Neutrophil transepithelial migration assay

Primary airway epithelial cells were obtained from nasal brushings, as described previously [21] or purchased from Epithelix Sàrl (Geneva, Switzerland). A diagram of our methodology is shown

in figure 1 and a detailed protocol for nAEC culture can be found in the supplementary methods. We found no difference in the cultures grown from a brushing or from commercial supplier (supplementary figure S1). Briefly, progenitor basal cells were propagated in co-culture with 3T3-JF mouse fibroblast feeder layers, as described previously [22]. Primary nAECs are seeded into feeder layers at a density of 5×10⁵ per flask in F-media (DMEM and Hams F12 media at a 3:1 ratio supplemented with 1×penicillin streptomycin, 7.5% fetal bovine serum (Gibco, Waltham, MA, USA), 5 mM Y-27632 (Abcam, Cambridge, UK), 25 ng·mL⁻¹ hydrocortisone/0.125 ng·mL⁻¹ epidermal growth factor (Sigma, St Louis, MO, USA), 5 mg·mL⁻¹ insulin (Sigma), 0.1 nM cholera toxin (Sigma) and amphotericin B (2.50 μg·mL⁻¹)) and cultured until confluent. Primary nAECs and 3T3-J2 fibroblasts were then separated by differential trypsin dissociation, as previously described [22]. Basal cells were collected in fresh F-media and centrifuged at 200×g for 3 min.

Collagen-coated 3-μm pore membrane inserts (ThinCert; Greiner, Kremsmunster, Austria, with culture surface of 33.6 mm²) were inverted and 300 000 cells·cm⁻² nAECs in 70 μL of F-media were seeded onto the bottom of the insert and incubated at 37°C 5% carbon diolxide (CO₂) for 4–6 h. After incubation, membrane inserts were inverted into a 24-well plate and 500 μL of fresh F-media added underneath and 100 μl of F-media supplemented with 30 μg·mL⁻¹ collagen I and 5% (v/v) Matrigel (Corning, Corning, NY, USA) added to the top of the membrane inserts. Cells were incubated for 24–48 h at 37°C 5% CO_2. After this period, cells were placed at ALI. Media was aspirated from both sides of the membrane insert and cells fed basolaterally with 100 μL of ALI media (1:1 DMEM: airway epithelial cell growth media (PromoCell, Heidelberg, Germany), with all supplements added, further supplemented with 2.5 μg·mL⁻¹ Amphotericin B, 1×penicillin/streptomycin and 1 μm retinoic acid (Sigma). The medium was replaced every 1–2 days, and cells incubated at 37°C 5% CO₂ in a high-humidity incubator for 4 weeks/28 days to allow cellular differentiation. Once differentiated, cells were infected with RSV as before [19].

Neutrophils were purified from 10 mL of peripheral venous blood using a negative immunoselection neutrophil isolation kit (Stemcell Technologies, Vancouver, Canada), as per the manufacturer's instructions. The mean number of neutrophils isolated from was 1.5×10⁷ cells with a purity of 99.8–99.9% as confirmed by flow cytometry (supplementary figure S1d). Neutrophils were stained with CellTrace Calcein Red-Orange cell stain (ThermoFisher, Waltham, MA, USA), as described previously [14].

For neutrophil transepithelial migration 400 μL of apical surface media was placed underneath the membrane insert for each experimental group: mock infected, RSV infected, mock infected exposed to apical surface media collected from RSV infected cells or N-formylmethionine-leucyl-phenylalanine (fMLP). To investigate the interaction of the integrin leukocyte function-associated antigen-1F (LFA1) with the intracellular cell adhesion molecule ICAM-1, we supplemented the apical surface media collected from RSV-infected cells with 1 μM (2E)-1-(4-acetyl-1-piperazinyl)-3-[4-[[2-(1-methylethyl)phenyl]thio]-3-nitrophenyl]-2-propen-1-one (A286982, TOCRIS BioTechne, Minneapolis, MN, USA), a potent antagonist (inhibitor) of the LFA-1 CD11a I domain [23]. A median inhibitory concentration of 35–44 nM was used, based on that used in other studies [24, 25]. Neutrophils (5×10⁵) were added to the basolateral side of membrane inserts and left to migrate for 1 or 4 h. After migration, neutrophils were collected from the apical side of the epithelial cells for quantification (see later). Apical surface media (containing epithelial secreted factors including cytokines) were collected and membrane inserts were fixed and stained for ICAM-1, acetylated tubulin (cilia) or adherent neutrophils (see later).