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

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.

Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

A non-olfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features

Adenosine receptors (ADORs) are G-protein coupled purinoceptors that have several functions including regulation of chloride secretion via CFTR in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic- and expression- analyses indicate that elasmobranch ADORs are non-olfactory, and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We co-expressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two electrode voltage clamping was performed and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR- co-injected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes (agonists: R-PIA > S-PIA > CGS21680 > CPA > 2ClADO > CV1808 = DPMA > NECA) and (antagonists: DPCPX > PD115199 > 8PT > CGC > CGS15943). Structures of human ADORs permitted a high-confidence homology model of the shark A0 core which revealed unique structural features of ancestral receptors. We conclude: (1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; (2) A0 likely activates CFTR in vivo and this receptor activates CFTR in oocytes indicating an evolutionary coupling between ADORs and chloride secretion; and (3) A0 appears to be a non-olfactory evolutionary ancestor of all four mammalian ADOR subtypes.

Clinical and molecular characterisation of the R751L-CFTR mutation

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterised. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harbouring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared to non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes together with folding and cell surface transport of R751L-CFTR were not different to wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had similar functional properties to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterisation of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and importantly inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.