Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype
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Evaluation Summary:
This paper is of interest to scientists and clinicians within the field of muco-obstructive diseases in the airways, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). It identifies the sodium-bicarbonate cotransporter SLC4A4 as a key component of the mechanism by which normal airways prevent the formation of sticky mucus and defend theirselves against bacterial and viral infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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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 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.
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Evaluation Summary:
This paper is of interest to scientists and clinicians within the field of muco-obstructive diseases in the airways, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). It identifies the sodium-bicarbonate cotransporter SLC4A4 as a key component of the mechanism by which normal airways prevent the formation of sticky mucus and defend theirselves against bacterial and viral infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Saint-Criq and colleagues reports an extensive study on the role of basolateral Na+-bicarbonate cotransport in bicarbonate secretion and pH regulation of human and mouse airway epithelium. The apical components of this process (CFTR, TMEM16A, SLC26A) have been extensively studied in the past, but the basolateral components are not understood. The present study shows that both human and mouse airway epithelial cells express the Na+-bicarbonate cotransporter SLC4A4, and that the protein localizes to the basolateral membrane. They further demonstrate that SLC4A4 function is essential for intracellular pH homeostasis, transepithelial bicarbonate secretion, and maintanance of pH of the airway surface liquid. Finally, knocking out SLC4A4 in the mouse results in a lung phenotype that resembles …
Reviewer #1 (Public Review):
The manuscript by Saint-Criq and colleagues reports an extensive study on the role of basolateral Na+-bicarbonate cotransport in bicarbonate secretion and pH regulation of human and mouse airway epithelium. The apical components of this process (CFTR, TMEM16A, SLC26A) have been extensively studied in the past, but the basolateral components are not understood. The present study shows that both human and mouse airway epithelial cells express the Na+-bicarbonate cotransporter SLC4A4, and that the protein localizes to the basolateral membrane. They further demonstrate that SLC4A4 function is essential for intracellular pH homeostasis, transepithelial bicarbonate secretion, and maintanance of pH of the airway surface liquid. Finally, knocking out SLC4A4 in the mouse results in a lung phenotype that resembles cystic fibrosis.
The experimens are carefully performed, the data seem to support the conclusions, and the findings represent a significant advance in understanding pulmonary anion secretion in health and disease. I have a number of suggestions for clarifications in figures and figure legends, to facilitate understanding of the presented complex sets of experiments for the reader.
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Reviewer #2 (Public Review):
In this study a human in vitro airway cell model. i.e. primary hAECs expanded by conditional reprogramming, seeded on semi-permeable supports and differentiated under air-liquid interface conditions, resulting in enrichment of ciliated cells, and trachea from a systemic null-mouse for the electrogenic Na+-dependent bicarbonate (HCO3-) transporter/importer NBCe1 (Slc4A4) were used to evaluate the importance of NBCe1 in transepithelial HCO3- secretion and in alkalinization of airways surface liquid (ASL).
The results demonstrate unequivocally that the B-splice variant of NBCe1 is expressed at the basolateral membrane of ciliated cells in humans and in CCSP+ (Club) cells in mice, and that its absence in Slc4A4-KO mice results in ASL acidification and the induction of a muco-obstructive, CF-like phenotype in the …
Reviewer #2 (Public Review):
In this study a human in vitro airway cell model. i.e. primary hAECs expanded by conditional reprogramming, seeded on semi-permeable supports and differentiated under air-liquid interface conditions, resulting in enrichment of ciliated cells, and trachea from a systemic null-mouse for the electrogenic Na+-dependent bicarbonate (HCO3-) transporter/importer NBCe1 (Slc4A4) were used to evaluate the importance of NBCe1 in transepithelial HCO3- secretion and in alkalinization of airways surface liquid (ASL).
The results demonstrate unequivocally that the B-splice variant of NBCe1 is expressed at the basolateral membrane of ciliated cells in humans and in CCSP+ (Club) cells in mice, and that its absence in Slc4A4-KO mice results in ASL acidification and the induction of a muco-obstructive, CF-like phenotype in the airways. The identification of Slc4A4 as the main HCO3- importer in airway epithelial cells and the demonstration of its pivotal role in the maintenance of ASL pH and mucociliary clearance (MCC) are novel findings (albeit not entirely unexpected) that contribute to the notion that alkalinizing the ASL pH (e.g. with nebulized bicarbonate), aside restoring ASL volume, is an important strategy as a supportive treatment of cystic fibrosis (CF) and other muco-obstructive airway diseases.
The methodology used is sound and the conclusions of this paper are mostly well supported by data, but some aspects need further clarification and discussion.
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Reviewer #3 (Public Review):
Saint-Criq et al have made some very nice and extensive studies of basolateral membrane bicarbonate transport in airway epithelial cells using both human primary cell cultures and mouse tissue derived from a wild type and/or an SL4A4 knockout line. The author's work points to an important role for the electrogenic SLC4A4 transporter in bicarbonate movement for both human and mouse airway epithelium. Although there is this similarity between mouse and human epithelia, the cell types in which this transporter operates differ in the two species and thus, the role of the transporter also seems likely to vary. A significant strength of this paper is the number of different approaches the authors take to investigating the function of this transporter, including immunohistochemistry, mucocilliary transport …
Reviewer #3 (Public Review):
Saint-Criq et al have made some very nice and extensive studies of basolateral membrane bicarbonate transport in airway epithelial cells using both human primary cell cultures and mouse tissue derived from a wild type and/or an SL4A4 knockout line. The author's work points to an important role for the electrogenic SLC4A4 transporter in bicarbonate movement for both human and mouse airway epithelium. Although there is this similarity between mouse and human epithelia, the cell types in which this transporter operates differ in the two species and thus, the role of the transporter also seems likely to vary. A significant strength of this paper is the number of different approaches the authors take to investigating the function of this transporter, including immunohistochemistry, mucocilliary transport measurements, Ussing chamber recordings and airway surface liquid pH measurements. One of the key findings, that of a CF-like mucus phenotype in the mouse, bears directly on a current, much debated, topic about whether the CF mucus rheology arises from an effect of pH or from an effect on airway surface liquid hydration. In relation to this topic, future, more direct measurements of ASL volume and pH would further strengthen the author's case.
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