In vitro Methods for the Development and Analysis of Human Primary Airway Epithelia

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors

Background: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. Methods: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. Results: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl(−) currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl(−) currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. Conclusions: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl(−) currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors

Dynamic regulation of airway surface liquid pH by TMEM16A and SLC26A4 in cystic fibrosis nasal epithelia with rare mutations

In cystic fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel lead to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy. However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation. The calcium-activated chloride channel, TMEM16A, and the anion exchanger, SLC26A4, have been proposed as targets for restoring ASL pH, but current results are contradictory and often utilise nonphysiological conditions. To provide better evidence for a role of these two proteins in ASL pH homeostasis, we developed an efficient CRISPR-Cas9-based approach to knock-out (KO) relevant transporters in primary airway basal cells lacking CFTR and then measured dynamic changes in ASL pH under thin-film conditions in fully differentiated airway cultures, which better simulate the in vivo situation. Unexpectantly, we found that both proteins regulated steady-state as well as agonist-stimulated ASL pH, but only under inflammatory conditions. Furthermore, we identified two Food and Drug Administration (FDA)-approved drugs which raised ASL pH by activating SLC26A4. While we identified a role for SLC26A4 in fluid absorption, KO had no effect on cyclic adenosine monophosphate (cAMP)-stimulated fluid secretion in airway organoids. Overall, we have identified a role of TMEM16A in ASL pH homeostasis and shown that both TMEM16A and SLC26A4 could be important alternative targets for ASL pH therapy in CF, particularly for those people who do not produce any functional CFTR

The SLC26A9 inhibitor S9‐A13 provides no evidence for a role of SLC26A9 in airway chloride secretion but suggests a contribution to regulation of ASL pH and gastric proton secretion

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl− transport under basal conditions is still unclear, due to the lack of a specific inhibitor for SLC26A9. In the present study, we report a novel potent and specific inhibitor for SLC26A9, identified by screening of a drug-like molecule library and subsequent chemical modifications. The most potent compound S9-A13 inhibited SLC26A9 with an IC50 of 90.9 ± 13.4 nM. S9-A13 did not inhibit other members of the SLC26 family and had no effects on Cl− channels such as CFTR, TMEM16A, or VRAC. S9-A13 inhibited SLC26A9 Cl− currents in cells that lack expression of CFTR. It also inhibited proton secretion by HGT-1 human gastric cells. In contrast, S9-A13 had minimal effects on ion transport in human airway epithelia and mouse trachea, despite clear expression of SLC26A9 in the apical membrane of ciliated cells. In both tissues, basal and stimulated Cl− secretion was due to CFTR, while acidification of airway surface liquid by S9-A13 suggests a role of SLC26A9 for airway bicarbonate secretion

Data for 2023 PNAS publication on Airway Surface Liquid (ASL) pH in Cystic Fibrosis (CF) human airway cells

In Cystic Fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel leads to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy (HEMT). However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation. The calcium-activated chloride channel, TMEM16A, and the anion exchanger, SLC26A4, have been proposed as targets for restoring ASL pH, but current results are contradictory and often utilise non-physiological conditions. To provide better evidence for a role of these two proteins in ASL pH homeostasis we developed an efficient CRISPR-Cas9-based approach to knock-out (KO) relevant transporters in primary airway basal cells lacking CFTR, and then measured dynamic changes in ASL pH under thin-film conditions in fully differentiated 2D airway cultures, which better simulate the in vivo situation. Unexpectantly, we found that both proteins regulated steady-state as well as agonist-stimulated ASL pH, but only under inflammatory conditions. Furthermore, we identified two FDA-approved drugs which raised ASL pH by activating SLC26A4. While we identified a role for SLC26A4 in fluid absorption, KO had no effect on cAMP-stimulated fluid secretion in 3D airway organoids. Overall, we have identified a novel role of TMEM16A in ASL pH homeostasis and shown that both TMEM16A and SLC26A4 could be important alternative targets for ASL pH therapy in CF, particularly for those people who do not produce any functional CFTR.</p

Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air&ndash;liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications

Different reactivity of primary fibroblasts and endothelial cells towards crystalline silica: A surface radical matter.

none8Quartz is a well-known occupational fibrogenic agent able to cause fibrosis and other severe pulmonary diseases such as silicosis and lung cancer. The silicotic pathology owes its severity to the structural and chemo-physical properties of the particles such as shape, size and abundance of surface radicals. In earlier studies, we reported that significant amounts of surface radicals can be generated on crystalline silica by chemical aggression with ascorbic acid (AA), a vitamin naturally abundant in the lung surfactant, and this reaction led to enhanced cytotoxicity and production of inflammatory mediators in a macrophage cell line. However in the lung, other cells acting in the development of silicosis, like fibroblasts and endothelial cells, can come to direct contact with inhaled quartz. We investigated the cytotoxic/pro-inflammatory effects of AA-treated quartz microcrystals (QA) in human primary fibroblasts and endothelial cells as compared to unmodified microcrystals (Q). Our results show that, in fibroblasts, the abundance of surface radicals on quartz microcrystals (Q vs QA) significantly enhanced cell proliferation (with or without co-culture with macrophages), reactive oxygen species (ROS) production, NF-κB nuclear translocation, smooth muscle actin, fibronectin, Bcl-2 and tissue inhibitor of metalloproteinase-1 expression and collagen production. Contrariwise, endothelial cells reacted to the presence of quartz microcrystals independently from the abundance of surface radicals showing similar levels of cytotoxicity, ROS production, cell migration, MCP-1, ICAM-I and fibronectin gene expression when challenged with Q or QA. In conclusion, our in vitro experimental model demonstrates an important and quite unexplored direct contribute of silica surface radicals to fibroblast proliferation and fibrogenic responses.Pozzolini, Marina; Vergani, Laura; Ragazzoni, Milena; Delpiano, Livia; Grasselli, Elena; Voci, Adriana; Giovine, Marco; Scarfì, SoniaPozzolini, Marina; Vergani, Laura; Ragazzoni, Milena; Delpiano, Livia; Grasselli, Elena; Voci, Adriana; Giovine, Marco; Scarfi', Soni

Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells

Esomeprazole increases airway surface liquid pH in primary cystic fibrosis epithelial cell

Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype

International audienceBicarbonate 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