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.

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.

Tissue traction microscopy to quantify muscle contraction within precision-cut lung slices.

In asthma, acute bronchospasm is driven by contractile forces of airway smooth muscle (ASM). These forces can be imaged in the cultured ASM cell or assessed in the muscle strip and the tracheal/bronchial ring, but in each case, the ASM is studied in isolation from the native airway milieu. Here, we introduce a novel platform called tissue traction microscopy (TTM) to measure ASM contractile force within porcine and human precision-cut lung slices (PCLS). Compared with the conventional measurements of lumen area changes in PCLS, TTM measurements of ASM force changes are 1) more sensitive to bronchoconstrictor stimuli, 2) less variable across airways, and 3) provide spatial information. Notably, within every human airway, TTM measurements revealed local regions of high ASM contraction that we call "stress hotspots". As an acute response to cyclic stretch, these hotspots promptly decreased but eventually recovered in magnitude, spatial location, and orientation, consistent with local ASM fluidization and resolidification. By enabling direct and precise measurements of ASM force, TTM should accelerate preclinical studies of airway reactivity.

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.

Lack of cystic fibrosis transmembrane conductance regulator disrupts fetal airway development in pigs.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.

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.

Systems-based discovery of tomatidine as a natural small molecule inhibitor of skeletal muscle atrophy.

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.

Clinical insights into pulmonary hypertension in chronic obstructive pulmonary disease.

Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD). Little is known about the prevalence and clinical profiles of patients with COPD-PH. We report the clinical characteristics, hemodynamic profiles, and prognosis in a large population of patients with COPD referred for right heart catheterization (RHC). We extracted data from all patients referred for RHC between 1997 and 2017 in Vanderbilt's deidentified medical record. PH was defined as mean pulmonary artery pressure >20 mmHg. Pre- and postcapillary PH were defined according to contemporary guidelines. COPD was identified using a validated rules-based algorithm requiring international classification of diseases codes relevant to COPD. We identified 6065 patients referred for RHC, of whom 1509 (24.9%) had COPD and 1213 had COPD and PH. Patients with COPD-PH had a higher prevalence of diabetes, atrial fibrillation, and heart failure compared with COPD without PH. Approximately 55% of patients with COPD-PH had elevated left ventricle (LV) filling pressure. Pulmonary function testing data from individuals with COPD-PH revealed subtype differences, with precapillary COPD-PH having lower diffusion capacity of the lungs for carbon monoxide (DLCO) values than the other COPD-PH subtypes. Patients with COPD-PH had significantly increased mortality compared with COPD alone (hazard ratio [HR]: 1.70, 95% confidence interval [CI]: 1.28-2.26) with the highest mortality among the combined pre- and postcapillary COPD-PH subgroup (HR: 2.39; 95% CI: 1.64-3.47). PH is common among patients with COPD referred for RHC. The etiology of PH in patients with COPD is often mixed due to multimorbidity and is associated with high mortality, which may have implications for risk factor management.

CF airway smooth muscle transcriptome reveals a role for PYK2.

Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

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.

Postnatal airway growth in cystic fibrosis piglets.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis.NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis.

Muscle satellite cells from GRMD dystrophic dogs are not phenotypically distinguishable from wild type satellite cells in ex vivo culture.

Duchenne muscular dystrophy is a neuromuscular degenerative disorder caused by the absence of dystrophin protein. It is characterized by progressive muscle weakness and cycles of degeneration/regeneration accompanying chronic muscle damage and repair. Canine models of muscular dystrophy, including the dystrophin-deficient golden retriever muscular dystrophy (GRMD), are the most promising animal models for evaluation of potential therapies, however canine-specific molecular tools are limited. In particular, few immune reagents for extracellular epitopes marking canine satellite cells (muscle stem cells) are available. We generated an antibody to the satellite cell marker syndecan-4 that identifies canine satellite cells. We then characterized isolated satellite cells from GRMD muscle and wildtype muscle by several in vitro metrics, and surprisingly found no significant differences between the two populations. We discuss whether accumulated adverse changes in the muscle environment rather than cell-intrinsic defects may be implicated in the eventual failure of satellite cell efficacy in vivo.