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. 2021 Jun 22;22(13):6657.
doi: 10.3390/ijms22136657.

Chr15q25 Genetic Variant rs16969968 Alters Cell Differentiation in Respiratory Epithelia

Zania Diabasana  1 , Jeanne-Marie Perotin  1   2 , Randa Belgacemi  1 , Julien Ancel  1   2 , Pauline Mulette  1   2 , Claire Launois  2 , Gonzague Delepine  1   3 , Xavier Dubernard  4 , Jean-Claude Mérol  1   4 , Christophe Ruaux  5 , Philippe Gosset  6 , Uwe Maskos  7 , Myriam Polette  1   8 , Gaëtan Deslée  1   2 , Valérian Dormoy  1
Affiliations

Chr15q25 Genetic Variant rs16969968 Alters Cell Differentiation in Respiratory Epithelia

Zania Diabasana et al. Int J Mol Sci. .

Abstract

The gene cluster region, CHRNA3/CHRNA5/CHRNB4, encoding for nicotinic acetylcholine receptor (nAChR) subunits, contains several genetic variants linked to nicotine addiction and brain disorders. The CHRNA5 single-nucleotide polymorphism (SNP) rs16969968 is strongly associated with nicotine dependence and lung diseases. Using immunostaining studies on tissue sections and air-liquid interface airway epithelial cell cultures, in situ hybridisation, transcriptomic and cytokines detection, we analysed rs16969968 contribution to respiratory airway epithelial remodelling and modulation of inflammation. We provide cellular and molecular analyses which support the genetic association of this polymorphism with impaired ciliogenesis and the altered production of inflammatory mediators. This suggests its role in lung disease development.

Keywords: airway epithelial cells; differentiation; inflammation; remodelling; rs16969968.

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Conflict of interest statement

Ruaux reports grants and personal fees from Sanofi-Aventis outside the submitted work. Deslée reports personal fees from Nuvaira, personal fees from BTG/PneumRx, personal fees from Chiesi, personal fees from Boehringer, personal fees from Astra Zeneca, outside the submitted work. Dormoy reports personal fees from Chiesi outside the submitted work. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
SNP rs16969968 is associated with a decrease in multiciliated cells in bronchi epithelia. (A) Representative micrographs showing the bronchial epithelia of α5WT- and α5SNP-expressing patients stained for haematoxylin and eosin. Magnification corresponding to the selected area is shown. (B) Box and whisker plots (median with min to max, the plus sign (+) represents the mean value) represent the measurements of epithelium height (µm), and the percentage of non-remodelled and remodelled (basal cell hyperplasia, goblet cell hyperplasia and metaplasia) epithelium in α5WT (black) and α5SNP-expressing (red) groups (n ≥ 7 for each group). (C) Representative micrographs showing the bronchial epithelia of α5WT and α5SNP-expressing patients stained for ciliated cells (Arl13b and FOXJ1, red), intermediate cells (uteroglobin, red), proliferative cells (Ki67, red), basal cells (P63, green), mucin-secreting cells (Muc5ac and Muc5b, green), intermediate filaments (vimentin, green) and cell nuclei (DAPI, blue). Magnification corresponding to the selected area is shown. (D) Box and whisker plots (median with min to max, the plus sign (+) represents the mean value) report the proportion of FOXJ1- and P63-positive cells per mm, the proliferative index, and the mean grey pixel values of mucin- and uteroglobin-associated fluorescence in α5WT (black) and α5SNP-expressing cells (red) (n ≥3 for each group). ** p < 0.01 α5WT vs. α5SNP.
Figure 2
Figure 2
SNP rs16969968 alters respiratory airway epithelial cell differentiation. (A) Curves represent the TEER of AEC (n = 13) measured at ALI-1, ALI-7, ALI-14, ALI-21, ALI-28, and ALI-35 in α5WT (black) and α5SNP-expressing (red) cells. Means ± SEM are shown for each ALI time point. * p < 0.05; *** p < 0.001 α5WT vs. α5SNP. (B) Box and whisker plots (median with min to max, the plus sign (+) represents the mean value) represent the relative mRNA levels during ALI cultures by RT-qPCR (n = 13) for differentiation markers (CK5, non-differentiated cells; FOXJ1, ciliated cells; MUC5AC/MUC5B, mucous-secreting cells) in α5SNP-expressing groups. * p < 0.05; ** p < 0.01; *** p < 0.001 α5WT vs. α5SNP. (C) Examples of micrographs taken from AEC cultures at ALI-14 and ALI-35 showing multiciliated (Arl13b, red) and mucin-secreting (Muc5ac, green) cells on the left, basal (CK13, red) and mucin-secreting (Muc5b, green) cells on the right. Nuclei are stained in blue (DAPI). Magnification corresponding to the selected area is shown. (D) Box and whisker plots (median with min to max, the plus sign (+) represents the mean value) represent the mean grey pixel values of Arl13b-, CK13-, muc5ac- and muc5b-associated fluorescence at ALI-14 and ALI-35 in α5WT (black) and α5SNP-expressing cells (red) (n ≥ 3 for each group). * p < 0.05 α5WT vs. α5SNP.
Figure 3
Figure 3
Cytokine and chemokine expression in AEC culture supernatants. Heat map colours correspond to the α5SNP/α5WT cytokine and chemokine ratios at ALI-7 from apical (left) and basal (right) supernatants obtained from AEC cultures (n = 4 α5WT and n = 9 α5SNP). Downregulated (blue) and upregulated (orange) proteins in supernatants from α5SNP-expressing cells are represented. Non-detected proteins are represented in white. * p < 0.05; ** p < 0.01; *** p < 0.001 α5WT vs. α5SNP.
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