressed as mean ± standard error of the mean, n = 12 donors per group in duplicate. PM2.5, particulate matter with an aerodynamic diameter of less than 2.5 μm; FITC, fluorescein isothiocyanate; ALI, air-liquid interface; TER, transepithelial resistance. *P < 0.05; †P < 0.01; ‡P < 0.001.

• 

  Fig. 3 PM2.5 undermined the barrier integrity of air-liquid interface cultures established from nasal epithelial cells, which could not be prevented by budesonide pretreatment. The relative TER of cultures exposed to 50 μg/mL PM2.5 (A) or 100 μg/mL PM2.5 (B) and (C) paracellular flux of FITC-dextran 4kDa at 72 hours in cultures incubated in the absence or presence of 50/100 μg/mL PM2.5 was not significantly altered by pretreatment of the cultures for 1 hour with budesonide (1 μmol/L). Data are expressed as mean ± standard error of the mean, n = 5 donors per group in duplicate. PM2.5, particulate matter with an aerodynamic diameter less than 2.5 μm; FITC, fluorescein isothiocyanate; TER, transepithelial resistance. *P < 0.05; †P < 0.001.

• 

  Fig. 4 Effect of PM2.5 on the expression of tight junction mRNAs in air-liquid interface cultures established from nasal epithelial cells. After incubation with PM2.5 for 72 hours, relative mRNA expression of claudin-1 was significantly decreased and expression of ZO-1 and claudin-7 significantly increased in cultures treated with 100 μg/mL PM2.5, compared to control cultures (n = 10 each for the control and 50-μg/mL PM2.5-treated groups, n = 9 for the 100-μg/mL PM2.5-treated group). PM2.5 with an aerodynamic diameter of less than 2.5 μm; mRNA, messenger RNA. *P < 0.05, †P < 0.01.

• 

  Fig. 5 (A) Representative images of claudin-1 (green), occludin (green) and ZO-1 (red) immunofluorescent staining in cultured nasal epithelial cells exposed to medium alone, 100 μg/mL PM2.5 or 100 μg/mL PM2.5 pretreated with budesonide. Cultures exposed to medium alone displayed distinct TJs at cell borders, whereas cultures treated with PM2.5 displayed diminished and less discernible TJs at cell-cell junctions. Cultures pre-treated with budesonide showed similar changes to those treated with just PM2.5. Representative images from 3 donors each are shown. Bar = 20 μm. (B) Fluorescence intensities were quantified using ImageJ software. PM2.5, particulate matter with an aerodynamic diameter less than 2.5 μm; N.S., not significant; TJ, tight junction; TRITC, tetramethylrhodamine. *P < 0.05.

• 

  Fig. 6 Cytokine concentrations (pg/mL) in ALI culture medium of NECs treated with medium alone, 50 μg/mL PM2.5 or 100 μg/mL PM2.5 intermittently for 3 days, respectively. Cultured NECs produced significantly more IL-8, TIMP-1 and TSLP after treatment with PM2.5, compared to cultures treated with medium alone. A dose-dependent effect was visible, although it was not statistically significant for all mediators. The secretion of other cytokines produced by NECs was not affected by PM2.5. Data are presented as mean ± standard error of the mean (n = 8 to 10 in each group). PM2.5, particulate matter with an aerodynamic diameter less than 2.5 μm; IL, interleukin; TIMP-1, TIMP metallopeptidase inhibitor 1; TSLP, thymic stromal lymphopoietin; MMP, matrix metalloproteinase; NEC, nasal epithelial cell. *P < 0.05; †P < 0.01; ‡P < 0.001.

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