PMN transepithelial migration and subsequent inflammation occur first in the small airways of CF patients, i.e., the bronchiolar region, which is lined with a microvilli-covered epithelial monolayer dominated by Club cells [3 (link)–5 (link)]. Therefore, to mimic PMN transmigration into the small airway lumen, we selected the H441 human Club cell line [20 (link)] to grow epithelial monolayers at air-liquid interface (ALI). To enable PMN loading in the lamina propria and transepithelial migration (Fig. 1A), we used Alvetex (Reinnervate) 200 μm-thick inert 3D scaffolds with >90% porosity (pore sizes of 36–40 μm, with interconnects of 12–14 μm). In brief, inserts were activated with 70% ethanol, coated overnight at 37ºC with rat-tail collagen I (3 mg/mL, Sigma) and seeded with H441 cells at 2.5×105 cells per 12-well insert. Cells were first grown in submerged cultures with DMEM/F12 supplemented with 10% heat-inactivated serum, penicillin, and streptomycin. After 2 days, cells were supplemented basally with serum-free DMEM/F12 with 10% Ultroser G (Pall Life Sciences) to establish ALI. Cultures were grown for 2 weeks at ALI and supplemented basally with fresh medium every 48 hours. For TM experiments, the ALI cultures were placed with the apical compartment exposed to RPMI, leukotriene B4 (LTB4, 100 nM), CXCL8 (100 ng/mL), formyl-methionine-leucine-phenylalanine (fMLF, 100 ng/mL), lipopolysaccharide (LPS, 500 ng/mL), or airway supernatant (ASN) from CF, HC, COPD, and LD subjects. TM experiments with 0.5–1 ×106 PMNs loaded onto the 200 μm-thick basal compartment of the Alvetex scaffold (situated upside), and allowed to migrate at 37°C at 5% CO2 through the collagen and epithelial layers into the apical compartment (situated downside, and bathed with either control medium with chemoattractant, or ASN). In some experiments, drugs were added to apical ASN and/or basal PMN suspensions. In other experiments, LPS-RS (competitive inhibitor of LPS binding to TLR4) was added to apical LPS or CF ASN. LPS and LPS-RS were purchased as ultrapure reagents from InvivoGen.