Figure 8.

Evaluation of the impact of gastric fluid samples and pepsin on neutrophil migration using a human basal stem cell derived airway mucosa model cultured at air-liquid interface. Inverted air-liquid interface (ALI) airway mucosa cultured on inverted 3.0 μm pore sized permeable transwell filters were wholemount stained to highlight ciliated cells (AcTub & Ezrin), tight junctions (ZO-1), and nuclei (DAPI). Scale bar, 20 μm for ZO-1 field, 50 μm for AcTub & Ezrin (A). Pepsin (29 µg/ml) set at pH 3 and a gastric fluid sample set at pH 3 and diluted 1:4 in pH matched HBSS derived from a patient on PPI therapy both induce a significant magnitude of neutrophil (PMN) migration across a human primary airway mucosa grown at air-liquid interface (B). HBSS set at pH 3 and a gastric fluid sample derived from the patient off PPI therapy set at pH 3 failed to induce neutrophil migration as did all conditions set at pH 7.4 (B). These effects observed with pepsin and the gastric sample derived from a patient on PPI therapy, both set at pH 3, are completely abrogated by the addition of Pepstatin A (20 µg/ml) (C). Panels A and B are separate internally controlled experiments. Each data point depicts the mean +/− SD of triplicate wells assayed (n = 3). Differences were considered significant at p ≤ 0.05 and noted by the symbol (*). The p values were determined using an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. The gastric sample from the patient on PPI therapy was patient #18 and the gastric sample from the patient off PPI therapy was patient #11 (Supplementary Table S1).