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

Abstract

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 <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.

Bei Mu Gua Lou San facilitates mucus expectoration by increasing surface area and hydration levels of airway mucus in an air-liquid-interface cell culture model of the respiratory epithelium

“…Next, we evaluated expression levels of MUC5AC and MUC5B including their ratio using RT-qPCR and our recently developed image-based analysis approach to assess protein levels (Groiss et al, 2022). BMGLS application significantly reduced mRNA expression of both MUC5AC and MUC5B (Figure 6D).…”

“…NHBE cells were isolated as previously described (Groiss et al, 2022). Briefly, the tracheal tissue was washed in sterile 0.9% NaCl / 3% penicillin-streptomycin before mechanically removing soft tissue or lung parenchyma and incubation in MEM (Gibco) supplemented with 1x MycoZAP (Lonza), 1x Antibiotic-Antimycotic (Anti-Anti, Gibco), 40 μg/mL ticarcillin (Merck), 500 μg/mL dithiothreitol and 10 μg/mL DNase (in PBS) for 4h at 4 °C followed by incubation in the aforementioned medium supplemented with collagenase solution (185 U/ml collagenase type II, 2 mg/ml BSA, 0.5 mM calcium chloride, 1 U amino acids 100x, 5% FCS in DMEM) and 10 μg/ml DNase (Gibco) over night at room temperature (RT).…”

“…Cell Profiler uses a number of consecutive modules to generate an algorithm known as pipeline to detect and analyze target areas. The pipelines for analysis of airway mucus and mucosal protein content were previously described in Groiss et al, 2021 (Groiss et al, 2022). The Cell-Profiler pipeline used within this study including template data sets are available at http://cellprofiler.org.…”

“…Therefore, the present study investigates the effects of BMGLS on goblet cell metaplasia (GCM) and goblet cell hyperplasia (GCH) on mucus production with special emphasis on the mucosal proteins MUC5AC and MUC5B in an air-liquid-interface (ALI) cell cultures system of Calu-3 and primary normal human bronchial epithelial cells (NHBE). We assessed cellular integrity and morphology including the differentiation state of key cellular components following three weeks of BMGLS application by immunostaining and electron microscopy, and evaluated the mucus quantity and composition on mRNA and protein level using our recently developed automated image-based analysis approach (Groiss et al, 2022). We further explored whether the potential changes in mucus quantity may attribute to regulation of key mucus-stimulating pathways.…”

Anti-Oxidative and Immune Regulatory Responses of THP-1 and PBMC to Pulsed EMF Are Field-Strength Dependent

“…In this study, we stimulated leukemic THP1 cells in comparison to peripheral blood mononuclear cells (PBMC) derived from healthy individuals with LPS for activation, and exposed them to an EMF equivalent of 16.7 Hz at different flux densities of <50 µT (termed weak EMF in this study; wkEMF), <250 µT (moderate EMF; mdEMF) and <4.8 mT (strong EMF; stEMF). We first assessed stEMF-induced ROS generation on a single cell level using our expertise on automated image analysis [32] and evaluated cellular NAD(P)H levels. Next, we screened for transcriptional anti-oxidative and immune regulatory responses of which key hits were probed in detail for field strength dependent activation, alongside typical key players in anti-oxidative defense and pro-and anti-inflammatory signaling.…”