Cadherin-26 (CDH26) regulates airway epithelial cell cytoskeletal structure and polarity.

Polarization of the airway epithelial cells (AECs) in the airway lumen is critical to the proper function of the mucociliary escalator and maintenance of lung health, but the cellular requirements for polarization of AECs are poorly understood. Using human AECs and cell lines, we demonstrate that cadherin-26 (CDH26) is abundantly expressed in differentiated AECs, localizes to the cell apices near ciliary membranes, and has functional cadherin domains with homotypic binding. We find a unique and non-redundant role for CDH26, previously uncharacterized in AECs, in regulation of cell-cell contact and cell integrity through maintaining cytoskeletal structures. Overexpression of CDH26 in cells with a fibroblastoid phenotype increases contact inhibition and promotes monolayer formation and cortical actin structures. CDH26 expression is also important for localization of planar cell polarity proteins. Knockdown of CDH26 in AECs results in loss of cortical actin and disruption of CRB3 and other proteins associated with apical polarity. Together, our findings uncover previously unrecognized functions for CDH26 in the maintenance of actin cytoskeleton and apicobasal polarity of AECs

Interleukin-13–Induced Mucous Metaplasia Increases Susceptibility of Human Airway Epithelium to Rhinovirus Infection

Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium exhibits mucous metaplasia, which increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium manifest rhinoviral infection at levels many times higher than in well-differentiated cultures of a mucociliary phenotype. Here we tested the hypothesis that mucous metaplasia is also associated with increased levels of rhinoviral infection. Mucous metaplasia was induced with IL-13, which doubled the numbers of goblet cells. In both control (mucociliary) and IL-13– treated (mucous metaplastic) cultures, goblet cells were preferentially infected by rhinovirus. IL-13 doubled the numbers of infected cells by increasing the numbers of infected goblet cells. Furthermore, IL-13 increased both the maturity of goblet cells and the probability that a goblet cell would be infected. The infection of cells other than goblet cells was unaltered by IL-13. Treatment with IL-13 did not alter the levels of rhinovirus receptor ICAM-1, nor did the proliferative effects of IL-13 enhance infection, because rhinovirus did not colocalize with dividing cells. However, the induction of mucous metaplasia caused changes in the apical membrane structure, notably a marked decrease in overall ciliation, and an increase in the overall flatness of the apical surface. We conclude that mucous metaplasia in asthma increases the susceptibility of airway epithelium to infection by rhinovirus because of changes in the overall architecture of the apical surface

Oxidation increases mucin polymer cross-links to stiffen airway mucus gels

Airway mucus in cystic fibrosis (CF) is highly elastic, but themechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates

IL1RL1 asthma risk variants regulate airway type 2 inflammation.

Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 - rs1420101 and rs11685480 - are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33-driven type 2 inflammation

Alternative splicing of interleukin-33 and type 2 inflammation in asthma

Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an “alarmin” during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1β (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways