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 (∆I_scF/I+V) experiments. CFTR expression was evaluated by apical membrane fluorescence semi-quantification. I_sc 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. ∆I_scF/I+V correlated with CFTR cellular apical expression and NPD measurements. The CFTR correctors lumacaftor and tezacaftor significantly increased the ∆I_scF/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.

Airway disease in cystic fibrosis (CF) is characterised by impaired mucociliary clearance, persistent bacterial infection and neutrophilic inflammation. Lipoxin A₄(LXA₄) initiates the active resolution of inflammation and promotes airway surface hydration in CF models. 15-Lipoxygenase (LO) plays a central role in the “class switch” of eicosanoid mediator biosynthesis from leukotrienes to lipoxins, initiating the active resolution of inflammation. We hypothesised that defective eicosanoid mediator class switching contributes to the failure to resolve inflammation in CF lung disease.Using bronchoalveolar lavage (BAL) samples from 46 children with CF and 19 paediatric controls we demonstrate that the ratio of LXA₄to leukotriene B₄(LTB₄) is depressed in CF BAL (p<0.01), even in the absence of infection (p<0.001).Furthermore, 15-LO2 transcripts were significantly less abundant in CF BAL samples (p<0.05). In control BAL, there were positive relationships between 15-LO2 transcript abundance and LXA₄/LTB₄ratio (p=0.01, r=0.66) and with percentage macrophage composition of the BAL fluid (p<0.001, r=0.82), which were absent in CF.Impoverished 15-LO2 expression and depression of the LXA₄/LTB₄ratio are observed in paediatric CF BAL. These observations provide mechanistic insights into the failure to resolve inflammation in the CF lung.

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways.

Lipoxin A₄(LXA₄) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, LXA₄production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of LXA₄on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to LXA₄(100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. LXA₄(100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 (LXA₄receptor antagonist). LXA₄also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that LXA₄plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the LXA₄receptor.

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl⁻ secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA₄ is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA₄ are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA₄ produced a rapid and transient increase in intracellular Ca²⁺. We have investigated, the effect of LXA₄ on Cl⁻ secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA₄ stimulated a rapid intracellular Ca²⁺ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA₄ stimulated whole-cell Cl⁻ currents which were inhibited by NPPB (calcium-activated Cl⁻ channel inhibitor), BAPTA-AM (chelator of intracellular Ca²⁺) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA₄ increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA₄ effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl⁻ secretion. The LXA₄ stimulation of intracellular Ca²⁺, whole-cell Cl⁻ currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA₄ in the stimulation of intracellular Ca²⁺ signalling leading to Ca²⁺-activated Cl⁻ secretion and enhanced ASL height in non-CF and CF bronchial epithelia.