Altered polarization of PAR-2 signaling during airway epithelial remodeling

Ryan M. Carey; Jenna R. Freund; Benjamin M. Hariri; Nithin D. Adappa; James N. Palmer; Robert J. Lee

To begin to understand how epithelial remodeling changes polarization of PAR-2 in primary sinonasal cells, we initially de-differentiated/remodeled primary ciliated air-liquid interface (ALI) cultures by two methods. The first was exposure to IL-13 (10 ng/ml, basolateral), which induces cilia loss and goblet cell metaplasia. 43, 44 The second was apical submersion, which induces a more squamous phenotype. 43, 45 ALIs were cultured for 21 days to establish a well-ciliated baseline (Fig 2A) . IL-13 or apical submersion both resulted in a decrease in the number of ciliated cells over 6 days, shown by a reduction in b-tubulin IV immunofluorescence (Fig 2A-B) . While IL-13 increased goblet cell specific Muc5AC intensity (Figs 2A and C) , submersion only minimally increased Muc5AC at 6 days (Figs 2A and C) . Submersion instead increased expression of transgutaminase 1 (TG-1; measured via ELISA), previously reported to reflect squamous differentiation 46, 47 (Fig 2D) . Transepithelial electrical resistance (TEER) remained intact up to 4 days (Fig 2E) , and maintenance of epithelial barrier function was confirmed by measurement of FITC-conjugated dextran flux at day 4 as well as ability of cells to maintain low apical glucose levels (Supplementary Fig S9A) . Evidence of cell death (LDH release) was not observed (Supplementary Fig S9B) .