Cystic fibrosis (CF) is caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR) anion channel. Since the initial characterization of CF in the early 20th century, advances in clinical management have reduced disease burden and increased longevity. These advances are amplified by the recent development of highly effective CFTR modulator therapies (HEMTs) that have provided remarkable clinical results in people with CF (pwCF). However, some pwCF do not benefit from HEMT due to several limiting factors. Genetic therapies have increasingly emerged as prospective, complementary treatments to HEMTs for those unable to benefit from HEMTs. Genetic therapies have yet to be approved for clinical use in pwCF, but multiple clinical trials are in progress. In anticipation of future approval of one or more of these candidates, it is essential to identify and discuss how these genetic therapies might be used in early life, when many CF lesions originate, especially for those with severe mutations. In this multidisciplinary review, we discuss several pertinent factors such as CFTR localization and function, CF disease origins, emerging developments in genetic therapies, ethical considerations, and other perspectives needed to guide future genetic therapies in early life.

Previous studies indicate that pigs with CFTR-null and CFTR-deltaF508 mutations develop multiorgan disease similar to that in people with cystic fibrosis (CF). At birth, their airways exhibit host defense defects that predispose to airway infection, inflammation, and mucus accumulation. The CFTR-G551D mutation causes CF by producing CFTR channels that localize correctly but have reduced channel activity. Ivacaftor (VX-770) is a small molecule drug developed to potentiate CFTR activity. To test the phenotype of the CFTR-G551D mutation in pigs and determine whether ivacaftor can rescue CF abnormalities, we developed CFTRG551D/G551D (CF-G551D) pigs through homologous recombination in fetal fibroblasts and somatic cell nuclear transfer. Newborn CF-G551D piglets exhibited phenotypes similar to CF-null piglets, including meconium ileus, exocrine pancreatic destruction, micro-gallbladder, vas deferens destruction, and airway structural abnormalities. Compared to wild-type pigs, CF-G551D pigs had reduced forskolin-stimulated short-circuit current in airway and intestinal tissues. Ivacaftor increased the single-channel open state probability of CFTR-G551D and increased short-circuit current to near wild-type levels. Similar to our other CF pig models, we found that 100% of CF-G551D pigs were born with meconium ileus. To test whether in utero ivacaftor treatment could prevent or alleviate meconium ileus, pregnant sows were treated with ivacaftor beginning at day 35 of gestation and continuing until delivery. This treatment rescued the pancreas, gallbladder, and vas deferens phenotype in the majority of CF-G551D pigs. Animals that were spared from meconium ileus were able to survive without ivacaftor treatment. Airway disease developed similar to other CF pig models. These findings indicate that this model may be useful for studies in which CFTR function can be reversed, for investigating in utero CFTR correction strategies, and for longitudinal studies in CF pigs. ### Competing Interest Statement The authors have declared no competing interest. Cystic Fibrosis Foundation, https://ror.org/00ax59295 National Institutes of Health, https://ror.org/01cwqze88

RATIONALE: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder resulting in nonfunctional cilia leading to chronic infections of the lungs. Recurrent infections are thought to be responsible for structural changes (air trapping, atelectasis, and bronchiectasis) seen in nearly all adults with PCD. Previously, we have shown that DNAI1 KO PCD pigs have defective cilia and develop lung disease similar to humans shortly after birth. To understand the progression of PCD lung disease we investigated PCD pigs at 6, 9, and 12 months of age. We hypothesized lung disease in older PCD pigs would mimic that of humans with PCD and worsen over time.METHODS: Thoracic computed tomography (CT) scans were acquired from PCD and WT pigs at 6, 9, and 12 months of age with a dual source high-resolution multi-row detector CT scanner (Canon Aquilion ONE, 120 kVp, 400 mA, slice thickness 0.5 mm, slice spacing 0.3 mm). At each time point, a total lung capacity (TLC) scan was acquired at 25 cm H2O and a functional residual capacity (FRC) scan was acquired at 0 cm H2O. We analyzed differences in lung densitometry using the free open-source software 3D Slicer (version 4.13.0, NA-MIC, NIH). Lung samples were collected for histological analysis, and bronchoalveolar lavage (BAL) was conducted to quantify cellular, cytokine, and bacterial components in both healthy and diseased lung regions.RESULTS: CT scans revealed atelectasis in PCD pig lungs, predominantly in dependent regions. Histology showed mucus accumulation in the airways, indicating mucus stasis and blockage as potential contributing factors. In some instances, mucus was observed moving into the respiratory bronchioles and alveolar ducts. BAL fluid analysis indicated elevated levels of IL-1α, IL-1β, IL-1ra, and IL-8 in diseased lung regions at all time points. At 9 months, cytokine profiles in healthy areas of PCD pig lungs resembled those of healthy controls, but by 12 months, these profiles resembled those of diseased lungs. Microbiological analyses corroborated these trends.CONCLUSIONS: Our study demonstrates a progression of lung disease in PCD pigs that mirrors aspects observed in human PCD patients. These findings underscore the utility of the PCD pig model in studying the early development and progression of PCD lung disease. Understanding these changes will be crucial for developing potential therapeutic strategies and interventions to manage or mitigate the progression of lung disease in PCD patients.

Patient-derived airway cell cultures are used in personalized medicine strategies for people with cystic fibrosis (pwCF) to predict potential clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. While bronchial epithelial cells from lung explants (HBEx) are the gold standard for CFTR functional measurements, nasal epithelial cells (HNE) are a more practical tissue source resulting in widespread use for preclinical functional platforms. HNE have so far not been rigorously validated against the gold standard for this purpose. In this study, we collected nasal and bronchial cells, and lung explants from pwCF undergoing lung transplantation as well as non-CF controls. Comparative studies in non-CF cells showed that while CFTR-mediated transepithelial currents in HNE underestimated those in HBEx, the magnitude of the CFTR modulator response was similar between CF HNE, brushed HBE (HBEb), and HBEx with significant correlation between matched HNE and HBEb from 16 pwCF. These findings confirm use of HNE as surrogate of bronchial airway for preclinical drug testing with report of drug responses in relation to the tissue-specific non-CF or baseline controls rather than as absolute results. Furthermore, CF centres offering HNE-based drug testing utilize different techniques, challenging the comparison of results between centres. We show how culture media, use of fresh or freeze-thawed cells as well as difference in Ussing technique impact the magnitude of measured CFTR function, which is why we suggest diligence in reporting of these factors when presenting CFTR modulator drug response results.

Type 2 inflammation and epithelial-to-mesenchymal transitions (EMTs) play critical roles in airway repair after damage from allergens or parasites. The matricellular protein periostin (POSTN) has increased expression in inflammatory conditions and has been implicated in fibrosis and EMT, suggesting a role in airway repair. This study investigates the role of periostin in airway epithelial and lung fibroblast wound repair using an in vitro wound model. Our results demonstrate that the type 2 cytokine IL-13 induces periostin secretion from primary human airway epithelial basal cells. Periostin knockdown in human airway epithelial cells (HAEs) and human lung fibroblasts (HLFs) impairs wound closure, indicating that periostin is required for airway repair. In a coculture model of HAE and HLFs, fibroblast-secreted POSTN is required for airway epithelial wound repair, suggesting that periostin is involved in paracrine signaling between the two cell types. These findings highlight periostin's critical function in epithelial and fibroblast-mediated wound repair, suggesting its potential as a therapeutic target for diseases characterized by aberrant wound healing and fibrosis, such as asthma and idiopathic pulmonary fibrosis.NEW & NOTEWORTHY This article highlights the critical role of periostin (POSTN) in airway epithelial and fibroblast-mediated wound repair. Moreover, the study reveals a paracrine signaling loop between airway epithelial basal cells and lung fibroblasts, emphasizing periostin's therapeutic potential for diseases like asthma and idiopathic pulmonary fibrosis.

Rationale:Cystic fibrosis is a genetic disorder characterized by recurrent airway infections, inflammation, and progressive decline in lung function. Autopsy and spirometry data suggest that cystic fibrosis may start in the small airways which, due to the fractal nature of the airways, account for most of the airway tree surface area. However, they are not easily accessible for testing. Objectives:Here, we tested the hypothesis that mucociliary clearance is abnormal in the small airways of newborn cystic fibrosis pigs. Methods:Current mucociliary clearance assays are limited therefore we developed a dynamic positron emission tomography scan assay with high spatial and temporal resolution. Each study was accompanied by a high-resolution computed tomography scan that helped identify the thin outer region of the lung that contained small airways. Measurements and Main Results:Clearance of aerosolized [ 68 Ga]macro aggregated albumin from distal airways occurred within minutes after delivery and followed a two-phase process. In cystic fibrosis pigs, both early and late clearance rates were slower. Stimulation of the cystic fibrosis airways with the purinergic agonist UTP further impaired late clearance. Only 1 cystic fibrosis pig treated with UTP out of 6 cleared more than 20% of the delivered dose. Conclusions:These data indicate that mucociliary transport in the small airways is fast and can easily be missed if the acquisition is not fast enough. The data also indicate that mucociliary transport is impaired in small airways of cystic fibrosis pigs. This defect is exacerbated by stimulation of mucus secretions with purinergic agonists.

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

Background:Many of those infected with COVID-19 experience long-term disability due to persistent symptoms known as Long-COVID, which include ongoing respiratory issues, loss of taste and smell, and impaired daily functioning. Research Question:This study aims to better understand the chronology of long-COVID symptoms. Study Design and Methods:We prospectively enrolled 403 adults from the University of Iowa long-COVID clinic (June 2020 to February 2022). Participants provided symptom data during acute illness, symptom progression, and other clinical characteristics. Patients in this registry received a survey containing questions including current symptoms and status since long-COVID diagnosis (sliding status scale, PHQ2, GAD2, MMRC). Those >12 months since acute-COVID diagnosis had chart review done to track their symptomology. Results:Of 403 participants contacted, 129 (32%) responded. The mean age (in years) was 50.17 +/-14.28, with 31.8% male and 68.2% female. Severity of acute covid treatment was stratified by treatment in the outpatient (70.5%), inpatient (16.3%), or ICU (13.2%) settings. 51.2% reported subjective improvement (sliding scale scores of 67-100) since long-COVID onset. Ages 18-29 reported significantly higher subjective status scores. Subjective status scores were unaffected by severity. 102 respondents were >12 months from their initial COVID-19 diagnosis and were tracked for longitudinal symptom persistence. All symptoms tracked had variance (mean fraction 0.58, range 0.34-0.75) in the reported symptoms at the time of long-COVID presentation when compared with patient survey report. 48 reported persistent dyspnea, 23 (48%) had resolved it at time of survey. For fatigue, 44 had persistence, 12 (27%) resolved. Interpretation:Overall, 51.2% respondents improved since their long-COVID began. Pulmonary symptoms were more persistent than neuromuscular symptoms (anosmia, dysgeusia, myalgias). Gender, time since acute COVID infection, and its severity didn't affect subjective status or symptoms. This study highlights recall bias that may be prevalent in other long-COVID research reliant on participant memory.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease. Historically, two COPD phenotypes have been described: chronic bronchitis and emphysema. Although these phenotypes may provide additional characterization of the pathophysiology of the disease, they are not extensive enough to reflect the heterogeneity of COPD and do not provide granular categorization that indicates specific treatment, perhaps with the exception of adding inhaled glucocorticoids (ICS) in patients with chronic bronchitis. In this review, we describe COPD phenotypes that provide prognostication and/or indicate specific treatment. We also describe COPD-like phenotypes that do not necessarily meet the current diagnostic criteria for COPD but provide additional prognostication and may be the targets for future clinical trials.

In contrast to pig large airways, the pH of airway surface liquid (ASL) in pig small airways is regulated by CFTR-mediated HCO-3 secretion and the vacuolar-type H+ ATPase (V-ATPase) proton secretion. We hypothesized that, in cystic fibrosis (CF), the ASL pH of small airways is acidic, and the V-ATPase is internalized. We quantified proton secretion during the addition of an alkaline test solution by measuring changes in a pH-dependent fluorescent dye generated by porcine small airway epithelia in the absence and presence of bafilomycin A1. The pH-dependent translocation of V-ATPase in ex vivo and in vivo preparations was measured using immunolocalization of V-ATPase. We found that bafilomycin-sensitive proton secretion stopped when the ASL pH was less than 7.10. In non-CF pigs and mice, we found that V-ATPase was localized in the apical membrane, and internalized when the lungs were instilled with a pH 6.8 solution. Studies in which we immediately fixed lungs from pigs revealed apical V-ATPase detection in non-CF piglets and less apical detection in CF piglets. Our data suggest that V-ATPase in small airways is internalized when the ASL pH is acidic. The decrease in apical localization of V-ATPase in CF pigs is consistent with an acidic ASL pH.NEW & NOTEWORTHY In this study, we describe that vacuolar-type H+ ATPase (V-ATPase) internalizes when the airway surface liquid (ASL) pH in pig small airways is less than 7.10. Furthermore, we found that V-ATPase is not localized to the apical membrane in the small airways of newborn cystic fibrosis pigs.

"ATP12A: Connecting Mucus and Fibrosis in IPF." American Journal of Respiratory Cell and Molecular Biology, 0(ja), pp.

Notre objectif était d’analyser la relation entre les profils d’excrétion de 6-sulfatoxymelatonin (aMT6s) urinaire sur 24 heures et le pronostic des patients hospitalisés en réanimation. Hypothèse : les dysrythmies circadiennes sont associées à un mauvais pronostic. Recueil rétrospectif de 37 patients en choc septique et/ou insuffisance respiratoire aiguë (35 sous ventilation invasive et 27 en choc septique). Une régression non linéaire permettait d’obtenir une courbe sinusoïdale pour chaque patient à partir des valeurs de aMT6s urinaire normalisées, collectées chaque heure, durant 24 heures. La rythmicité de la courbe était obtenue par le test zéro-amplitude. Le rapport nuit (23h–07 h) sur jour (07h–23 h) de aMT6s était déterminé par le rapport des aires sous chaque courbe. Nous avons réalisé des analyses uni- et multivariées afin d’identifier les variables prédictives du retour à domicile en comparaison au décès ou transfert en unité de long séjour. Dans l’ensemble, l’acrophase de la courbe présentait un retard de phase (7h18 ± 14 min). L’amplitude était plus grande dans le groupe retour à domicile que dans le groupe décès/transfert (p = 0,005). 2/18 patients avaient un profil arythmique dans le groupe retour à domicile, comparé à 10/19 dans le groupe décès/transfert (p = 0,013). Le retour à domicile était fortement associé à un rapport nuit/jour de aMT6s élevé en analyse multivariée (OR : 2,4, IC95 % : 1,2, 6,8 ; p = 0,037). L’absence de rythmicité et la faible amplitude du profil de aMT6s sur 24 h étaient associées à un mauvais pronostic chez les patients hospitalisés en réanimation.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

Circadian dysrhythmias occur commonly in critically ill patients reflecting variable effects of underlying illness, ICU environment, and treatments. We retrospectively analyzed the relationship between clinical outcomes and 24-h urinary 6-sulfatoxymelatonin (aMT6s) excretion profiles in 37 critically ill patients with shock and/or respiratory failure. Nonlinear regression was used to fit a 24-h cosine curve to each patient's aMT6s profile, with rhythmicity determined by the zero-amplitude test. From these curves we determined acrophase, amplitude, phase, and night/day ratio. After assessing unadjusted relationships, we identified the optimal multivariate models for hospital survival and for discharge to home (vs. death or transfer to another facility). Normalized aMT6s rhythm amplitude was greater (p = 0.005) in patients discharged home than in those who were not, while both groups exhibited a phase delay. Patients with rhythmic aMT6s excretion were more likely to survive (OR 5.25) and be discharged home (OR 8.89; p < 0.05 for both) than patients with arrhythmic profiles, associations that persisted in multivariate modelling. In critically ill patients with shock and/or respiratory failure, arrhythmic and/or low amplitude 24-h aMT6s rhythms were associated with worse clinical outcomes, suggesting a role for the melatonin-based rhythm as a novel biomarker of critical illness severity.

Background: In humans and other large mammals, airway submucosal glands (SMGs) secrete mucus involved in bacterial killing and mucociliary transport.Regulating the function and secretion of SMGs is key to respiratory host defense, but in CF, SMG hypertrophy and hypersecretion generate large amounts of elastic mucus that obstructs the airway.Understanding the pathophysiology of SMGs is critical to treating mucus obstruction in CF, but little is known about the cellular and molecular mechanisms of SMG mucus secretion and SMG hypertrophy in normal and CF lungs.Methods: We used single-cell RNA sequencing, single-molecule fluorescence in situ hybridization, and immunofluorescence staining to build a single-cell atlas of SMGs in newborn normal and CF pigs.We investigated how SMGs sense and respond to airway surface inflammatory signals by SMG live imaging, Ca2+ imaging, and primary SMG cell culture.Results: Single-cell atlas identified cellular and molecular features of newborn pig SMGs.Cell types and gene expression were the same in normal and CF SMGs, suggesting no developmental defects of CF SMGs at birth.Mucous and serous cells expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties.We discovered that SMGs contain pulmonary neuroendocrine cells (PNECs), a rare chemosensory cell type previously detected only on the airway surface.PNECs express SUCNR1 to detect succinate, a stress signal that accumulates on the airway surface in response to inflammation and infection.Succinate activates SUCNR1 in PNECs to trigger ATP release, which stimulates P2Y1 purinergic receptors in myoepithelial cells to facilitate SMG contraction.This process is disrupted in CF when mucus plugs the gland ducts, impairing access from the airway surface.In CF SMGs with lung disease, PNEC hyperplasia released calcitonin gene-related peptide, which might trigger inflammation and SMG hypertrophy.Conclusions: These findings increase our understanding of SMGs at singlecell resolution and reveal a local circuit in which rare PNECs within SMGs sense an environmental cue to modulate the function of airway SMGs.They also shed light on the mechanisms of SMG hypertrophy in CF with lung disease.