The Nicotinic Receptor Polymorphism rs16969968 Is Associated with Airway Remodeling and Inflammatory Dysregulation in COPD Patients

Abstract

Genome-wide association studies unveiled the associations between the single nucleotide polymorphism rs16969968 of CHRNA5, encoding the nicotinic acetylcholine receptor alpha5 subunit (α5SNP), and nicotine addiction, cancer, and COPD independently. Here, we investigated α5SNP-induced epithelial remodeling and inflammatory response in human COPD airways. We included 26 α5SNP COPD patients and 18 wild-type α5 COPD patients in a multi-modal study. A comparative histologic analysis was performed on formalin-fixed paraffin-embedded lung tissues. Isolated airway epithelial cells from bronchial brushings were cultivated in the air-liquid interface. Broncho-alveolar fluids were collected to detect inflammatory mediators. Ciliogenesis was altered in α5SNP COPD bronchial and bronchiolar epithelia. Goblet cell hyperplasia was exacerbated in α5SNP small airways. The broncho-alveolar fluids of α5SNP COPD patients exhibited an increase in inflammatory mediators. The involvement of the rs16969968 polymorphism in airway epithelial remodeling and related inflammatory response in COPD prompts the development of innovative personalized diagnostic and therapeutic strategies.

Keywords: COPD; airways; epithelial remodeling; inflammation; nicotinic receptors; rs16969968.

Figure 1

Bronchial epithelial remodeling in rs16969968 (α5SNP) COPD patients. (A): Hematoxylin and eosin staining showing the epithelial height of α5SNP and α5WT COPD patients. (B): Examples of the microscopic acquisition of immunofluorescent stainings for basal cells (P63, red), ciliated cells (Arl13b, red), proliferative cells (Ki67, green), mucins secretory cells (Muc5ac, green; Muc5b, red), and intermediate cells (Uteroglobin, red). Nuclei are stained in blue (DAPI). Magnification corresponding to the selected area is represented. (C): Dot plots (means with SEM) representing measurements of the epithelial height, the number of basal, proliferative, and primary ciliated cells per mm, motile cilia recovery (%), and the mean grey values of mucins (Muc5ac, Muc5b) and uteroglobin-associated fluorescence of α5SNP and α5WT COPD patients. **, p <0.01 α5WT vs. α5SNP; ns, non-significant.

Figure 2

Bronchiolar epithelial remodeling in rs16969968 (α5SNP) COPD patients. (A): Hematoxylin and eosin staining showing the epithelial height of α5SNP and α5WT COPD patients. (B): Examples of the microscopic acquisition of immunofluorescent stainings for basal cells (P63, red), ciliated cells (Arl13b, red), proliferative cells (Ki67, green), mucins secretory cells (Muc5ac, green; Muc5b, red), and intermediate cells (Uteroglobin, red). Nuclei are stained in blue (DAPI). Magnification corresponding to the selected area is represented. (C): Dot plots (means with SEM) representing measurements of the epithelial height, the number of basal, proliferative, and primary ciliated cells per mm, motile cilia recovery (%), and the mean grey values of mucins (Muc5ac, Muc5b) and uteroglobin-associated fluorescence of α5SNP and α5WT COPD patients. *, p < 0.05 α5WT vs. α5SNP; ns, non-significant.

Figure 3

Lung inflammatory response in rs16969968 (α5SNP) COPD patients. (A): Microscopic acquisitions showing peribronchial recruitment of immune populations in α5SNP and α5WT COPD patients. (B): Dot plot showing the number of eosinophils, basophils, neutrophils, and lymphocytes per mm of epithelium in α5SNP vs. α5WT COPD patients. (C): Heatmap presenting the ratios of inflammatory mediators’ expression in broncho-alveolar lavage fluids of α5SNP vs. α5WT COPD patients. Downregulated inflammatory mediators are presented in blue, and upregulated ones are in red. The inflammatory mediators whose expression is lower than the detection cut-off value (5% of positive control) are identified in white. The inflammatory mediators are categorized according to their detected abundance in the broncho-alveolar lavage fluids of COPD patients (from very high, >50% of the detection of the positive control; to very low, <5% of the detection of the positive control). ns, non-significant.