Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus.

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

Airway Gland Structure and Function.

Submucosal glands contribute to airway surface liquid (ASL), a film that protects all airway surfaces. Glandular mucus comprises electrolytes, water, the gel-forming mucin MUC5B, and hundreds of different proteins with diverse protective functions. Gland volume per unit area of mucosal surface correlates positively with impaction rate of inhaled particles. In human main bronchi, the volume of the glands is ∼ 50 times that of surface goblet cells, but the glands diminish in size and frequency distally. ASL and its trapped particles are removed from the airways by mucociliary transport. Airway glands have a tubuloacinar structure, with a single terminal duct, a nonciliated collecting duct, then branching secretory tubules lined with mucous cells and ending in serous acini. They allow for a massive increase in numbers of mucus-producing cells without replacing surface ciliated cells. Active secretion of Cl(-) and HCO3 (-) by serous cells produces most of the fluid of gland secretions. Glands are densely innervated by tonically active, mutually excitatory airway intrinsic neurons. Most gland mucus is secreted constitutively in vivo, with large, transient increases produced by emergency reflex drive from the vagus. Elevations of [cAMP]i and [Ca(2+)]i coordinate electrolyte and macromolecular secretion and probably occur together for baseline activity in vivo, with cholinergic elevation of [Ca(2+)]i being mainly responsive for transient increases in secretion. Altered submucosal gland function contributes to the pathology of all obstructive diseases, but is an early stage of pathogenesis only in cystic fibrosis.

Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin.

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance.

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea.

In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends on gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho HJ, Joo NS, Wine JJ. Am J Physiol Lung Cell Mol Physiol 299: L124-L136, 2010). Stimulated MCC velocities (mm/min, means ± SE for 10- to 35-min period poststimulation) were as follows: 1 µM carbachol: 19.1 ± 3.3 > 10 µM phenylephrine: 15.3 ± 2.4 ≈ 10 µM isoproterenol: 15.0 ± 1.9 ≈ 10 µM forskolin: 14.6 ± 3.1 > 1 µM vasoactive intestinal peptide (VIP): 10.2 ± 2.2 >> basal (t15): 1.8 ± 0.3; n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70% and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3 (-)-free solutions did not significantly inhibit MCC to either intracellular Ca(2+) concentration or intracellular cAMP concentration ([cAMP]i)-elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion, and bumetanide almost completely inhibited [cAMP]i-stimulated MCC while it had a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this.

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC(50), 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.

How the sweat gland reveals levels of CFTR activity.

CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·PO·Î³) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the simple and widely used sweat chloride test can be calibrated to provide more accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects (hereafter 'CFTR activity'). Sweating stimulated by ß-adrenergic agonists (ß-sweat) is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity with carriers = 50% and CF = 0% of HC values set = 100%, but the ß-sweat assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting mean sweat chloride values, taken from numerous studies and the CFTR2 database against mean ß-sweat rates for CF, carriers and HC. The resulting inverse logarithmic relations indicate that sweat chloride values ≥60 mmol/L occur when CFTR activity is below 1.2% -10% of HC. These are lower than most previous estimates, which resulted from setting nasal potential difference (NPD) as linear rather than logarithmic measures of CFTR activity. Features of the sweat gland coil and duct are used to explain why readouts of CFTR activity are linear for ß-sweat and logarithmic for sweat chloride. Sweat chloride values fall steeply for small increments of CFTR activity above zero-the most clinically relevant region. Thus, large health benefits can be achieved by restoring low levels of CFTR activity, especially if this is done before irreversible lung damage. Truncated Abstract: CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·PO·Î³) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the sweat chloride test can be calibrated to provide accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects. Sweating stimulated by ß-adrenergic agonists is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity, but the assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting it against mean ß-sweat rates for different groups. The resulting logarithmic relations indicate that CF sweat chloride values occur when CFTR activity is below 1.2% -10% of HC, and that large health benefits can be achieved by restoring low levels of CFTR activity if this is done early.

Variation in CFTR-dependent 'ß-sweating' among healthy adults.

The genetic disease cystic fibrosis (CF) results when mutations in the gene for the anion channel CFTR reduce CFTR's activity below a critical level. CFTR activity = N·PO·Î³ (number of channels x open probability x channel conductance). Small molecules are now available that partially restore CFTR function with dramatic improvements in health of CF subjects. Continued evaluation of these and other compounds in development will be aided by accurate assessments of CFTR function. However, measuring CFTR activity in vivo is challenging and estimates vary widely. The most accurate known measure of CFTR activity in vivo is the 'ß/M' ratio of sweat rates, which is produced by stimulation with a ß-adrenergic agonist cocktail referenced to the same individual's methacholine-stimulated sweat rate. The most meaningful metric of CFTR activity is to express it as a percent of normal function, so it is critical to establish ß/M carefully in a population of healthy control subjects. Here, we analyze ß/M from a sample of 50 healthy adults in which sweat rates to cholinergic and ß-adrenergic agonists were measured repeatedly (3 times) in multiple, (~50) identified sweat glands from each individual (giving ~20,000 measurements). The results show an approximately 7-fold range, 26-187% of the WT average set to 100%. These provide a benchmark against which other measures of CFTR activity can be compared. Factors contributing to ß/M variation in healthy controls are discussed.

Properties of substance P-stimulated mucus secretion from porcine tracheal submucosal glands.

Human and pig airway submucosal glands secrete mucus in response to substance P (SubP), but in pig tracheal glands the response to SubP is >10-fold greater than in humans and shares features with cholinergically produced secretion. CFTR-deficient pigs provide a model for human cystic fibrosis (CF), and in newborn CF pigs the response of tracheal glands to SubP is significantly reduced (Joo et al. J Clin Invest 120: 3161-3166, 2010). To further define features of SubP-mediated gland secretion, we optically measured secretion rates from individual adult porcine glands in isolated tracheal tissues in response to mucosal capsaicin and serosal SubP. Mucosal capsaicin (EC(50) = 19 µM) stimulated low rates of secretion that were partially inhibited by tetrodotoxin and by inhibitors for muscarinic, VIP, and SubP receptors, suggesting reflex stimulation of secretion by multiple transmitters. Secretion in response to mucosal capsaicin was inhibited by CFTR(inh)-172, but not by niflumic acid. Serosal SubP (EC(50) = 230 nM) stimulated 10-fold more secretion than mucosal capsaicin, with a V(max) similar to that of carbachol. Secretion rates peaked within 5 min and then declined to a lower sustained rate. SubP-stimulated secretion was inhibited 75% by bumetanide, 53% by removal of HCO(3)(-), and 85% by bumetanide + removal of HCO(3)(-); it was not inhibited by atropine but was inhibited by niflumic acid, clotrimazole, BAPTA-AM, nominally Ca(2+)-free bath solution, and the adenylate cyclase inhibitor MDL-12330A. Ratiometric measurements of fura 2 fluorescence in dissociated gland cells showed that SubP and carbachol increased intracellular Ca(2+) concentration by similar amounts. SubP produced rapid volume loss by serous and mucous cells, expansion of gland lumina, mucus flow, and exocytosis but little or no contraction of myoepithelial cells. These and prior results suggest that SubP stimulates pig gland secretion via CFTR- and Ca(2+)-activated Cl(-) channels.

Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model.

Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a ß adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases ~ 55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30 min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.

Mucus secretion from individual submucosal glands of the ferret trachea.

Mucus secretion from individual tracheal glands in adult ferrets was studied with time-lapse optical imaging of mucus droplets under an oil layer. Density of functional glands (determined by responses to 1 muM carbachol) was 1.5 +/- 0.3 per mm(2) (n = 6). Secretion rates (in pl.min(-1).gland(-1)) were as follows: 4.1 +/- 0.7 basal (unstimulated; n = 27, 669 glands), 338 +/- 70 to 10 microM forskolin (n = 8, 90 glands), 234 +/- 13 to 1 microM VIP (n = 6, 57 glands), 183 +/- 92 to 10 microM isoproterenol (n = 3, 33 glands), 978 +/- 145 to 1 microM carbachol (n = 11, 131 glands), and 1,348 +/- 325 to 10 muM phenylephrine (n = 7, 74 glands). The potency (EC(50), in microM) and efficacy (V(max), in pl x min(-1) x gland(-1)) were 7.6 (EC(50)) and 338 +/- 16 (V(max)) to forskolin, 1.0 (EC(50)) and 479 +/- 19 (V(max)) to VIP, 0.6 (EC(50)) and 1,817 +/- 268 (V(max)) to carbachol, and 3.7 (EC(50)) and 1,801 +/- 95 (V(max)) to phenylephrine. Although carbachol and phenylephrine were equally effective secretagogues, only carbachol caused contractions of the trachealis muscle. Synergy was demonstrated between 300 nM isoproterenol and 100 nM carbachol, which, when combined, produced a secretion rate almost fourfold greater than predicted from their additive effect. The dependence of fluid secretion on Cl(-) and HCO(3)(-) varied depending on the mode of stimulation. Secretion stimulated by VIP or forskolin was reduced by approximately 60% by blocking either anion, while carbachol-stimulated secretion was blocked 68% by bumetanide and only 32% by HEPES replacement of HCO(3)(-). These results provide parametric data for comparison with fluid secretion from glands in ferrets lacking CFTR.

Synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis.

Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl(-) and HCO(3) (-), and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.

High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia.

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

Ivacaftor restores CFTR-dependent sweat gland fluid secretion in cystic fibrosis subjects with S945L alleles.

BACKGROUND: To determine in vivo effects of CFTR modulators on mutation S945L. METHODS: We measured effects of CFTR modulators on CFTR-dependent sweating ('C-sweat') in two pancreatic sufficient cystic fibrosis (CF) subjects. S1 (S945L/G542X) took ivacaftor and S2 (S945L/F508del) took ivacaftor+tezacaftor. Sweating was stimulated pharmacologically to produce sequentially both CFTR-independent (methacholine stimulated) M-sweat and C-sweat; and the ratio of these was compared. Sweat secretion was measured with two methods: real time secretory rate quantitative recording and by optically measuring the growth of sweat bubbles under oil from multiple identified glands. RESULTS: Using the quantitative recorder, we saw zero C-sweat secretion off-drug, but when on-drug the C-sweat responses for both subjects were comparable to those seen in carriers. The on-drug response was further quantified using the sweat bubble method. Each subject again showed robust C-sweat responses, with C-sweat/M-sweat ratios~half of the ratio determined for a cohort of 40 controls tested under identical conditions. CONCLUSION: These in vivo results, consistent with prior in vitro findings, indicate that the drug treatments restore near-normal function to S945L-CFTR, and support the use of ivacaftor as a treatment for CF patients who carry this allele.

Progress in understanding mucus abnormalities in cystic fibrosis airways.

Normal airways below the carina maintain an essentially sterile environment via a multi-pronged innate defence system that includes mucus clearance via mucociliary clearance and cough, multiple antimicrobials and cellular components including macrophages and neutrophils. In cystic fibrosis (CF), loss of CFTR function compromises these defences, and with present standard of care virtually all people with CF eventually develop mucus accumulation, plugging and chronic infections. This review focuses on how mucus is affected by CFTR loss.

Sweat rate analysis of ivacaftor potentiation of CFTR in non-CF adults.

To determine if ivacaftor (Kalydeco) influences non-CF human CFTR function in vivo, we measured CFTR-dependent (C-sweat) and CFTR-independent (M-sweat) rates from multiple identified sweat glands in 8 non-CF adults. The two types of sweating were stimulated sequentially with intradermal injections of appropriate reagents; each gland served as its own control via alternating off-on drug tests on both arms, given at weekly intervals with 3 off and 3 on tests per subject. We compared drug effects on C-sweating stimulated by either high or low concentrations of ß-adrenergic cocktail, and on methacholine-stimulated M-sweating. For each subject we measured ~700 sweat volumes from ~75 glands per arm (maximum 12 readings per gland), and sweat volumes were log-transformed for statistical analysis. T-tests derived from linear mixed models (LMMs) were more conservative than the familiar paired sample t-tests, and show that ivacaftor significantly increased C-sweating stimulated by both levels of agonist, with a larger effect in the low cocktail condition; ivacaftor did not increase M-sweat. Concurrent sweat chloride tests detected no effect of ivacaftor. We conclude that ivacaftor in vivo increases the open channel probability (PO) of WT CFTR, provided it is not already maximally stimulated.

Parasympathetic control of airway submucosal glands: central reflexes and the airway intrinsic nervous system.

Airway submucosal glands produce the mucus that lines the upper airways to protect them against insults. This review summarizes evidence for two forms of gland secretion, and hypothesizes that each is mediated by different but partially overlapping neural pathways. Airway innate defense comprises low level gland secretion, mucociliary clearance and surveillance by airway-resident phagocytes to keep the airways sterile in spite of nearly continuous inhalation of low levels of pathogens. Gland secretion serving innate defense is hypothesized to be under the control of intrinsic (peripheral) airway neurons and local reflexes, and these may depend disproportionately on non-cholinergic mechanisms, with most secretion being produced by VIP and tachykinins. In the genetic disease cystic fibrosis, airway glands no longer secrete in response to VIP alone and fail to show the synergy between VIP, tachykinins and ACh that is observed in normal glands. The consequent crippling of the submucosal gland contribution to innate defense may be one reason that cystic fibrosis airways are infected by mucus-resident bacteria and fungi that are routinely cleared from normal airways. By contrast, the acute (emergency) airway defense reflex is centrally mediated by vagal pathways, is primarily cholinergic, and stimulates copious volumes of gland mucus in response to acute, intense challenges to the airways, such as those produced by very vigorous exercise or aspiration of foreign material. In cystic fibrosis, the acute airway defense reflex can still stimulate the glands to secrete large amounts of mucus, although its properties are altered. Importantly, treatments that recruit components of the acute reflex, such as inhalation of hypertonic saline, are beneficial in treating cystic fibrosis airway disease. The situation for recipients of lung transplants is the reverse; transplanted airways retain the airway intrinsic nervous system but lose centrally mediated reflexes. The consequences of this for gland secretion and airway defense are poorly understood, but it is possible that interventions to modify submucosal gland secretion in transplanted lungs might have therapeutic consequences.