A high-capacity air sampler was employed to capture particulate matter with a diameter of 2.5 μm or less in Seoul. The collected PM_2.5 with phosphate-buffered saline (PBS) was filtered through a 2.5 μm pore filter, effectively sieving out particles with a diameter of 2.5 μm or less. The particles smaller than 2.5 μm were separated through centrifugation. Subsequently, the collected particulate matter was transferred to a tube, subjected to a 15 minute heating at 121 °C, and then stored at –20 °C. Quantification of 19 trace elements (PM_2.5, Al, Ti, V, Mn, Fe, Ni, Co, Cu, Zn, As, Sr, Mo, Cd, Ba, Pb, P, Cr, Si) was performed using atomic absorption spectroscopy (GBC Avanta PM, Australia). The entire process was designed to ensure the accurate measurement of trace elements associated with PM_2.5 in the air quality assessment in Seoul.

The inferior turbinate tissues were harvested from individuals who exhibited no indications of inflammation, allergic reactions, asthma, or sensitivity to aspirin. during rhinoplasty. Patients were recruited from the Department of Otorhinolaryngology at Korea University Medical Center, following the principles outlined in the Declaration of Helsinki, with prior informed consent obtained. This study received approval from the Institutional Review Board for Bioethics at Korea University Medical Center (No. 2023GR0179). Ethical handling of patients and their tissue specimens followed established protocols and guidelines.

Air-liquid interface (ALI) culture in 12 well transwell plates were placed on VITROCELL Cloud12 (VITROCELL Systems) and treated with the exposure system as an aerosol that exposes cells to 200 µL of PM_2.5 and ZnCl₂ (Sigma-Aldrich) (Fig. 1A). The exposure to aerosol was conducted single time, and real-time measurements of the deposited particles were performed using a quartz microbalance.

Primary nasal epithelial cells were collected using a brush and incubated with PneumaCult-Ex Plus medium (Stemcell Technologies) on plates coated with collagen type 1 (Corning). After cells were adherent, red blood cells were removed by washing with PBS. When cells reached >90% confluence, cells were isolated using an animal-free cell isolation kit (Stemcell Technologies) and cultured in 75T flasks.

Human nasal epithelial cells (HNECs) were expanded, washed with Dulbecco’s Phosphate-Buffered Saline, and dissociated using the Animal Component-Free Cell Dissociation Kit. For ALI culture, 0.5 mL of PneumaCult-Ex medium (Stemcell Technologies) containing HNECs is seeded into the upper chamber of a 0.4 μm Transwell insert (Corning) and only medium is added to the lower chamber. The media in both the upper and lower chambers were refreshed every 2 to 4 days. Upon reaching 100% cellular confluence, the medium in both the upper and lower chambers was aspirated. Subsequently, 1 mL of PneumaCult-ALI Maintenance Medium (Stemcell Technologies) was added only to the lower chamber, with subsequent medium changes every 2 days.

The inferior turbinate, obtained from the patient, are finely chopped and placed in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen). The chopped tissue is then evenly distributed onto a plate to isolate. The cells around the tissue are dissociated using 0.05% trypsin and cultured to isolate fibroblasts. When the fibroblasts reach 100% confluence, they are detached from the plate using trypsin and cultured at a density of 5×10⁴ cells per well in a 96-well round-bottom ultra-low attachment plate (Corning). Each well formed a single sphere and was cultured for 7 days, with the medium changed every 3 days. For co-culture with ALI and nasal fibroblast spheroids, spheroids were transferred to Transwell bottom plates, inserted into the upper chamber containing ALI culture, and incubated at 37 °C with 5% CO₂ (Fig. 1B). RPMI 1640 medium (Hyclone) was added only to the bottom plate.

Cellular cytotoxicity induced by PM_2.5, ZnCl₂, and SiO₂ (Sigma-Aldrich) was evaluated using a WST-1 assay. Nasal epithelial cells or nasal fibroblasts were plated in 96-well plates and exposed to various concentrations of PM_2.5, ZnCl₂, or SiO₂ for 72 hours. WST-1 solution is then added to the medium. Incubation at 37 °C facilitated the enzymatic conversion of WST-1 to formazan by metabolically active cells. The absorbance at 450 nm (with reference to 650 nm) was measured using a microplate reader (Bio-Rad).

PM_2.5, ZnCl₂, and SiO₂ were treated with epithelial cells and fibroblasts for 24 hours. RNA was isolated from the samples using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Concentration and purity of the extracted RNA were determined using a NanoDrop spectrophotometer. For complementary DNA (cDNA) synthesis, 2 μg of total RNA was reverse transcribed into cDNA using a mixture of M-MLV reverse transcriptase (Invitrogen), oligo dT primers, and a ribonuclease inhibitor. The synthesized cDNA was used for gene expression analysis by real-time polymerase chain reaction (RT-PCR). Power SYBR Green PCR Master Mix (Applied Biosystems) was mixed with the target-specific primers and a cDNA template. The target-specific primers are described in Table 1. RT-PCR was performed using Quantstudio3 to measure PCR amplification. Gene expression levels were quantified using the ΔΔCt method. The Ct values obtained from target gene amplification were normalized to the expression of the reference gene, GAPDH. The relative fold change in gene expression was calculated by comparing the normalized target gene Ct values between experimental and control samples.

PM_2.5, ZnCl₂, and SiO₂ were treated with epithelial cells and fibroblasts for 72 hours. The concentrations of IL-6, IL-25, IL-33, thymic stromal lymphopoietin (TSLP), and periostin in the culture media were determined using enzyme-linked immunosorbent assays (R&D Systems). Standards and samples were added to the wells and incubated at room temperature for 2 hours. After washing the wells three times, the appropriate conjugate (IL-6, IL-25, IL-33, TSLP, or periostin) was added and incubated for 2 hours at room temperature. The reaction was then stopped with a stop solution, and the optical densities of the standards and samples were measured at 450 nm using a microplate reader (Bio-Rad).

PM_2.5, ZnCl₂, and SiO₂ were treated with epithelial cells and fibroblasts for 72 hours. Proteins extracted using radioimmunoprecipitation assay (RIPA) buffer were measured for concentration using the Bradford assay. Samples were prepared by mixing equal amounts of proteins with 5×sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and resolved by electrophoresis on 10% SDS-PAGE gels. The proteins were transferred to polyvinyl difluoride membranes and then blocked in 3% skim milk. The membranes were probed with anti–alpha-smooth muscle actin (α-SMA; Abcam), anti-E-cadherin, anti-fibronectin, anti-GAPDH (Santa Cruz Biotechnology), and anti-vimentin (Cell Signaling Technology) primary antibodies overnight at 4 °C. The horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories) were diluted in 3% skim milk for 1 hour before measurement using an ECL system (Pierce). Subsequently, acquired images were analyzed with ImageJ software (National Institutes of Health) to quantify the observed signals.

Cellular fixation was achieved by treating cells with 4% paraformaldehyde for 30 minutes, followed by permeabilization using 0.1% Triton X-100 for 10 minutes. Subsequently, a blocking step with 5% BSA for 1 hour was implemented before incubating the cells with primary antibodies against E-cadherin, α-SMA, or periostin (Santa Cruz Biotechnology, 1:100 dilution in blocking buffer) at 4 °C overnight. Goat anti-mouse IgG (H+L) Alexa 488 or goat anti-rabbit IgG (H+L) Alexa 555 (Invitrogen) was diluted 1:200 in blocking solution and cells were incubated in this mixture for 1 hour at room temperature. Nuclei were counterstained with DAPI (4´,6-diamidino-2-phenylindole, dihydrochloride; Invitrogen) for 10 minutes. After washing with PBS, the coverslips were mounted on glass slides using a mounting medium. A confocal laser scanning microscope LSM700 (Zeiss) was used to capture images of the stained cells.

Epithelial electrical resistance (transepithelial electrical resistance [TEER]) measurements were performed by inserting sterile electrodes of an EVOM2 epithelial volt-ohmmeter (World Precision Instruments) into both the upper and lower chambers of the transwell system. The background resistance of the cell-free insert membrane devoid of cells was subtracted from each measurement. TEER values were recorded in ohms (Ω), to provide insight into the barrier integrity of the epithelial cell layer.

The data are presented as the mean±standard deviation of at least three experiments performed in duplicate. Statistical analysis was performed by unpaired t-test or one-way analysis of variance followed by Tukey’s test using GraphPad Prism 5 (GraphPad). Statistical significance was set at P<0.05.

Correlations were observed between concentrations of PM_2.5 and concentrations of heavy metals. Among the elements comprising PM_2.5, the most abundant was Si, followed by aluminum (Al) (Fig. 1C). The correlation coefficient between PM_2.5 and heavy metal concentrations was the highest for Zn, followed by iron (Fe) and nickel (Ni). The correlation coefficients between PM_2.5 and Zn and Si concentrations were 0.8 and 0.54, respectively, which were statistically significant (Fig. 1D and E). Considering these results, Zn and Si were selected as target heavy metals for further research.

The primary nasal epithelial cells treated with different concentrations of PM_2.5 showed no cytotoxicity until 1,000 μg/mL (Fig. 2A). We evaluated the mRNA and protein expression of markers associated with allergic inflammation, including IL-6, IL-25, IL-33, TSLP, and periostin. The results showed that IL-6, IL-25, IL-33, and TSLP levels significantly increased in a concentration-dependent manner for both mRNA (Fig. 2B-E) and protein expression (Fig. 2G-J) in response to PM_2.5. The expression of periostin mRNA and protein slightly increased at a PM_2.5 concentration of 100 μg/mL (Fig. 2F and K). Under ALI culture conditions (Fig. 2L), the expression levels of IL-6, IL-25, IL-33, and TSLP increased in response to PM_2.5 exposure, whereas periostin expression did not change (Fig. 2M-V).

Primary nasal epithelial cells treated with various concentrations of ZnCl₂ showed no cytotoxicity at concentrations <100 μM (Fig. 3A). We evaluated the mRNA and protein expression of markers associated with allergic inflammation, including IL-6, IL-25, IL-33, TSLP, and periostin. Significant increases were observed for IL-6, IL-25, IL-33, and TSLP in both mRNA (Fig. 3B-E) and protein expression (Fig. 3G-J) in a concentration-dependent manner in response to ZnCl₂ exposure. Periostin expression weakly increased at a ZnCl₂ concentration of 50 μg/mL, but significantly increased at a concentration of 100 μg/mL (Fig. 3F and K). In ALI culture conditions, ZnCl₂ did not lead to cytotoxicity at concentrations less than 100 μM (Fig. 3L). The expression of IL-6, IL-25, IL-33, and TSLP increased in response to ZnCl₂ exposure, while periostin expression remained unchanged (Fig. 3M-V).

Primary nasal epithelial cells treated with various concentrations of SiO₂ showed no cytotoxicity until a concentration of 100 μM (Fig. 4A). We evaluated the mRNA and protein expression of markers associated with allergic inflammation, including IL-6, IL-25, IL-33, TSLP, and periostin. The results showed that IL-6, IL-25, IL-33, and TSLP exhibited significant increases in both mRNA (Fig. 4B-E) and protein expression (Fig. 4G-J) in a concentration-dependent manner in response to SiO₂ exposure. However, the periostin mRNA and protein expression levels did not change (Fig. 4F and K). Consistently, in ALI culture conditions, SiO₂ did not lead to cytotoxicity at concentrations <40 μM (Fig. 4L), and the expression levels of IL-6, IL-25, IL-33, and TSLP increased in response to SiO₂ exposure, whereas periostin expression remained unchanged (Fig. 4M-V).

The effects of PM_2.5 and Zn on nasal fibroblast spheroids from the perspective of allergic inflammation were examined. Fibroblast spheroids were exposed to PM_2.5 and ZnCl₂ by aerosolization under two conditions (with and without ALI on top) to examine the expression of alpha-smooth muscle actin (α-SMA), a marker associated with myofibroblast differentiation, and fibronectin, an extracellular matrix (ECM) component. In the presence of ALI, ALI-cultured cells were exposed to aerosolized PM_2.5 (24.2 μg/cm²) and ZnCl₂ (46.3 ng/cm²) to investigate their effects on nasal fibroblast spheroids and allergic inflammatory responses. Both the mRNA and protein expression of α-SMA and fibronectin significantly increased following exposure to PM_2.5 or ZnCl₂ aerosols (Fig. 5A-C). Additionally, both mRNA and protein levels of periostin, an important factor in allergic inflammatory responses, dramatically increased after exposure to PM_2.5 or ZnCl₂ aerosols (Fig. 5D and E). Immunofluorescence staining confirmed the increased expression of α-SMA and periostin, with increased periostin expression in α-SMA-positive cells (Fig. 5F). Furthermore, direct aerosol exposure to PM_2.5 or ZnCl₂ significantly elevated periostin mRNA and protein expression in nasal fibroblast spheroids without ALI (Fig. 5G and H). These results suggest that exposure to PM_2.5 and ZnCl₂, whether directly or indirectly, increases the activation of nasal fibroblast spheroids and, consequently, upregulates periostin expression.

Experiments conducted in ALI culture and nasal fibroblast spheroids examined the effect of exposure to aerosolized PM_2.5 and ZnCl₂ on the expression of epithelial cell-derived cytokines and epithelial-mesenchymal transition (EMT). TEER, which was measured to quantify the barrier integrity of the cells, decreased upon exposure to PM_2.5 and ZnCl₂ (Fig. 6A). Both mRNA and protein expression levels of IL-6, IL-25, IL-33, and TSLP significantly increased in response to PM_2.5 and ZnCl₂ exposure (Fig. 6B-E and G-J). Furthermore, periostin mRNA expression was upregulated by exposure to PM_2.5 and ZnCl₂ (Fig. 6F). Notably, exposure to PM_2.5 and ZnCl₂ reduced the expression of E-cadherin, a marker of epithelial cells, and increased the expression of mesenchymal cell markers, including vimentin and fibronectin (Fig. 6K-N). Immunofluorescence staining supported these findings and revealed decreased E-cadherin and increased periostin expression (Fig. 6O). Aerosol treatment with PM_2.5 or ZnCl₂ showed similar trends in their effect on ALI in the presence of nasal fibroblast spheroids, but with increased reactivity.