Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells.

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl-/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A randomised trial of oral prednisone for cystic fibrosis pulmonary exacerbation treatment.

BACKGROUND: Elevated markers of systemic and pulmonary inflammation are associated with failure to recover lung function following pulmonary exacerbations in people with cystic fibrosis (pwCF). Our aim was to determine whether adjuvant oral prednisone treatment would improve recovery of forced expiratory volume in 1 s (FEV1) % pred in CF pulmonary exacerbations not responding to antibiotic therapy. METHODS: This was a randomised, double-blind, placebo-controlled trial in pwCF treated with intravenous antibiotics for a pulmonary exacerbation. At day 7, those who had not returned to >90% baseline FEV1 % pred were randomised to adjuvant prednisone 1 mg·kg-1 twice daily (maximum 60 mg·day-1) or placebo for 7 days. The primary outcome was the difference in proportion of subjects who recovered >90% baseline FEV1 % pred at day 14 of i.v. antibiotic therapy. RESULTS: 173 subjects were enrolled, with 76 randomised. 50% of subjects in the prednisone group recovered baseline FEV1 on day 14 compared with 39% of subjects in the placebo group (difference of 11%, 95% CI -11-34%; p=0.34). The mean±sd change in FEV1 % pred from day 7 to day 14 was 6.8±8.8% predicted in the prednisone group and 4.6±6.9% predicted in the placebo group (mean difference 2.2% predicted, 95% CI -1.5-5.9%; p=0.24). Time to subsequent exacerbation was not prolonged in prednisone-treated subjects (hazard ratio 0.83, 95% CI 0.45-1.53; p=0.54). CONCLUSIONS: This study failed to detect a difference in FEV1 % pred recovery between adjuvant oral prednisone and placebo treatment in pwCF not responding at day 7 of i.v. antibiotic therapy for pulmonary exacerbations.

Airway submucosal glands from cystic fibrosis swine suffer from abnormal ion transport across the serous acini, collecting duct, and ciliated duct.

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.

A Mixed Methods Assessment of Home-Based Video Pretransplant Lung Education.

Introduction: Patients awaiting lung transplantation must learn new information to successfully navigate the transplant process. A supplemental video series was piloted to patients at home during the Covid-19 pandemic to improve pre-transplant education. Methods: A mixed methods study was undertaken to assess patient experiences with this method of education, confirm the ideal timing of the education, and identify gaps that require further attention. Semi-structured interviews were conducted with 17 one-on-one or dyadic (patients and caregivers) who viewed the video series at home. A third-party researcher (not involved in creation of the educational materials) conducted the interviews by phone, which were audio recorded and then transcribed verbatim. NVivo 12 Pro for Windows software was used to code the data and identify emerging themes. Results: Participants indicated that home-based videos were applicable, and informative and helpful (4.7 on 5-point Likert scale) and appreciated the advice and experiences of real patients. They were satisfied with their transplant education (4.2/5). While there were few aspects that the participants disliked about the videos, the interviews elicited outstanding questions about the transplant process (eg, logistical aspects of travel) and transplant concerns (eg, medications, expenses, and precautions in daily life). Conclusion: Patients being assessed or listed for lung transplant valued the novel electronic video education, and we will implement the home-based process into standard of care after the patient's initial visit with the transplant respirologist. Pre-transplant education will be tailored to help address the outstanding gaps identified in this program evaluation.

Pharmacokinetics of apixaban and tacrolimus or cyclosporine in kidney and lung transplant recipients.

Apixaban is frequently used off-label in transplant recipients. However, a potential drug interaction exists with the calcineurin inhibitors. We conducted an open-label drug-drug interaction study to determine the pharmacokinetics of apixaban in lung and kidney transplant recipients who were taking a calcineurin inhibitor. A single dose of apixaban 10 mg was administered orally to kidney and lung transplant recipients maintained on either tacrolimus or cyclosporine, and pharmacokinetic parameters were compared to a reference cohort of 12 healthy subjects who used the same apixaban dose and pharmacokinetic blood sampling. Fourteen participants were enrolled (n = 6 kidney, n = 8 lung), with 10 maintained on tacrolimus and four on cyclosporine. Data from 13 participants was usable. Participants were taking triple therapy immunosuppression and had a mean (SD) of 12 (3) medications. Participants receiving tacrolimus and cyclosporine had area under the plasma concentration-time curve from time zero to infinity (AUC0-inf ) geometric least square means (90% confidence interval [CI]) of 4312 (95% CI 3682, 5049) and 5388 (95% CI 3277, 8858), respectively. Compared to healthy subjects, the associated geometric mean ratios (GMRs) for apixaban maximum plasma concentration (Cmax ), AUC from time zero to the last quantifiable concentration (AUC0-tlast ) and AUC0-inf were 197% (95% CI 153, 295), 244% (95% CI 184, 323), and 224% (95% CI 170, 295) for transplant recipients on tacrolimus. The GMR (90% CI) Cmax , AUC0-tlast , and AUC0-inf of apixaban for patients on cyclosporine were 256% (95% CI 184, 358), 287% (95% CI 198, 415), and 280% (95% CI 195, 401). Kidney and lung transplant recipients receiving tacrolimus had higher apixaban exposure. A similar trend was noted for patients receiving cyclosporine, but additional patients are needed to confirm this interaction. Future studies are needed before apixaban can be safely recommended in this population, and the impact of dose staggering should be investigated. This study highlights the importance of pharmacokinetic studies in actual patient populations.

Lipopolysaccharides induce a RAGE-mediated sensitization of sensory neurons and fluid hypersecretion in the upper airways.

Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

cAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

Nebulized hypertonic saline triggers nervous system-mediated active liquid secretion in cystic fibrosis swine trachea.

Inhaled hypertonic saline (HTS) treatment is used to improve lung health in patients with cystic fibrosis (CF). The current consensus is that the treatment generates an osmotic gradient that draws water into the airways and increases airway surface liquid (ASL) volume. However, there is evidence that HTS may also stimulate active secretion of ASL by airway epithelia through the activation of sensory neurons. We tested the contribution of the nervous system and airway epithelia on HTS-stimulated ASL height increase in CF and wild-type swine airway. We used synchrotron-based imaging to investigate whether airway neurons and epithelia are involved in HTS treatment-triggered ASL secretion in CFTR-/- and wild-type swine. We showed that blocking parasympathetic and sensory neurons in airway resulted in ~50% reduction of the effect of HTS treatment on ASL volume in vivo. Incubating tracheal preparations with inhibitors of epithelial ion transport across airway decreased secretory responses to HTS treatment. CFTR-/- swine ex-vivo tracheal preparations showed substantially decreased secretory response to HTS treatment after blockage of neuronal activity. Our results indicated that HTS-triggered ASL secretion is partially mediated by the stimulation of airway neurons and the subsequent activation of active epithelia secretion; osmosis accounts for only ~50% of the effect.

Cystic fibrosis swine fail to secrete airway surface liquid in response to inhalation of pathogens.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR-/- swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.