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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.sup.-] 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.sup.-] channels in their basolateral membrane. Disrupting barttin/[Cl.sup.-] channel function impaired liquid absorption, and overexpressing barttin/[Cl.sup.-] channels increased absorption. Together, apical CFTR and basolateral barttin/[Cl.sup.-] channels provide an electrically conductive pathway for [Cl.sup.-] flow through ionocytes, and the transepithelial voltage generated by apical [Na.sup.+] 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., Introduction Pulmonary ionocytes are a rare cell type accounting for approximately 1% of airway epithelial cells. Ionocytes have attracted attention because, although rare, in airway epithelia they contain about half [...]

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. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a genetic disorder characterized by recurrent airway infections, inflammation, impaired mucociliary clearance, and progressive decline in lung function. The disease may start in the small airways; however, this is difficult to prove due to the limited accessibility of the small airways with the current single-photon mucociliary clearance assay. Here, we developed a dynamic positron emission tomography assay with high spatial and temporal resolution. We tested that mucociliary clearance is abnormal in the small airways of newborn cystic fibrosis pigs. Clearance of [68Ga]-tagged macroaggregated albumin from small airways started immediately after delivery and continued for the duration of the study. Initial clearance was fast but slowed down a few minutes after delivery. Cystic fibrosis pigs' small airways cleared significantly less than non-CF pigs' small airways (non-CF 25.1 ± 3.1% vs. CF 14.6 ± 0.1%). Stimulation of the cystic fibrosis airways with the purinergic secretagogue uridine-5′-triphosphate (UTP) further impaired clearance (non-CF with UTP 20.9 ± 0.3% vs. CF with UTP 13.0 ± 1.8%). None of the cystic fibrosis pigs treated with UTP (n = 6) cleared more than 20% of the delivered dose. These data indicate that mucociliary clearance in the small airways is fast and can easily be missed if the assay is not sensitive enough. The data also indicate that mucociliary clearance is impaired in the small airways of cystic fibrosis pigs. This defect is exacerbated by stimulation of mucus secretions with purinergic agonists. NEW & NOTEWORTHY: We developed a novel positron emission tomography scan assay with unprecedented temporal and spatial resolution to measure mucociliary clearance in the small airways. We proved a long-standing but unproven assertion that mucociliary clearance is inherently abnormal in the small airways of newborn cystic fibrosis piglets that are otherwise free of infection or inflammation. This technique can be easily extended to other airway diseases such as asthma, idiopathic pulmonary fibrosis, or chronic obstructive pulmonary disease. [ABSTRACT FROM AUTHOR]

In this paper, I will discuss recent studies using a cystic fibrosis pig model to better understand the origins of cystic fibrosis lung disease. Specifically, I will review our work investigating how loss of the cystic fibrosis transmembrane conductance regulator function (CFTR) impairs mucociliary transport in the cystic fibrosis airway. These studies reveal new insights into the early, underlying mechanisms of cystic fibrosis lung disease and could lead to novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HC[O.sub.3]-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-[alpha] and IL-17 (TNF-[alpha]+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-[alpha]+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl/HC[O.sub.3]-exchanger. Moreover, when CF epithelia were exposed to TNF-[alpha]+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 HC[O.sub.3]-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., Introduction Cystic fibrosis (CF) is the most common life-shortening inherited disorder among White individuals (1). CF is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), [...]

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. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non- CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HC[O.sub.3.sup.-] concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator-dependent alkalinization to abnormal CF ASL. In addition, we found that increasing [Ca.sup.2+] concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing [Ca.sup.2*] concentration, and/or altering electrostatic interactions in ASL might benefit early CF., Introduction Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel (1-3). Lung disease is the major [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and [...]

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1). Although bacterial lung infection and the resulting inflammation cause most [...]

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. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing--deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing--dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality., Introduction The paraoxonase (PON) family in mammals is made up of 3 members: PON1, PON2, and PON3. PON-like proteins are evolutionarily conserved, including in Caenorhabditis elegans, but interestingly are absent [...]

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation ([DELTA]F508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice., Introduction Animals are an important resource in biomedical research. Mice with genetic alterations, in particular, have been responsible for many advances in our understanding of human disease pathogenesis and in [...]

Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa's virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC 12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2, but not PON1 or PON3, knockout mice had impaired 3OC12-HSL inactivation compared with wild-type mice. In contrast, PON1-, PON2-, or PON3-targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC 12-HSL degradation by human airway epithelial cell lysates but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2-deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC 12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule. N-3-oxododecanoyl homoserine lactone; cystic fibrosis; innate immunity; paraoxonase