General study design

This was an experimental study based on cell cultures of epithelial cells obtained from asthma, COPD patients, and healthy subjects. The study protocol was approved by the Ethics Committee of the Medical University of Warsaw (KB/58/A2021) and informed written consent was obtained from all the participants.

Patient characteristics

The study involved 5 controls, 8 asthma patients, and 8 COPD patients. In all patients, the diagnosis of asthma or COPD was previously established according to the current recommendations of the Global Initiative for Asthma (GINA) and the Global Initiative for Chronic Obstructive Lung Disease (GOLD), respectively. Inclusion criteria for COPD group were as follows: (1) the age of 40 years and over, (2) diagnosis of COPD based on the past medical history, history of smoking ≥ 10 packyears, typical signs and symptoms and irreversible airway obstruction found in spirometry (z-score of the post-bronchodilator FEV1/FVC below − 1.645). Inclusion criteria for asthma were as follows: (1) the age of 18 years and over, (2) diagnosis of asthma based on past medical history, typical signs and symptoms and demonstration of variable expiratory airflow limitation. The exclusion criteria included: (1) severe asthma or severe COPD, (2) unstable or uncontrolled disease (3) a concomitant COPD and asthma diagnosis, or any other chronic or acute lung disease, autoimmune and haematological diseases, malignancies, severe cardiovascular diseases (4) treatment with systemic or nasal corticosteroids within 4 weeks before the study enrolment (5) asthma, or COPD exacerbation within 3 months from mucosal sampling as well as symptoms of respiratory tract infection in the preceding 3 months. The control group consisted of non-smoking participants, with normal spirometry, chronic diseses and without symptoms of respiratory tract infection in the preceding 3 months.

Cell culture

Epithelial cells were obtained by nasal brushing (Cytobrush Plus GT, CooperSurgical), cultured to 80% of confluency in Airway Epithelial Growth Medium (PromoCell, Germany) with antibiotics (Thermo Fisher Scientific) and then seeded into sterile plastic T25 bottles (Thermo Fisher Scientific).

The spheroid culture was performed according to methodology described before [16]. Matrigel Basement Membrane Matrix (354234, Corning) and Airway Epithelial Growth Medium (PromoCell) were mixed to obtain protein concentration at least 5 mg/ml, according to manufacturer protocol. 1.5-1.9 × 104 cells per single well were added and seeded into 24-well plate (Greiner Bio-One). The spheroids were cultivated in Matrigel on basal side of the well covered with 500 µl of Pneumacult Airway Seeding Medium (StemCell) with antibiotics. Seeding stage lasts for the first 4–6 days and afterwards the spheroids were differentiated for 21 days in Pneumacult Airway Differentiating Medium (StemCell). After full differentiation, the spheroids were exposed for 24 h to 50 µg PM2.5/well (100 µg/ml) added directly into culture medium (apical side).

Spheroids were recovered from Matrigel according to the adapted manufacturer’s protocol. Culture medium was removed, and 1 ml of cold gentle cell dissociation reagent (StemCell) was added into every single well. Plates were put on tilting platform with agitation 30 rpm for 30 min on ice. Matrigel domes were triturated with 1 ml pipette tip pre-rinsed in anti-adherence rinsing solution (StemCell) and transferred into pre-rinsed 15 ml tube (Corning). Tubes were put on ice and tilted for 30 min, 30 rpm. Tubes were placed in stand and left for 10 min to settle by gravity, supernatants were aspirated, and spheroids were washed with 5 ml of PBS and gently centrifuged (60 rpm, 5 min, 4 °C). The diameter of 20 spheroids of each group were measured three times based on the spheroid width on the image (using ImageJ software). The scale was set according to the microscope image scale bar.

Particle preparation

Urban particulate matter was obtained from the Silesian University of Technology. The samples were collected with a low-volume PM sampler type PNS-15 (Atmoservice) 1.5 m above the ground, at a flow rate of 2.3m3/ h in Gliwice during heating season. Daily PM2.5 samples were collected continuously on high-purity quartz (SiO2) microfiber filters (QM-A Whatman) with 47 mm diameter. The filters were weighed, cut into small pieces, suspended in PBS; the particles were detached from filters by sonication, filtrated through strainers with 70 μm pores (Corning). The sediment of the particles was dried at 96 °C to dry mass, weighted, resuspended in PBS into stock solution containing 5 µg/µL PM2.5, and autoclaved. Quartz microfiber filters without particles were processed the same way and constituted a control suspension for cell treatment.

PM2.5 trace elements analysis

The QM-A filter was mineralized under high pressure and high temperature in a system for microwave mineralization in 8 mL HNO3 and 2 mL H2O2 (Merck). Elemental concentrations of nine elements (Zn, Fe, Mn, Pb, Cd, Cu, Cr, Ni and Co) were analysed for each sample by atomic absorption spectrometry (Avanta PM, GBC Scientific Equipment Pty Ltd) [17, 18].

Cell viability

To evaluate viability of nasal epithelial cells treated with PM2.5 lactate dehydrogenase (LDH) release was measured with Cytotox96 assay (Promega).

Immunofluorescence staining

Immunofluorescence staining was performed on 3D spheroid cell cultures. Spheroids were fixed with 4% paraformaldehyde at room temperature. Antigen retrieval was performed in 98 °C using citrate buffer pH6 for 20 min. Spheroids were then washed in 0.3 M glycine, permeabilized and blocked with 5% goat serum with 1% triton X-100 for 24 h (ThermoFisher Scientific). The blocking solution was aspired, spheroids were divided into two separate tubes. For half of the tubes, mouse monoclonal pan-cytokeratin antibody (cocktail which recognizes acidic (Type I or LMW) and basic (Type II or HMW) cytokeratins) (1:100) (Novus Biologicals) was added, while the other half received mouse monoclonal MUC5AC antibody (1:100) (Novus Biologicals). The addition took place overnight at room temperature with gentle agitation at 20 rpm. After washing, second primary antibodies rabbit polyclonal anti-alpha tubulin (1:150) (Abbexa) and rabbit polyclonal anti-tight junctions’ protein 1 (1:150) (Novus Biologicals) were added respectively, overnight at room temperature with agitation 20 rpm. After triple washing, secondary antibody anti-mouse Alexa Fluor 488 (1:500, ThermoFisher Scientific) was added to all tubes for overnight at room temperature with agitation 20 rpm. Further, after washing, additional incubation of all tubes with secondary antibody anti-rabbit Alexa Fluor 555 (1:500, ThermoFisher Scientific) was carried out overnight at room temperature with agitation 20 rpm. Spheroids were then washed with PBS and transferred onto slides (Surgipath Snowcoar Pear Slides, Leica Biosystems). Slides were mounted with Fluoroshield mounting medium with DAPI (Abcam) and images were captured using fluorescence microscope Nikon ECLIPSE Ts2R equipped with Hamamatsu ORCA-Flash4.0 camera and analysed with Nikon NIS-Elements and ImageJ software.

RT-qPCR

Total RNA was isolated from the cells using Tri reagent (Sigma Aldrich). The concentration and purity of isolated RNA was assessed by DU650 spectrophotometer (Beckman Coulter). cDNA synthesis was carried out with 1 µg of total RNA using cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). cDNA amplification was performed in ABI-Prism 7500 Sequence Detector System (Applied Biosystems, Thermo Fisher Scientific). For quantitative PCR (qPCR) reaction 0.7 µl of cDNA was amplified in 14 µl PCR volume, containing a TaqMan master mix (Thermo Fisher Scientific). By real-time qPCR the expression of IL-1β, IL-6, IL-8, TNF-α, MMP7, MMP9, MUC5AC, BPIFA2, ENPEP TIPARP, TGF-β and EGF was quantified. 18 S rRNA served as a reference gene. Assay ID of primers or their sequence used for real-time qPCR are shown in Table S1. Relative quantification values were calculated by the 2−∆∆CT method (7500 Software, Thermo Fisher Scientific), the unstimulated spheroids of each individual were used as a calibrator.

Protein concentration measurements

The levels of IL-8, IL-1β, IL-6, IL-10, TNF-α, IL-12p70 were measured in cell supernatants by BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit (BD Biosciences, Catalog No: 551811) according to manufacturer’s procedure. As concentrations of IL-8 exceeded the kit’s detection threshold, additional measurements for the cytokine concentrations were performed using ELISA (Thermo Fisher). The MMP-9 level was measured by Human MMP9 ELISA (Biorbyt) according to manufacturers’ instruction. The sensitivity of kits was 20 pg/ml for CBA and 2 pg/ml for ELISA.

Flow cytometry analysis

After spheroid recovering from matrigel cells were dissociated into single cell suspension by Accutase (BD Biosciences) treatment at 37 °C, shaking at 1400 RPM for 15 min and twice PBS rinsing and centrifugation at 1800 RPM. The cell suspension was filtrated through strainers with 70 μm pores (Corning). Human TruStain FcX (Biolegend) (5 µl per 100 µl of sample) was added to block non-specific bindings. Cells were stained with antibodies against the surface binding molecules, for epithelial cells: Tetraspanin-8 (TSPAN8) (BV786, rat anti-human cat. no. 748226), CD49f (BV650, rat anti-human, cat. no. 563706), epidermal growth factor receptor (EGFR) (BV421, mouse anti-human, cat no. 566254), CD151 (BB700, mouse anti-huma, cat. no. 746219), Toll-like receptor 4 (TLR4) (BV605, mouse anti-human, cat. no. 743692), Aryl Hydrocarbon Receptor (AHR) (PE-CF594, mouse anti-AhR cat. no. 565790) (BD Biosciences), in BD Horizon Brilliant Stain Buffer (BD Biosciences), and incubated for 20 min in the dark at room temperature. After washing away, the reagents, the cells were fixed and permeabilizated using lysis buffer and permeabilizations solution 2 (BD Biosciences) then stained with intracellular marker: β-tubulin (Alexa Fluor 488 Mouse anti-β-Tubulin, Class III, cat. no. 560381) and Transforming Growth Factor beta-1 (TGF-β) (PE, mouse anti-huma, cat. no. 562339, BD Biosciences, ) for 20 min in the dark.

Cells were analysed by flow cytometry using a FACSCelesta instrument (BD Biosciences) equipped with blue (488-nm), violet (405-nm), and red (640-nm) lasers. Unstained cells and compensation beads (BD Biosciences) were used to set voltages and create single stain negative and positive controls. Compensation was set to account for spectral overlap between the seven fluorescent channels used in the study. Samples were examined by side scatter area (SSC-A) versus forward scatter area (FSC-A), then using forward scatter height (FSC-H) versus FSC-A to select single cells, eliminating debris and clumped cells from the analysis.

The basal, secretory and ciliated epithelial cells were identified using markers CD49f, β-Tubulin, TSPAN described previously [19]. The epithelial cell subpopulations were defined as follows:

Epithelial cells with basal phenotype: CD49f + β-Tubulin- TSPAN-.

Epithelial cells with secretory phenotype: TSPAN + CD49f- β-Tubulin-.

Epithelial cells with ciliated phenotype: β-Tubulin + TSPAN-CD49f-.

Reactive oxygen species (ROS) analysis

The ROS identification was performed using the total ROS assay kit (Invitrogen, Thermo Fisher) according to manufacturer’s instruction. The cells were analyzed on FACSCelesta instrument (BD Biosciences) at 520 nm (FITC channel).

Statistical analysis

Statistical analyses were performed using Statistica 13.3 software (StatSoft Inc.) and GraphPad Prism software (version 9.3.1) (GraphPad Software). Data are presented as median and interquartile range (IQR) or number and percentage. Differences between continuous variables in 3 groups were tested using Kruskal-Wallis test and between 2 groups using nonparametric Mann–Whitney U test. Fisher’s exact test was used to test the differences between nominal variables. A p-value of less than 0.05 was taken as the threshold of statistical significance.