Clin Exp Otorhinolaryngol.  2024 May;17(2):147-159. 10.21053/ceo.2023.00079.

Effect of Air Pollutants on Allergic Inflammation in Structural Cells of the Nasal Mucosa

• Park JH
• Moon JW
• Yang HW
• Song DJ
• Park IH

• KMID: 2556005
• DOI: http://doi.org/10.21053/ceo.2023.00079

Abstract

Objectives
. Air pollution is an increasing global concern, and its effect on allergic inflammation has attracted the attention of many researchers. Particulate matter (PM) is a major component of ambient air pollution, and heavy metals are the primary toxic constituents of PM. As previous studies on the impact of air pollutants on allergic inflammation did not adequately mimic real-world atmospheric exposure, we developed an experimental model to investigate the effects of aerosolized air pollutants on nasal epithelial cells and fibroblasts.
Methods
. We collected particulate matter 2.5 (PM_2.5) samples from ambient 24-hour air samples obtained in Seoul from August 2020 to August 2022, and then conducted component analysis for metallic constituents. Primary nasal epithelial cells and nasal fibroblasts, obtained and cultured from the turbinate tissues of human participants, were treated with PM_2.5. The associations of heavy metals identified from the component analysis with cytokine expression were investigated. A three-dimensional (3D)-hybrid culture model, consisting of co-culture of an air-liquid interface and nasal fibroblast spheroids, was constructed to observe the impact of aerosolized air pollutants.
Results
. Among the heavy metals, Si was the predominant component of PM_2.5, and Zn showed the highest correlation with the concentration of PM_2.5 in Seoul. PM_2.5, Zn, and Si increased the production of epithelial cell-derived cytokines, and PM_2.5 and Zn exhibited similar trends with one another. Exposure of the 3D-hybrid model to aerosolized PM_2.5 and Zn resulted in elevated periostin, alpha-smooth muscle actin, and fibronectin expression in fibroblast spheroids, and those without an epithelial barrier exhibited a similar increase in periostin expression.
Conclusion
. Ambient air pollutants in the form of aerosols increase the expression of allergic inflammatory cytokines in both nasal epithelial cells and fibroblasts. Regulations on air pollution will help reduce the global burden of allergic diseases in the future.

Keyword

Allergic Rhinitis; Air Pollution; Particulate Matter; Epithelial Cell; Fibroblasts

Figure

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  Fig. 1. Schematic drawing of aerosolized particulate matter 2.5 (PM2.5) and ZnCl2 treatment of a co-culture of air-liquid interface (ALI) and fibroblast spheroids. (A) Aerosol exposure of epithelial cells to PM2.5 and ZnCl2, (B) co-culture of ALI and nasal fibroblast spheroids. Illustration of the correlation between PM2.5 and the heavy metals Zn and Si. (C) The list of the order of abundance of heavy metals in PM2.5. (D) Heatmap of correlation coefficients between PM2.5 concentrations and heavy metal concentrations. (E) The correlation coefficients between Zn concentrations and PM2.5 concentrations and between Si concentrations and PM2.5 concentrations. SD, standard deivation.

• 

  Fig. 2. The effects of particulate matter 2.5 (PM2.5) on allergic inflammation in primary nasal epithelial cells. (A) Primary nasal epithelial cells were treated by different concentrations of PM2.5 (0–1,000 μg/mL) and cytotoxicity was measured using WST-1. (B-E) The mRNA levels of allergic inflammation markers, including interleukin (IL)-6, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), were measured by real-time polymerase chain reaction after treatment with PM2.5. (G-J) Protein expression of these markers was measured by enzyme-linked immunosorbent assays. (F, K) The expression of periostin mRNA and protein was measured. (L) Air-liquid interface (ALI) culture treated with different concentrations of PM2.5 (0–1,000 μg/mL) was analyzed for cytotoxicity using WST-1. (M-V) IL-6, IL-25, IL-33, TSLP, and periostin mRNA and protein expression levels were measured after treatment with PM2.5. Values are presented as mean±standard deviation of three independent experiments. *P<0.05 compared to control.

• 

  Fig. 3. The effects of ZnCl2 on allergic inflammation in primary nasal epithelial cells. (A) Primary nasal epithelial cells treated with different concentrations of ZnCl2 (0–1,000 μM) were measured for cytotoxicity using WST-1. (B-E) The mRNA levels of allergic inflammation markers including interleukin (IL)-6, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) were measured by real-time polymerase chain reaction after treatment with ZnCl2. (G-J) Protein expression of these markers was measured by enzyme-linked immunosorbent assays. (F, K) The expression levels of periostin mRNA and protein were measured. (L) Air-liquid interface (ALI) culture treated with different concentrations of ZnCl2 (0–1,000 μM) were measured for cytotoxicity using WST-1. (M-V) IL-6, IL-25, IL-33, TSLP, and periostin mRNA and protein expression levels were measured after treatment with ZnCl2. Values are presented as mean±standard deviation of three independent experiments. *P<0.05 compared to control.

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  Fig. 4. Effects of SiO2 on allergic inflammation in primary nasal epithelial cells. (A) Primary nasal epithelial cells treated with different concentrations of SiO2 (0–100 μM) were measured for cytotoxicity using WST-1. (B-E) The mRNA levels of allergic inflammation markers including interleukin (IL)-6, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) were measured by real-time polymerase chain reaction after treatment with SiO2. (G-J) Protein expression of these markers was measured by enzyme-linked immunosorbent assays. (F, K) The expression of periostin mRNA and protein was measured. (L) Air-liquid interface (ALI) culture treated with different concentrations of ZnCl2 (0–100 μM) was measured for cytotoxicity using WST-1. (M-V) IL-6, IL-25, IL-33, TSLP, and periostin mRNA and protein expression levels were measured after treatment with SiO2. Values are presented as mean±standard deviation of three independent experiments. *P<0.05 compared to control.

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  Fig. 5. Aerosolized particulate matter 2.5 (PM2.5) and ZnCl2 induced fibroblast activation and an allergic inflammatory response in spheroids of nasal fibroblasts co-cultured with air-liquid interface (ALI) cultures. (A-C) The mRNA and protein levels of alpha-smooth muscle actin (α-SMA) and fibronectin were determined after exposure to PM2.5 or ZnCl2 aerosols. (D, E) The mRNA and protein expression of periostin was measured after exposure to PM2.5 or ZnCl2 aerosols. (F) Immunofluorescence staining data confirmed the expression of α-SMA (green) and periostin (red). The nuclei of the cells were stained with DAPI (blue). Scale bar=50 μM. (G, H) Periostin mRNA and protein expression levels were measured in nasal fibroblast spheroids upon direct aerosol exposure to PM2.5 or ZnCl2. Values are presented as mean±standard deviation of three independent experiments. 3D, three-dimensional; DW, distilled water; DAPI, 4´,6-diamidino-2-phenylindole, dihydrochloride. *P<0.05 compared to control.

• 

  Fig. 6. Aerosolized particulate matter 2.5 (PM2.5) and ZnCl2 induced an allergic inflammatory response in epithelial cells in co-cultures of air-liquid interface (ALI) and nasal fibroblast spheroids. (A) Transepithelial electrical resistance (TEER), which reflects cell barrier integrity, was measured. Cell exposure to aerosolized PM2.5 or ZnCl2 was examined for (B-E) mRNA and (G-J) protein expression of interleukin (IL)-6, epithelial cell-derived cytokines IL-25, IL-33, thymic stromal lymphopoietin (TSLP), (F) mRNA of periostin. (K-N) The mRNA and protein expression levels of E-cadherin, vimentin, and fibronectin involved in epithelial-mesenchymal transition were measured after exposure to aerosolized PM2.5 or ZnCl2. (O) Immunofluorescence staining data confirmed the expression of E-cadherin (green) and periostin (red). The nuclei of the cells were stained with DAPI (blue). Scale bar=50 μM. Values are presented as mean±standard deviation of three independent experiments. 3D, three-dimensional; DW, distilled water; DAPI, 4´,6-diamidino-2-phenylindole, dihydrochloride. *P<0.05 compared to control.

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