Clinical studies with modulators of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein have demonstrated that functional restoration of the mutated CFTR can lead to substantial clinical benefit. However, studies have shown highly variable patient responses. The objective of this study was to determine a biomarker predictive of the clinical response. CFTR function was assessed in vivo via nasal potential difference (NPD) and in human nasal epithelial (HNE) cultures by the response to Forskolin/IBMX and the CFTR potentiator VX-770 in short-circuit-current (∆IscF/I+V) experiments. CFTR expression was evaluated by apical membrane fluorescence semi-quantification. Isc measurements discriminated CFTR function between controls, healthy heterozygotes, patients homozygous for the severe F508del mutation and patients with genotypes leading to absent or residual function. ∆IscF/I+V correlated with CFTR cellular apical expression and NPD measurements. The CFTR correctors lumacaftor and tezacaftor significantly increased the ∆IscF/I+V response to about 25% (SEM = 4.4) of the WT-CFTR level and the CFTR apical expression to about 22% (SEM = 4.6) of the WT-CFTR level in F508del/F508del HNE cells. The level of CFTR correction in HNE cultures significantly correlated with the FEV1 change at 6 months in 8 patients treated with CFTR modulators. We provide the first evidence that correction of CFTR function in HNE cell cultures can predict respiratory improvement by CFTR modulators.

Available drugs are unable to effectively rescue the folding defects in vitro and ameliorate the clinical-phenotype of cystic fibrosis (CF), caused by deletion of F508 (ΔF508 or F508del) and some point mutations in the CF transmembrane conductance regulator (CFTR), a plasma membrane (PM) anion channel. To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutants expression and function at the PM. High throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at the nucleotide binding domain (NBD1), NBD2 and their membrane spanning domains (MSDs) interfaces. While individually these compounds marginally improve ΔF508-CFTR folding efficiency, function, and stability, their combinations lead to ~50–100% of wild type-level correction in immortalized and primary human airway epithelia, and in mouse nasal epithelia. Likewise, corrector combinations were effective for rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

Renal ammonium (NH 3 ؉ NH 4 ؉ ) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow NH 4 ؉ transmembrane translocation without high specificity for NH 4 ؉ , but it is still debated whether NH 3 , and more generally, gas, may be transported by transmembrane proteins.

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

The ubiquitous ClC-2 Cl(-) channel is thought to contribute to epithelial Cl(-) secretion, but the distribution of the ClC-2 protein in human epithelia has not been investigated. We have studied the distribution of ClC-2 in adult human and rat intestine and airways by immunoblotting and confocal microscopy. In the rat, ClC-2 was present in the lateral membranes of villus enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells. We found significant expression of ClC-2 at the apex of ciliated cells in both rat and human airways. These results show that the distribution of ClC-2 in airways is consistent with participation of ClC-2 channels in Cl(-) secretion and indicate that extrapolation of results from studies of ClC-2 function in rat intestine to human intestine is not straightforward.

An improved understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein structure and the consequences of CFTR gene mutations have allowed the development of novel therapies targeting specific defects underlying CF. Some strategies are mutation specific and have already reached clinical development; some strategies include a read-through of the specific premature termination codons (read-through therapies, nonsense mediated decay pathway inhibitors for Class I mutations); correction of CFTR folding and trafficking to the apical plasma membrane (correctors for Class II mutations); and an increase in the function of CFTR channel (potentiators therapy for Class III mutations and any mutant with a residual function located at the membrane). Other therapies that are in preclinical development are not mutation specific and include gene therapy to edit the genome and stem cell therapy to repair the airway tissue. These strategies that are directed at the basic CF defects are now revolutionizing the treatment for patients and should positively impact their survival rates.

Background Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity.ObjectiveWe evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo.MethodsWild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models.ResultsWe showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways.ConclusionsOur data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.

1. Hyperpolarization‐activated Cl‐ currents (ICl,hyp) were investigated in the T84 human adenocarcinoma cell line, using the patch‐clamp whole‐cell configuration. 2. During whole‐cell recording with high‐chloride and ATP‐containing internal solutions, hyperpolarizing jumps from a holding potential of 0 mV elicited slow inward current relaxations, carried by Cl‐ and detected at membrane potentials more negative than ‐40 mV. Analysis of the relative permeabilities to monovalent anions gave the following sequence: Cl‐ > Br‐ > I‐ > glutamate. 3. ICl,hyp was partially inhibited by 1 mM diphenylamine‐2‐carboxylic acid or 0.1 mM 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate, and was completely blocked by Cd2+ (> 300 microM). It was insensitive to 1 mM external 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid or 1 mM Ba2+. 4. ICl,hyp was inhibited by external application of 500 microM cptcAMP (8‐(4‐chlorophenylthio)‐adenosine 3':5'‐cyclic monophosphate) or 500 nM of the protein kinase C activator, phorbol 12‐myristate, 13‐acetate. 5. (i) Omission of ATP from the pipette solution, (ii) ATP replacement by the non‐hydrolysable ATP analogue 5'‐adenylylimidodiphosphate, and (iii) inhibition of protein kinase C by staurosporine or calphostin C accelerated the activation kinetics of the current and increased its amplitude, but did not alter its pharmacological properties. 6. We conclude that hyperpolarization‐activated Cl‐ channels similar to those of ClC‐2 channels (mammalian homologue of Torpedo chloride channel ClC‐0) are present in T84 cells, and that their gating properties are modulated by phosphorylation.