Interactions of nasal epithelium with macrophages and dendritic cells variously alter urban PM-induced inflammation in healthy, asthma and COPD

Urban particulate matter (UPM) is an important trigger of airway inflammation. The cross-talk between the external and internal matrix in the respiratory tract occurs due to the transepithelial network of macrophages/dendritic cells. This study characterized the immune processes induced by the epithelium after UPM exposure in special regard to interactions with monocyte-derived dendritic cells (moDCs) and monocyte-derived macrophages (moMφs) in obstructive lung diseases. A triple-cell co-culture model (8 controls, 10 asthma, and 8 patients with COPD) utilized nasal epithelial cells, along with moMφs, and moDCs was exposed to UPM for 24 h. The inflammatory response of nasal epithelial cells to UPM stimulation is affected differently by cell–cell interactions in healthy people, asthma or COPD patients of which the interactions with DCs had the strongest impact on the inflammatory reaction of epithelial cells after UPM exposure. The epithelial remodeling and DCs dysfunction might accelerate the inflammation after air pollution exposure in asthma and COPD.


Patients' characteristics
This was a prospective, cross-sectional study which involved 8 healthy controls, 10 asthma patients, and 8 patients with COPD. In all patients, the diagnosis of asthma or COPD was previously established according to current Global Initiative for Asthma (GINA) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommendations, respectively [14,15]. The following examinations were performed after patient enrolment: medical history, physical examination, spirometry with flowvolume curve, airway obstruction reversibility test (when applicable, performed according to the recommendations of the European Respiratory Society (ERS)), allergy skin prick tests with a panel of fifteen aeroallergens. Disease control was assessed by Asthma Control Test (ACT) in asthmatics and by the COPD Assessment Test (CAT) in patients with COPD. The nasal brushing and peripheral blood sample were obtained from each patient. Exclusion criteria for all asthma and COPD patients were systemic or nasal steroid treatment, disease exacerbation or symptoms of respiratory tract infection in the previous 3 months. The control group consisted of smoking and non-smoking volunteers, without with normal spirometry.

The culture of epithelial cells in air liquid interface (ALI)
Epithelial cells were obtained by nasal brushing (Cytobrush Plus GT, CooperSurgical, Germany). The cells were detached from the brush by gentle agitation, centrifuged (300 g, 10 min, room temperature) and treated with Accutase (BD Biosciences, San Jose, CA, USA) for 10 min. in 37°C. The cell pellet was suspended in a total volume of 5 ml of airway epithelial growth medium (Promocell, Germany) containing antibiotics, and seeded into sterile plastic T25 bottles (Thermo Fisher Scientific, MA, USA). Cells were incubated in a plastic dish for 24 hours at 37°C. The undetached cells were removed and the cell medium changed. The adhered cells were cultured until reached 80% of confluency. 1x10 5 cells/cm 2 were seeded on the apical surface of 6.5 mm trans well Thin Certs with 0.4 µm pore size and placed in 24 well flatbottomed plates (Greiner Bio-One, Austria) with epithelial growth medium (Promocell, Germany) in the basal (0.5 ml) and upper (0.2 ml) compartments. After 100% of confluence was reached, cells in the upper chamber were exposed to air, the medium in basal chamber was changed for ALI Maintenance Medium (Stemcell, Canada) and exchanged every 2 day. The ALI epithelium was generated for 21 days.

Triple Co-Culture
Each triple-cell co-culture model will contain three different cell types obtained from one patient. The fully specialized ALI epithelial cells were supplemented with moMφs and moDCs as follows: moDCs were harvested, washed, centrifuged, re-suspended in X-Vivo-20 medium and 2x10 5 cells in 50 μl medium was added to the basal side of the inserts placed upside down. The dish with the inserts was covered and placed in the incubator for 1.5h-2h. MoMφs were harvested by washing with PBS followed by addition of Cellstripper (Corning, New York, USA) and detached with cell scrapers. A volume of 35 μl Macrophage Medium DXF (Promocell, Germany) containing 1.0x10 5 cells was added on the apical side of the epithelial monolayer on the insert, forming the upper chamber.

RNA isolation, cDNA synthesis and real time PCR
Total RNA was isolated from the cells using Trizol (Sigma Aldrich, MO, USA). The concentration and purity of isolated RNA was measured on a DU650 spectrophotometer (Beckman Coulter, Brea, CA, USA). Eight microliters of total RNA was used for reverse transcription (Thermo Fisher Scientific, MA, USA). Real-time PCR measurements were performed with an ABI-Prism 7500 Sequence Detector System (Applied Biosystems, Thermo Fisher, MA, USA). For PCR reaction 0.8µl of cDNA was amplified in 16 µl PCR volume, containing a TaqMan master mix (Thermo Fisher Scientific, MA, USA) with 150 nM of specific primers and 100 nM of probe (Table S1 Thermo Fisher Scientific, MA, USA). Each sample was measured in duplicate. The results were expressed as relative quantification units (fold change). Relative quantification values were calculated by the 2 -∆∆CT method. 18S rRNA was applied for each sample as an internal control in order to normalize gene expression levels. The mean ΔCT of unstimulated epithelial cells from controls was used as a calibrator for all groups.

Flow cytometry
Human TruStain FcX (Biolegend, San Diego, CA, USA) (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: CD326, CD45, EGF, MUC1 (BD Biosciences, San Jose, CA, USA), ST2 (Biotechne, R&D Systems, MN, USA) (Table S2) in BD Horizon Brilliant Stain Buffer (BD Biosciences, San Jose, CA, USA), and incubated for 20 minutes 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, San Jose, CA, USA), then stained with intracellular marker (TGF-β1, β-tubulin (BD Biosciences, San Jose, CA, USA)) for 20 minutes in the dark.
Figure S1 Transepithelial electrical resistance (TEER) after 24h of nasal epithelial cells from healthy donors cultured in air-liquid interface (ALI) conditions in multi co-culture models and stimulated with UPM. Figure S2. Transepithelial electrical resistance (TEER) after 24h of nasal epithelial cells from asthma patients cultured in air-liquid interface (ALI) conditions in multi co-culture models and stimulated with UPM. Figure S3. Transepithelial electrical resistance (TEER) after 24h of nasal epithelial cells from COPD patients cultured in air-liquid interface (ALI) conditions in multi co-culture models and stimulated with UPM.