Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image‐based approach

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco‐obstructive diseases. Currently used image‐based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image‐based, and high‐processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air–liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18‐fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to