ABSTRACT Bicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that it’s pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4 -null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function.
ABSTRACT The respiratory tract possesses a highly regulated innate defense system which includes efficient cilia-mediated mucus transport or mucociliary clearance (MCC). This essential process relies on appropriate hydration of airway surfaces which is controlled by a blend of transepithelial sodium and liquid absorption via the epithelial sodium channel (ENaC), and anion and liquid secretion, primarily regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. MCC is tightly regulated by second messenger signalling pathways. Succinate is derived from parasites, microorganisms and inflammatory cells, and its concentration increases in the airway surface liquid (ASL) during infections. Increases in ASL succinate activates the G-protein coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Here, we tested the hypothesis that succinate signalling was linked to CFTR activity, ASL hydration and increased MCC. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport and induced bronchoconstriction in mouse airways. In parallel, stimulation of human bronchial epithelial cells (HBEC) with succinate activated anion secretion and increased ASL height. All functions activated by succinate/SUCNR1 were impeded when working with tissues and cells isolated from animal models or individuals affected cystic fibrosis (CF) or when CFTR was inhibited. Moreover, when HBECs derived from ΔF508 individuals were incubated with the triple drug combination of elexacaftor/tezacaftor/ivacaftor (ETI), succinate-induced anion secretion was restored, confirming the tight relationship between SUCNR1 signalling and CFTR function. Our results identify a novel activation pathway for CFTR that participates in the defence response of the airways, which is defective in CF. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways.
Bicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that it’s pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells cultures, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4 -null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function.
Ionocytes are a new type of rare airway epithelial cells expressing Ascl3 and Foxi1 transcription factors, as well as 50% of Cftr-transcripts in mouse airways. However, their role and precise location remain mostly unexplored. This research aimed to shed light on ionocyte ontogeny and distribution within the mouse airway epithelium. Mice were bred in the C57Bl6/J background and maintained in the Specific Pathogen Free mouse facility of Centro de Estudios Científicos (CECs) with access to food and water ad libitum. We used 1,7, 10, 14,21 and 28-days-old wild-type mice, 6 and 8-week-old, and 1 year wild-type and 14-days-old 6 and 8-week-old Cftr ΔF508/ΔF508 animals. The tracheas were sliced in frontal sections, and the ionocytes were identified by immunofluorescence against the FOXI1 transcription factor. All values were expressed as mean ± S.E.M. All animal procedures were approved by the institutional IACUC (CECS-2022-03). We found that FOXI1+cells had a triangular shape with a basolateral process. Unexpectedly, we observed that FOXI1+cells appear postnatally close to the submucosal glands (SMG), and its number increases drastically between 6 and 8-week-old wild type mice. The quantity of FOXI1+cells cells in the adult trachea decreased towards the distal part (proximal=11.2±1.2 vs distal=4.0±0.9 FOXI1+cells/mm basal lamina, n=7, P=<0,001, t-test), and we did not find significative differences between WT and Cftr ΔF508/ΔF508 adult mouse. FOXI1+cells were often observed in the epithelia around the collecting duct exit and in the collecting duct epithelium of the SMG. In conclusion, our study provides new insights into the localization and distribution of ionocytes in the mouse airway epithelium. Our results indicate that ionocytes may play a role in regulating mucus composition in upper airways. We suggest that age-dependent changes in cell quantity might reflect the need of increased CFTR function in adult stages. Further research is needed to fully understand the function of ionocytes in the airway and their potential role in respiratory diseases such as cystic fibrosis. Proyecto Postdoctoral 3220672 Proyecto FONDECYT 1221257. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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