[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H+ secretion by the nongastric H+/K+ adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H+; consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human
and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.
[Show abstract][Hide abstract] ABSTRACT: 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. In order 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 to 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 to 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 mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.
Full-text · Article · Jan 2016 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: Lung transplantation is an effective therapy for many patients with end-stage lung disease. Few centers across the United States offer this therapy, as a successful lung transplant program requires significant institutional resources and specialized personnel. Analysis of the United Network of Organ Sharing database reveals that the failure rate of new programs exceeds 40%. These data suggest that an accurate assessment of program viability as well as a strategy to continuously assess defined quality measures is needed. As part of strategic planning, regional availability of recipient and donors should be assessed. Additionally, analysis of institutional expertise at the physician, support staff, financial, and administrative levels is necessary. In May of 2007, we started a new lung transplant program at the University of Iowa Hospitals and Clinics and have performed 101 transplants with an average recipient 1-year survival of 91%, placing our program among the top in the country for the past 5 years. Herein, we review internal and external factors that impact the viability of a new lung transplant program. We discuss the use of four prospectively identified quality measures: volume, recipient outcomes, financial solvency, and academic contribution as one approach to achieve programmatic excellence.
[Show abstract][Hide abstract] ABSTRACT: 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 β-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.
Full-text · Article · Nov 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl− channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary “conformational protein-associated” diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.
No preview · Article · Oct 2014 · Journal of Cystic Fibrosis
[Show abstract][Hide abstract] ABSTRACT: Background
Chronic sinusitis is universal in cystic fibrosis (CF) and our current treatments are ineffective in reversing sinus disease. The objective of this work was to determine if increasing CF transmembrane conductance regulator (CFTR) activity by ivacaftor could treat CF sinus disease and assess its effect on primary sinus epithelial cultures.Methods
Case report of 1 patient with long-standing chronic sinus disease and a new diagnosis of CF with a mild mutation (P205S) and a severe mutation (G551D). We discuss clinical changes in symptoms, radiographic findings, nasal potential difference testing, and nasal pH values before and after treatment with ivacaftor. We then developed primary sinonasal epithelial cell cultures from a biopsy of the patient to determine changes in airway surface liquid (ASL) pH and ASL viscosity after ivacaftor treatment.ResultsIvacaftor treatment reversed CT findings of CF sinus disease, increased nasal voltage and pH, and resolved sinus symptoms after 10 months of therapy. Ivacaftor significantly increased ASL pH and decreased ASL viscosity in primary airway cultures.Conclusion
This report documents the reversal of CF sinus disease. Based on our in vivo and in vitro results, we speculate that ivacaftor may reverse CF sinusitis by increasing ASL pH and decreasing ASL viscosity. These studies suggest that CFTR modulation may be effective in treating CF and perhaps non-CF sinusitis.
Full-text · Article · Oct 2014 · International Forum of Allergy and Rhinology
[Show abstract][Hide abstract] ABSTRACT: Lung disease in people with cystic fibrosis (CF) is initiated by defective host defense that predisposes airways to bacterial
infection. Advanced CF is characterized by a deficit in mucociliary transport (MCT), a process that traps and propels bacteria
out of the lungs, but whether this deficit occurs first or is secondary to airway remodeling has been unclear. To assess MCT,
we tracked movement of radiodense microdisks in airways of newborn piglets with CF. Cholinergic stimulation, which elicits
mucus secretion, substantially reduced microdisk movement. Impaired MCT was not due to periciliary liquid depletion; rather,
CF submucosal glands secreted mucus strands that remained tethered to gland ducts. Inhibiting anion secretion in non-CF airways
replicated CF abnormalities. Thus, impaired MCT is a primary defect in CF, suggesting that submucosal glands and tethered
mucus may be targets for early CF treatment.
[Show abstract][Hide abstract] ABSTRACT: Background Asthma is a disease of acute and chronic inflammation in which cytokines play a critical role in orchestrating the allergic inflammatory response. Interleukin 13 (IL-13) and transforming growth factor β (TGFβ) promote fibrotic airway remodeling, a major contributor to disease severity. Improved understanding is needed because current therapies are inadequate for suppressing development of airway fibrosis. IL-13 is known to stimulate respiratory epithelial cells to produce TGFβ, but the mechanism through which this occurs is unknown. Here we tested the hypothesis that reactive oxygen species (ROS) are a critical signaling intermediary between IL-13 or allergen stimulation and TGFβ dependent airway remodeling. Methods We used cultured human bronchial epithelial cells and an in vivo mouse model of allergic asthma to map a pathway where allergens enhanced mitochondrial ROS, which is an essential upstream signal for TGFβ activation and enhanced collagen production and deposition in airway fibroblasts. Results We show that mitochondria in airway epithelium are an essential source of ROS that activate TGFβ expression and activity. TGFβ from airway epithelium stimulates collagen expression in fibroblasts, contributing to an early fibrotic response to allergen exposure in cultured human airway cells and in ovalbumin-challenged mice. Treatment with the mitochondrial-targeted antioxidant MitoTEMPO significantly attenuated mitochondrial ROS, TGFβ, and collagen deposition in OVA-challenged mice and in cultured human epithelial cells. Conclusions Our findings suggest that mitochondria are a critical source of ROS for promoting TGFβ activity that contributes to airway remodeling in allergic asthma. Mitochondrial-targeted antioxidants may be a novel approach for future asthma therapies.
No preview · Article · Jul 2014 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Given the increased use of iron-containing nanoparticles in a number of applications, it is important to understand any effects that iron-containing nanoparticles can have on the environment and human health. Since iron concentrations are extremely low in body fluids, there is potential that iron-containing nanoparticles may influence the ability of bacteria to scavenge iron for growth, affect virulence and inhibit antimicrobial peptide (AMP) function. In this study, Pseudomonas aeruginosa (PA01) and AMPs were exposed to iron oxide nanoparticles, hematite (α-Fe2O3), of different sizes ranging from 2 to 540 nm (2 ± 1, 43 ± 6, 85 ± 25 and 540 ± 90 nm) in diameter. Here we show that the greatest effect on bacterial growth, biofilm formation, and AMP function impairment is found when exposed to the smallest particles. These results are attributed in large part to enhanced dissolution observed for the smallest particles and an increase in the amount of bioavailable iron. Furthermore, AMP function can be additionally impaired by adsorption onto nanoparticle surfaces. In particular, lysozyme readily adsorbs onto the nanoparticle surface which can lead to loss of peptide activity. Thus, this current study shows that co-exposure of nanoparticles and known pathogens can impact host innate immunity. Therefore, it is important that future studies be designed to further understand these types of impacts.
[Show abstract][Hide abstract] ABSTRACT: Disrupted HCO3(-) transport and reduced airway surface liquid (ASL) pH in cystic fibrosis (CF) may initiate airway disease. We hypothesized that ASL pH is reduced in neonates with CF.
In neonates with and without CF, we measured pH of nasal ASL. We also measured nasal pH in older children and adults.
In neonates with CF, nasal ASL (pH5.2±0.3) was more acidic than in non-CF neonates (pH6.4±0.2). In contrast, nasal pH of CF children and adults was similar to values measured in people without CF.
At an age when infection, inflammation and airway wall remodeling are minimal, neonates with CF had an acidic nasal ASL compared to babies without CF. The CF:non-CF pH difference disappeared in older individuals, perhaps because secondary manifestations of disease increase ASL pH. These results aid understanding of CF pathogenesis and suggest opportunities for therapeutic intervention and monitoring of disease.
Full-text · Article · Jan 2014 · Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society
[Show abstract][Hide abstract] ABSTRACT: Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury.
Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded.
Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection.
Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection.
[Show abstract][Hide abstract] ABSTRACT: Massively parallel DNA sequencing data suggest the presence of a diverse bacterial community in healthy lungs, yet dogma dictates that immune mechanisms keep lungs sterile. Whether all bacterial DNA in bronchoalveolar lavage (BAL) corresponds to live bacteria or equals diversity in lung parenchyma is unknown. We developed a DNase treatment-based method for bacterial DNA processing that discerns between intact cell DNA and DNA from bacteria with damaged cell walls. We investigated bacterial DNA load and diversity in transoral BAL samples and in transbronchial BAL and lung tissue samples obtained under sterile conditions in 6-week-old pigs. We hypothesized that bacterial DNA would correspond to dead bacteria and/or sample contamination from mouth bacteria and that lung tissue would be sterile. In both transoral and transbronchial BAL samples, DNase-resistant DNA was about 35% of total DNA. Most 16s rRNA gene sequences corresponded to the order Sphingobacteriales and to the families Mycoplasmataceae, Pasteurellaceae, and Xanthomonadaceae, with variable proportions and DNase sensitivity in each compartment. We were unable to culture bacteria from distal lung samples, and DNase-resistant DNA corresponded to approximately 4% of the total bacterial DNA load. Most 16s rRNA gene sequences corresponded to the Bacteroidaceae, Lactobacillaceae, Lachnospiraceae, and Ruminococcaceae families with similar proportions in DNase-untreated and -treated samples. These data suggest that normal distal lungs in pigs are mostly sterile and that antimicrobial mechanisms tend to keep the airways free from frequently inhaled and aspirated bacteria, but a low biomass of intact bacteria may persist in airways sampled by BAL. Careful consideration of these factors is therefore important when studying bacterial communities present in diseased lungs and comparing them to healthy lungs.
No preview · Article · Jan 2014 · Annals of the American Thoracic Society
[Show abstract][Hide abstract] ABSTRACT: To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4(+) cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4(+) epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4(-) epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4(+) epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4(+) cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4(+) epithelial cells significantly rescued defects in Cl(-) transport. Therefore, targeting the α6β4(+) epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.
[Show abstract][Hide abstract] ABSTRACT: Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
Full-text · Article · Jul 2013 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to discern between DNA from live bacteria and bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) and lung samples in young healthy pigs. Live bacterial DNA was DNase I-resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA load in porcine BAL and lung tissue by comparing DNase I treated vs. untreated samples. In contrast to BAL, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL and lung homogenates. In BAL, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive suggesting predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive and bacterial diversity determined by 16s rRNA gene sequencing was similar in DNase I-treated and untreated samples.Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.