Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs.

SignificanceIn many lung diseases, increased amounts of and/or abnormal mucus impair mucociliary clearance, a key defense against inhaled and aspirated material. Submucosal glands lining cartilaginous airways secrete mucus strands that are pulled by cilia until they break free from the duct and sweep upward toward the larynx, carrying particulates. In cystic fibrosis (CF) pigs, progressive clearance of insufflated microdisks was repeatedly interrupted as microdisks abruptly recoiled. Aerosolizing a reducing agent to break disulfide bonds linking mucins ruptured mucus strands, freeing them from submucosal gland ducts and allowing cilia to propel them up the airways. These findings highlight the abnormally increased elasticity of CF mucus and suggest that agents that break disulfide bonds might have value in lung diseases with increased mucus.

Early Lung Disease Exhibits Bacteria-Dependent and -Independent Abnormalities in Cystic Fibrosis Pigs.

Rationale: Although it is clear that cystic fibrosis (CF) airway disease begins at a very young age, the early and subsequent steps in disease pathogenesis and the relative contribution of infection, mucus, and inflammation are not well understood. Objectives: As one approach to assessing the early contribution of infection, we tested the hypothesis that early and continuous antibiotics would decrease the airway bacterial burden. We believed that, if they do, this might reveal aspects of the disease that are more or less sensitive to decreasing infection. Methods: Three groups of pigs were studied from birth until ∼3 weeks of age: 1) wild-type, 2) CF, and 3) CF pigs treated continuously with broad-spectrum antibiotics from birth until study completion. Disease was assessed with chest computed tomography, histopathology, microbiology, and BAL. Measurements and Main Results: Disease was present by 3 weeks of age in CF pigs. Continuous antibiotics from birth improved chest computed tomography imaging abnormalities and airway mucus accumulation but not airway inflammation in the CF pig model. However, reducing bacterial infection did not improve two disease features already present at birth in CF pigs: air trapping and submucosal gland duct plugging. In the CF sinuses, antibiotics did not prevent the development of infection or disease or the number of bacteria but did alter the bacterial species. Conclusions: These findings suggest that CF airway disease begins immediately after birth and that early and continuous antibiotics impact some, but not all, aspects of CF lung disease development.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

Polarized AAVR expression determines infectivity by AAV gene therapy vectors.

Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.

Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung.

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.

Solitary Cholinergic Stimulation Induces Airway Hyperreactivity and Transcription of Distinct Pro-inflammatory Pathways.

Airway hyperreactivity is a hallmark feature of asthma and can be precipitated by airway insults, such as ozone exposure or viral infection. A proposed mechanism linking airway insults to airway hyperreactivity is augmented cholinergic transmission. In the current study, we tested the hypothesis that acute potentiation of cholinergic transmission is sufficient to induce airway hyperreactivity. We atomized the cholinergic agonist bethanechol to neonatal piglets and forty-eight hours later measured airway resistance. Bethanechol-treated piglets displayed increased airway resistance in response to intravenous methacholine compared to saline-treated controls. In the absence of an airway insult, we expected to find no evidence of airway inflammation; however, transcripts for several asthma-associated cytokines, including IL17A, IL1A, and IL8, were elevated in the tracheas of bethanechol-treated piglets. In the lungs, prior bethanechol treatment increased transcripts for IFNγ and its downstream target CXCL10. These findings suggest that augmented cholinergic transmission is sufficient to induce airway hyperreactivity, and raise the possibility that cholinergic-mediated regulation of pro-inflammatory pathways might contribute.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

RATIONALE: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

pH modulates the activity and synergism of the airway surface liquid antimicrobials ß-defensin-3 and LL-37.

The pulmonary airways are continuously exposed to bacteria. As a first line of defense against infection, the airway surface liquid (ASL) contains a complex mixture of antimicrobial factors that kill inhaled and aspirated bacteria. The composition of ASL is critical for antimicrobial effectiveness. For example, in cystic fibrosis an abnormally acidic ASL inhibits antimicrobial activity. Here, we tested the effect of pH on the activity of an ASL defensin, human ß-defensin-3 (hBD-3), and the cathelicidin-related peptide, LL-37. We found that reducing pH from 8.0 to 6.8 reduced the ability of both peptides to kill Staphylococcus aureus. An acidic pH also attenuated LL-37 killing of Pseudomonas aeruginosa. In addition, we discovered synergism between hBD-3 and LL-37 in killing S. aureus. LL-37 and lysozyme were also synergistic. Importantly, an acidic pH reduced the synergistic effects of combinations of ASL antibacterials. These results indicate that an acidic pH reduces the activity of individual ASL antimicrobials, impairs synergism between them, and thus may disrupt an important airway host defense mechanism.

Electrolyte transport properties in distal small airways from cystic fibrosis pigs with implications for host defense.

While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl(-) and bicarbonate (HCO3 (-)) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl(-) and HCO3 (-) transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.

Air trapping and airflow obstruction in newborn cystic fibrosis piglets.

RATIONALE: Air trapping and airflow obstruction are being increasingly identified in infants with cystic fibrosis. These findings are commonly attributed to airway infection, inflammation, and mucus buildup. OBJECTIVES: To learn if air trapping and airflow obstruction are present before the onset of airway infection and inflammation in cystic fibrosis. METHODS: On the day they are born, piglets with cystic fibrosis lack airway infection and inflammation. Therefore, we used newborn wild-type piglets and piglets with cystic fibrosis to assess air trapping, airway size, and lung volume with inspiratory and expiratory X-ray computed tomography scans. Micro-computed tomography scanning was used to assess more distal airway sizes. Airway resistance was determined with a mechanical ventilator. Mean linear intercept and alveolar surface area were determined using stereologic methods. MEASUREMENTS AND MAIN RESULTS: On the day they were born, piglets with cystic fibrosis exhibited air trapping more frequently than wild-type piglets (75% vs. 12.5%, respectively). Moreover, newborn piglets with cystic fibrosis had increased airway resistance that was accompanied by luminal size reduction in the trachea, mainstem bronchi, and proximal airways. In contrast, mean linear intercept length, alveolar surface area, and lung volume were similar between both genotypes. CONCLUSIONS: The presence of air trapping, airflow obstruction, and airway size reduction in newborn piglets with cystic fibrosis before the onset of airway infection, inflammation, and mucus accumulation indicates that cystic fibrosis impacts airway development. Our findings suggest that early airflow obstruction and air trapping in infants with cystic fibrosis might, in part, be caused by congenital airway abnormalities.

Cystic Fibrosis Carrier States Are Associated With More Severe Cases of Bronchiectasis.

Background: People with cystic fibrosis (CF) are at increased risk for bronchiectasis, and several reports suggest that CF carriers may also be at higher risk for developing bronchiectasis. The purpose of this study was to determine if CF carriers are at risk for more severe courses or complications of bronchiectasis. Methods: Using MarketScan data (2001-2021), we built a cohort consisting of 105 CF carriers with bronchiectasis and 300 083 controls with bronchiectasis but without a CF carrier diagnosis. We evaluated if CF carriers were more likely to be hospitalized for bronchiectasis. In addition, we examined if CF carriers were more likely to be infected with Pseudomonas aeruginosa or nontuberculous mycobacteria (NTM) or to have filled more antibiotic prescriptions. We considered regression models for incident and rate outcomes that controlled for age, sex, smoking status, and comorbidities. Results: The odds of hospitalization were almost 2.4 times higher (95% CI, 1.116-5.255) for CF carriers with bronchiectasis when compared with non-CF carriers with bronchiectasis. The estimated odds of being diagnosed with a Pseudomonas infection for CF carriers vs noncarriers was about 4.2 times higher (95% CI, 2.417-7.551) and 5.4 times higher (95% CI, 3.398-8.804) for being diagnosed with NTM. The rate of distinct antibiotic fill dates was estimated to be 2 times higher for carriers as compared with controls (95% CI, 1.735-2.333), and the rate ratio for the total number of days of antibiotics supplied was estimated as 2.8 (95% CI, 2.290-3.442). Conclusions: CF carriers with bronchiectasis required more hospitalizations and more frequent administration of antibiotics as compared with noncarriers. Given that CF carriers were also more likely to be diagnosed with Pseudomonas and NTM infections, CF carriers with bronchiectasis may have a phenotype more resembling CF-related bronchiectasis than non-CF bronchiectasis.

Mucociliary Clearance is Impaired in Small Airways of Cystic Fibrosis Pigs.

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.

Tromethamine improves mucociliary clearance in cystic fibrosis pigs.

In cystic fibrosis (CF), the loss of cystic fibrosis transmembrane conductance regulator (CFTR) mediated Cl-  and HCO3 -  secretion across the epithelium acidifies the airway surface liquid (ASL). Acidic ASL alters two key host defense mechanisms: Rapid ASL bacterial killing and mucociliary transport (MCT). Aerosolized tromethamine (Tham) increases ASL pH and restores the ability of ASL to rapidly kill bacteria in CF pigs. In CF pigs, clearance of insufflated microdisks is interrupted due to abnormal mucus causing microdisks to abruptly recoil. Aerosolizing a reducing agent to break disulfide bonds that link mucins improves MCT. Here, we are interested in restoring MCT in CF by aerosolizing Tham, a buffer with a pH of 8.4. Because Tham is hypertonic to serum, we use an acidified formulation as a control. We measure MCT by tracking the caudal movement of individual tantalum microdisks with serial chest computed tomography scans. Alkaline Tham improves microdisk clearance to within the range of that seen in non-CF pigs. It also partially reverses MCT defects, including reduced microdisk recoil and elapse time until they start moving after methacholine stimulation in CF pig airways. The effect is not due to hypertonicity, as it is not seen with acidified Tham or hypertonic saline. This finding indicates acidic ASL impairs CF MCT and suggests that alkalinization of ASL pH with inhaled Tham may improve CF airway disease.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Mucociliary Transport in Healthy and Cystic Fibrosis Pig Airways.

Cystic fibrosis (CF) lung disease is the major cause of morbidity and mortality in people with CF. Abnormal mucociliary transport has been the leading hypothesis for the underlying pathogenesis of CF airway disease. However, this has been difficult to investigate at very early time points. A porcine CF model, which recapitulates many features of CF disease in humans, enables studies to be performed in non-CF and CF pigs on the day that they are born. In newborn CF pigs, we found that under basal conditions, mucociliary transport rates in non-CF and CF pigs are similar. However, after cholinergic stimulation, which stimulates submucosal gland secretion, particles become stuck in the CF airways owing to a failure of mucus strands to release from submucosal glands. In this review, we summarize these recent discoveries and also discuss the morphology, composition, and function of mucins in the porcine lung.

Ivacaftor-induced sweat chloride reductions correlate with increases in airway surface liquid pH in cystic fibrosis.

BACKGROUND: Disruption of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF), and lung disease produces most of the mortality. Loss of CFTR-mediated HCO3- secretion reduces the pH of airway surface liquid (ASL) in vitro and in neonatal humans and pigs in vivo. However, we previously found that, in older children and adults, ASL pH does not differ between CF and non-CF. Here, we tested whether the pH of CF ASL increases with time after birth. Finding that it did suggested that adaptations by CF airways increase ASL pH. This conjecture predicted that increasing CFTR activity in CF airways would further increase ASL pH and also that increasing CFTR activity would correlate with increases in ASL pH. METHODS: To test for longitudinal changes, we measured ASL pH in newborns and then at 3-month intervals. We also studied people with CF (bearing G551D or R117H mutations), in whom we could acutely stimulate CFTR activity with ivacaftor. To gauge changes in CFTR activity, we measured changes in sweat Cl- concentration immediately before and 48 hours after starting ivacaftor. RESULTS: Compared with that in the newborn period, ASL pH increased by 6 months of age. In people with CF bearing G551D or R117H mutations, ivacaftor did not change the average ASL pH; however reductions in sweat Cl- concentration correlated with elevations of ASL pH. Reductions in sweat Cl- concentration also correlated with improvements in pulmonary function. CONCLUSIONS: Our results suggest that CFTR-independent mechanisms increase ASL pH in people with CF. We speculate that CF airway disease, which begins soon after birth, is responsible for the adaptation. FUNDING: Vertex Inc., the NIH (P30DK089507, 1K08HL135433, HL091842, HL136813, K24HL102246), the Cystic Fibrosis Foundation (SINGH17A0 and SINGH15R0), and the Burroughs Wellcome Fund.

Monocyte derived macrophages from CF pigs exhibit increased inflammatory responses at birth.

BACKGROUND: We sought to address whether CF macrophages have a primary functional defect as a consequence of CFTR loss and thus contribute to the onset of infection and inflammation observed in CF lung disease. METHODS: Monocyte derived macrophages (MDMs) were prepared from newborn CF and non-CF pigs. CFTR mRNA expression was quantified by rtPCR and anion channel function was determined using whole cell patch clamp analysis. IL8 and TNFα release from MDMs in response to lipopolysaccharide stimulation was measured by ELISA. RESULTS: CFTR was expressed in MDMs by Q-rtPCR at a lower level than in epithelial cells. MDMs exhibited functional CFTR current at the cell membrane and this current was absent in CF MDMs. CF MDMs demonstrated an exaggerated response to lipopolysaccharide stimulation. CONCLUSIONS: In the absence of CFTR function, macrophages from newborn CF pigs exhibit an increased inflammatory response to a lipopolysaccharide challenge. This may contribute to the onset and progression of CF lung disease.

Mounier-Kuhn syndrome: a case of tracheal smooth muscle remodeling.

Mounier-Kuhn syndrome is a rare clinical disorder characterized by tracheobronchial dilation and recurrent lower respiratory tract infections. While the etiology of the disease remains unknown, histopathological analysis of Mounier-Kuhn airways demonstrates that the disease is, in part, characterized by cellular changes in airway smooth muscle.