Pancreatic enzymes digest obstructive meconium from cystic fibrosis pig intestines.

Introduction: Meconium ileus (MI) is a life-threatening obstruction of the intestines affecting ∼15% of newborns with cystic fibrosis (CF). Current medical treatments for MI often fail, requiring surgical intervention. MI typically occurs in newborns with pancreatic insufficiency from CF. Meconium contains mucin glycoprotein, a potential substrate for pancreatic enzymes or mucolytics. Our study aim was to determine whether pancreatic enzymes in combination with mucolytic treatments dissolve obstructive meconium using the CF pig model. Methods: We collected meconium from CF pigs at birth and submerged it in solutions with and without pancreatic enzymes, including normal saline, 7% hypertonic saline, and the reducing agents N-acetylcysteine (NAC) and dithiothreitol (DTT). We digested meconium at 37 °C with agitation, and measured meconium pigment release by spectrophotometry and residual meconium solids by filtration. Results and discussion: In CF pigs, meconium appeared as a solid pigmented mass obstructing the ileum. Meconium microscopically contained mucus glycoprotein, cellular debris, and bile pigments. Meconium fragments released pigments with maximal absorption at 405 nm after submersion in saline over approximately 8 h. Pancreatic enzymes significantly increased pigment release and decreased residual meconium solids. DTT did not improve meconium digestion and the acidic reducing agent NAC worsened digestion. Pancreatic enzymes digested CF meconium best at neutral pH in isotonic saline. We conclude that pancreatic enzymes digest obstructive meconium from CF pigs, while hydrating or reducing agents alone were less effective. This work suggests a potential role for pancreatic enzymes in relieving obstruction due to MI in newborns with CF.

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.

Injectable long-acting ivacaftor-loaded poly (lactide-co-glycolide) microparticle formulations for the treatment of cystic fibrosis: In vitro characterization and in vivo pharmacokinetics in mice.

Optimizing a sustained-release drug delivery system for the treatment of cystic fibrosis (CF) is crucial for decreasing the dosing frequency and improving patients' compliance with the treatment regimen. In the current work, we developed an injectable poly(D,L-lactide-co-glycolide) (PLGA) microparticle formulation loaded with ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases the open probability of the CFTR anion channel, using a single emulsion solvent evaporation technique. We aimed to study the effect of different parameters on the characteristics of the prepared formulations to select an optimized microparticle formulation to be used in an in vivo pharmacokinetic study in mice. First, a suite of ivacaftor-loaded microparticles were prepared using different formulation parameters in order to study the effect of varying these parameters on microparticle size, morphology, drug loading, encapsulation efficiency, and in vitro release profiles. Prepared microparticles were spherical with diameters ranging from 1.91-6.93 µm, percent drug loading (% DL) of 3.91-10.3%, percent encapsulation efficiencies (% EE) of 26.6-100%, and an overall slow cumulative release profile. We selected the formulation that demonstrated optimal combined % DL and % EE values (8.25 and 90.7%, respectively) for further studies. These microparticles had an average particle size of 6.83 µm and a slow tri-phasic in vitro release profile (up to 6 weeks). In vivo pharmacokinetic studies in mice showed that the subcutaneously injected microparticles resulted in steady plasma levels of ivacaftor over a period of 28 days, and a 6-fold increase in AUC 0 - t (71.6 µg/mL*h) compared to the intravenously injected soluble ivacaftor (12.3 µg/mL*h). Our results suggest that this novel ivacaftor-loaded microparticle formulation could potentially eliminate the need for the frequent daily administration of ivacaftor to people with CF thus improving their compliance and ensuring successful treatment outcomes.

CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

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.

Cystic Fibrosis Reprograms Airway Epithelial IL-33 Release and Licenses IL-33-Dependent Inflammation.

Rationale: Type 2 inflammation has been described in people with cystic fibrosis (CF). Whether loss of CFTR (cystic fibrosis transmembrane conductance regulator) function contributes directly to a type 2 inflammatory response has not been fully defined. Objectives: The potent alarmin IL-33 has emerged as a critical regulator of type 2 inflammation. We tested the hypothesis that CFTR deficiency increases IL-33 expression and/or release and deletion of IL-33 reduces allergen-induced inflammation in the CF lung. Methods: Human airway epithelial cells (AECs) grown from non-CF and CF cell lines and Cftr+/+ and Cftr-/- mice were used in this study. Pulmonary inflammation in Cftr+/+ and Cftr-/- mice with and without IL-33 or ST2 (IL-1 receptor-like 1) germline deletion was determined by histological analysis, BAL, and cytokine analysis. Measurements and Main Results: After allergen challenge, both CF human AECs and Cftr-/- mice had increased IL-33 expression compared with control AECs and Cftr+/+ mice, respectively. DUOX1 (dual oxidase 1) expression was increased in CF human AECs and Cftr-/- mouse lungs compared with control AECs and lungs from Cftr+/+ mice and was necessary for the increased IL-33 release in Cftr-/- mice compared with Cftr+/+ mice. IL-33 stimulation of Cftr-/- CD4+ T cells resulted in increased type 2 cytokine production compared with Cftr+/+ CD4+ T cells. Deletion of IL-33 or ST2 decreased both type 2 inflammation and neutrophil recruitment in Cftr-/- mice compared with Cftr+/+ mice. Conclusions: Absence of CFTR reprograms airway epithelial IL-33 release and licenses IL-33-dependent inflammation. Modulation of the IL-33/ST2 axis represents a novel therapeutic target in CF type 2-high and neutrophilic inflammation.

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.

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction.

Pulmonary neuroendocrine cells (PNECs) are rare airway cells with potential sensory capacity linked to vagal neurons and immune cells. How PNECs sense and respond to external stimuli remains poorly understood. We discovered PNECs located within pig and human submucosal glands, a tissue that produces much of the mucus that defends the lung. These PNECs sense succinate, an inflammatory molecule in liquid lining the airway surface. The results indicate that succinate migrates down the submucosal gland duct to the acinus, where it triggers apical succinate receptors, causing PNECs to release ATP. The short-range ATP signal stimulates the contraction of myoepithelial cells wrapped tightly around the submucosal glands. Succinate-triggered gland contraction may complement the action of neurotransmitters that induce mucus release but not gland contraction to promote mucus ejection onto the airway surface. These findings identify a local circuit in which rare PNECs within submucosal glands sense an environmental cue to orchestrate the function of airway glands.

Anion Transport Across Human Gallbladder Organoids and Monolayers.

Fluid and anion secretion are important functions of the biliary tract. It has been established that cAMP regulates Na+ absorption through NHE3. However, mechanisms of gallbladder anion transport are less defined. We created organoids and organoid-derived monolayers from human gallbladder tissue to measure organoid swelling and transepithelial electrophysiology. In our in vitro models, forskolin-stimulation caused organoid swelling and increased transepithelial anion transport. Full organoid swelling required Cl-while changes in short-circuit current were HCO3 --dependent. Organoids and monolayers from an individual homozygous for the cystic fibrosis-causing ΔF508 CFTR mutation had no apical expression of CFTR and minimal changes in transepithelial current and conductance with forskolin treatment. However, organoid swelling remained intact. Dilution potential studies revealed that forskolin treatment increased the paracellular permeability to anions relative to cations. These data suggest a novel paracellular contribution to forskolin-stimulated fluid transport across the gallbladder epithelium.

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.

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

The Rapid Reduction of Infection-Related Visits and Antibiotic Use Among People With Cystic Fibrosis After Starting Elexacaftor-Tezacaftor-Ivacaftor.

BACKGROUND: People with cystic fibrosis (CF) routinely suffer from recurrent sinopulmonary infections. Such infections require frequent courses of antimicrobials and often involve multidrug-resistant organisms. The goal of this study was to identify real-world evidence for the effectiveness of elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA) in decreasing infection-related visits and antimicrobial use in people with CF. METHODS: Using IBM MarketScan data, we identified 389 enrollees with CF who began taking ELX/TEZ/IVA before 1 December 2019 and were enrolled from 1 July 2019 to 14 March 2020. We also identified a comparison population who did not begin ELX/TEZ/IVA during the study period. We compared the following outcomes in the 15 weeks before and after medication initiation: total healthcare visits, inpatient visits, infection-related visits, and antimicrobial prescriptions. We analyzed outcomes using both a case-crossover analysis and a difference-in-differences analysis, to control for underlying trends. RESULTS: For the case-crossover analysis, ELX/TEZ/IVA initiation was associated with the following changes over a 15-week period: change in overall healthcare visit dates, -2.5 (95% confidence interval, -3.31 to -1.7); change in inpatient admissions, -0.16 (-.22 to -.10); change in infection-related visit dates, -0.62 (-.93 to -.31); and change in antibiotic prescriptions, -0.78 (-1.03 to -.54). Results from the difference-in-differences approach were similar. CONCLUSIONS: We show a rapid reduction in infection-related visits and antimicrobial use among people with CF after starting a therapy that was not explicitly designed to treat infections. Currently, there are >30 000 people living with CF in the United States alone. Given that this therapy is effective for approximately 90% of people with CF, the impact on respiratory infections and antimicrobial use may be substantial.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Effects of Tham Nasal Alkalinization on Airway Microbial Communities: A Pilot Study in Non-CF and CF Adults.

OBJECTIVES: In cystic fibrosis (CF), loss of CFTR-mediated bicarbonate secretion reduces the airway surface liquid (ASL) pH causing airway host defense defects. Aerosolized sodium bicarbonate can reverse these defects, but its effects are short-lived. Aerosolized tromethamine (THAM) also raises the ASL pH but its effects are much longer lasting. In this pilot study, we tested the hypothesis that nasally administered THAM would alter the nasal bacterial composition in adults with and without CF. METHODS: Subjects (n = 32 total) received intranasally administered normal saline or THAM followed by a wash out period prior to receiving the other treatment. Nasal bacterial cultures were obtained prior to and after each treatment period. RESULTS: At baseline, nasal swab bacterial counts were similar between non-CF and CF subjects, but CF subjects had reduced microbial diversity. Both nasal saline and THAM were well-tolerated. In non-CF subjects, nasal airway alkalinization decreased both the total bacterial density and the gram-positive bacterial species recovered. In both non-CF and CF subjects, THAM decreased the amount of Corynebacterium accolens detected, but increased the amount of Corynebacterium pseudodiphtheriticum recovered on nasal swabs. A reduction in Staphylococcus aureus nasal colonization was also found in subjects who grew C. pseudodiphtheriticum. CONCLUSIONS: This study shows that aerosolized THAM is safe and well-tolerated and that nasal airway alkalinization alters the composition of mucosal bacterial communities. CLINICAL TRIAL REGISTRATION: NCT00928135 (https://clinicaltrials.gov/ct2/show/NCT00928135).

Combining Ivacaftor and Intensive Antibiotics Achieves Limited Clearance of Cystic Fibrosis Infections.

Drugs called CFTR modulators improve the physiologic defect underlying cystic fibrosis (CF) and alleviate many disease manifestations. However, studies to date indicate that chronic lung infections that are responsible for most disease-related mortality generally persist. Here, we investigated whether combining the CFTR modulator ivacaftor with an intensive 3.5-month antibiotic course could clear chronic Pseudomonas aeruginosa or Staphylococcus aureus lung infections in subjects with R117H-CFTR, who are highly ivacaftor-responsive. Ivacaftor alone improved CFTR activity, and lung function and inflammation within 48 h, and reduced P. aeruginosa and S. aureus pathogen density by ∼10-fold within a week. Antibiotics produced an additional ∼10-fold reduction in pathogen density, but this reduction was transient in subjects who remained infected. Only 1/5 P. aeruginosa-infected and 1/7 S. aureus-infected subjects became persistently culture-negative after the combined treatment. Subjects appearing to clear infection did not have particularly favorable baseline lung function or inflammation, pathogen density or antibiotic susceptibility, or bronchiectasis scores on CT scans, but they did have remarkably low sweat chloride values before and after ivacaftor. All persistently P. aeruginosa-positive subjects remained infected by their pretreatment strain, whereas subjects persistently S. aureus-positive frequently lost and gained strains. This work suggests chronic CF infections may resist eradication despite marked and rapid modulator-induced improvements in lung infection and inflammation parameters and aggressive antibiotic treatment. IMPORTANCE Recent work shows that people with CF and chronic lung infections generally remain persistently infected after treatment with drugs that target the CF physiological defect (called CFTR modulators). However, changes produced by modulators could increase antibiotic efficacy. We tested the approach of combining modulators and intensive antibiotics in rapid succession and found that while few subjects cleared their infections, combined treatment appeared most effective in subjects with the highest CFTR activity. These findings highlight challenges that remain to improve the health of people with CF.

Early intrahepatic duct defects in a cystic fibrosis porcine model.

Hepatobiliary disease causes significant morbidity and mortality in people with cystic fibrosis (CF), yet this problem remains understudied. Previous studies in the newborn CF pig demonstrated decreased bile flow into the small intestine and a microgallbladder with increased luminal mucus and fluid secretion defects. In this study, we examined the intrahepatic bile ducts of the newborn CF pig. We assessed whether our findings from the gallbladder are present elsewhere in the porcine biliary tract and if CF pig cholangiocytes have fluid secretion defects. Immunohistochemistry demonstrated apical CFTR expression in non-CF pig intrahepatic bile ducts of a variety of sizes; CF pig intrahepatic bile ducts lacked CFTR expression. Assessment of serum markers did not reveal significant signs of hepatobiliary disease except for an elevation in direct bilirubin. Quantitative histology demonstrated that CF pigs had smaller bile ducts that more frequently contained luminal mucus. CF intrahepatic cholangiocyte organoids were smaller and lacked cAMP-mediated fluid secretion. Together these data suggest that cholangiocyte fluid secretion is decreased in the CF pig, contributing to structural changes in bile ducts and decreased biliary flow.

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.

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators.

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HCO3- secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl-/HCO3- exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3- secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.

Transduction of Pig Small Airway Epithelial Cells and Distal Lung Progenitor Cells by AAV4.

Cystic fibrosis (CF) is caused by genetic mutations of the CF transmembrane conductance regulator (CFTR), leading to disrupted transport of Cl- and bicarbonate and CF lung disease featuring bacterial colonization and chronic infection in conducting airways. CF pigs engineered by mutating CFTR develop lung disease that mimics human CF, and are well-suited for investigating CF lung disease therapeutics. Clinical data suggest small airways play a key role in the early pathogenesis of CF lung disease, but few preclinical studies have focused on small airways. Efficient targeted delivery of CFTR cDNA to small airway epithelium may correct the CFTR defect and prevent lung infections. Adeno-associated virus 4 (AAV4) is a natural AAV serotype and a safe vector with lower immunogenicity than other gene therapy vectors such as adenovirus. Our analysis of AAV natural serotypes using cultured primary pig airway epithelia showed that AAV4 has high tropism for airway epithelia and higher transduction efficiency for small airways compared with large airways. AAV4 mediated the delivery of CFTR, and corrected Cl- transport in cultured primary small airway epithelia from CF pigs. Moreover, AAV4 was superior to all other natural AAV serotypes in transducing ITGα6ß4+ pig distal lung progenitor cells. In addition, AAV4 encoding eGFP can infect pig distal lung epithelia in vivo. This study demonstrates AAV4 tropism in small airway progenitor cells, which it efficiently transduces. AAV4 offers a novel tool for mechanistical study of the role of small airway in CF lung pathogenesis in a preclinical large animal model.

V-Type ATPase Mediates Airway Surface Liquid Acidification in Pig Small Airway Epithelial Cells.

In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO3- transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 µm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3-, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.

Sustained Coinfections with Staphylococcus aureus and Pseudomonas aeruginosa in Cystic Fibrosis.

Rationale: Staphylococcus aureus and Pseudomonas aeruginosa often infect the airways in cystic fibrosis (CF). Because registry studies show higher prevalence of P. aeruginosa versus S. aureus in older patients with CF, a common assumption is that P. aeruginosa replaces S. aureus over time. In vitro, P. aeruginosa can outgrow and kill S. aureus. However, it is unknown how rapidly P. aeruginosa replaces S. aureus in patients with CF.Methods: We studied a longitudinal cohort of children and adults with CF who had quantitative sputum cultures. We determined the abundance of P. aeruginosa and S. aureus in cfu/ml. We determined the duration and persistence of infections and measured longitudinal changes in culture positivity and abundance for each organism.Measurements and Main Results: Between 2004 and 2017, 134 patients had ≥10 quantitative cultures, with median observation time of 10.15 years. One hundred twenty-four patients had at least one positive culture for P. aeruginosa, and 123 had at least one positive culture for S. aureus. Both species had median abundance of >106 cfu/ml. Culture abundance was stable over time for both organisms. There was an increase in the prevalence of S. aureus/P. aeruginosa coinfection but no decrease in S. aureus prevalence within individuals over time.Conclusions: S. aureus and P. aeruginosa are abundant in CF sputum cultures. Contrary to common assumption, we found no pattern of replacement of S. aureus by P. aeruginosa. Many patients with CF have durable long-term coinfection with these organisms. New strategies are needed to prevent and treat these infections.