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A7192 - The Proteome of Airway Epithelial Mucus and Apical Secretions is Altered by IL-13 Stimulation and Results in Changes to Ciliary Beat Dynamics
Author Block: J. L. Everman1, L. Ringel1, M. Chioccioli2, R. Powell3, C. Cruickshank-Quinn3, C. Rios1, N. Jackson1, L. Feriani2, M. Armstrong3, J. Gomez3, X. Zhang3, H. Chu4, P. Cicuta2, N. Reisdorph3, M. A. Seibold1; 1Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, United States, 2Biological Soft Systems Sector, University of Cambridge, Cambridge, United Kingdom, 3Department of Pharmaceutical Sciences, University of Colorado, Aurora, CO, United States, 4Department of Medicine, National Jewish Health, Denver, CO, United States.
RATIONALE Excessive type 2 cytokine airway inflammation is found in ~50% of asthmatics and is associated with greater airflow obstruction. The mechanistic basis for this obstruction is unclear, but may be related to Interleukin 13 (IL-13) stimulating the airway epithelium to undergo mucus metaplasia and mucus hypersecretion. How the airway epithelial apical protein and mucus secretome changes globally with chronic IL-13 stimulation is unknown. Moreover, it is unclear whether IL-13-induced changes of secreted apical and mucus proteins alter airway epithelial ciliary beat dynamics.
METHODS Human airway epithelial cells (AEC) were obtained from bronchial brushings of 19 donors and differentiated at air-liquid interface (ALI) for 21 days. Paired ALI cultures were mock-treated or IL-13-treated daily during the final 10 days of cellular differentiation. An unbiased proteomic screen of both the soluble and mucus fraction of apical washes was performed by LC/MS/MS. Ciliary motion analyses were performed. Human tracheal AECs were used for CRISPR gene knockout.
RESULTS IL-13 dramatically increased the apically secreted levels of 13 proteins including many mucin/mucin-like proteins (MUC5AC, FCGBP) and proteins involved in modification, cross-linking, and secretion of mucins (ITLN1, SIAT1, CLCA1). IL-13 stimulation also reduced secreted levels of 19 proteins, including SCGB1A1 and other proteins characteristic of club secretory cells. Proteomic analysis of the mucus fraction demonstrated a significant increase of the same mucin and mucin-related proteins identified in the soluble apical washes. High resolution video microscopy analysis shows a distinct decrease in ciliary beat frequency (CBF) in IL-13 stimulated (7.05 +/- 3.36 Hz) compared to mock-treated airway epithelial cultures (9.12 +/-1.54 Hz). This CBF decrease requires an intact mucus layer, and is abrogated upon chemical removal of the mucus layer. Additionally, the IL-13 induced change in the airway epithelial mucus layer affects a range of other cilia movement properties. Given Intelectin-1 (ITLN1) is reported to cross-link mucins and is prominently upregulated by IL-13 in our dataset, we examined whether knockout of this gene could perturb the IL-13 driven changes in CBF detailed above. We find the IL-13-mediated decrease in CBF is restored in muco-ciliary epithelial cultures knocked-out for the ITLN1 gene (97% decrease in ITLN1 protein).
CONCLUSIONS Our data suggests the IL-13-mediated changes in secreted mucin and mucin-related proteins drives changes in mucus physical properties, leading to dysregulation of ciliary beat dynamics. Preliminary experiments suggest targeting of IL-13-induced proteins involved in mucus formation may disrupt these IL-13 mediated changes in mucus properties and ciliary function.