Background: Hedgehog (HH) pathway is constantly under scrutiny in the context of organ development. Lung morphogenesis requires HH signalling which participates thereafter to the pulmonary homeostasis by regulating epithelial cell quiescence and repair. Since epithelial remodelling is a hallmark of Chronic Obstructive Pulmonary Disease (COPD), we investigated whether the main molecular actors of HH pathway participate to airway epithelial cell differentiation and we analysed their alterations in COPD patients. Methods: Sonic HH (Shh) secretion was assessed by ELISA in airway epithelial cell (AEC) air-liquid interface culture supernatants. HH pathway activation was evaluated by RT-qPCR, western blot and immunostaining. Inhibition of HH signalling was achieved upon Shh chelation during epithelial cell differentiation. HH pathway core components localization was investigated in lung tissues from non-COPD and COPD patients. Findings: We demonstrate that progenitors of AEC produced Shh responsible for the activation of HH signalling during the process of differentiation. Preventing the ligand-induced HH activation led to the establishment of a remodelled epithelium with increased number of basal cells and reduced ciliogenesis. Gli2 activating transcription factor was demonstrated as a key-element in the regulation of AEC differentiation. More importantly, Gli2 and Smo were lost in AEC from COPD patients. Interpretation: Our data suggest that HH pathway is crucial for airway epithelial cell differentiation and highlight its role in COPD-associated epithelial remodelling.
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels responsible for rapid neural and neuromuscular signal transmission. Although it is well documented that 16 subunits are encoded by the human genome, their presence in airway epithelial cells (AECs) remains poorly understood, and contribution to pathology is mainly discussed in the context of cancer. We analysed nAChR subunit expression in the human lungs of smokers and non-smokers using transcriptomic data for whole-lung tissues, isolated large AECs, and isolated small AECs. We identified differential expressions of nAChRs in terms of detection and repartition in the three modalities. Smoking-associated alterations were also unveiled. Then, we identified an nAChR transcriptomic print at the single-cell level. Finally, we reported the localizations of detectable nAChRs in bronchi and large bronchioles. Thus, we compiled the first complete atlas of pulmonary nAChR subunits to open new avenues to further unravel the involvement of these receptors in lung homeostasis and respiratory diseases.
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.
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