Clinical and Biological Features of a Thickened Basement Membrane Zone in Asthma.

Background: A subset of asthma patients have airway pathology characterized by a thickened subepithelial basement membrane zone ("BMZ-thick asthma").

Objectives: To characterize the clinical features of BMZ-thick asthma and to determine if BMZ thickness accompanies specific patterns of inflammation in the airway epithelium.

Methods: Design-based stereology was used to quantify BMZ thickness in endobronchial biopsy tissue sections from 109 asthma patients and 41 healthy controls from the Severe Asthma Research Program-3 whose participants had undergone spirometry and gene expression profiling in airway epithelial brushings.

Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements.

A simple method to generate human airway epithelial organoids with externally orientated apical membranes.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa.

The Type 2 Asthma Mediator IL-13 Inhibits Severe Acute Respiratory Syndrome Coronavirus 2 Infection of Bronchial Epithelium.

Asthma is associated with chronic changes in the airway epithelium, a key target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many epithelial changes, including goblet cell metaplasia, are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We found that IL-13 stimulation of differentiated human bronchial epithelial cells (HBECs) cultured at air-liquid interface reduced viral RNA recovered from SARS-CoV-2-infected cells and decreased double-stranded RNA, a marker of viral replication, to below the limit of detection in our assay.

Epithelial miR-141 regulates IL-13-induced airway mucus production.

IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation.

The type 2 asthma mediator IL-13 inhibits SARS-CoV-2 infection of bronchial epithelium.

Rationale: Asthma is associated with chronic changes in the airway epithelium, a key target of SARS-CoV-2. Many epithelial changes are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown.

Objectives: We sought to discover how IL-13 and other cytokines affect expression of genes encoding SARS-CoV-2-associated host proteins in human bronchial epithelial cells (HBECs) and determine whether IL-13 stimulation alters susceptibility to SARS-CoV-2 infection.

Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets.

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures.

Comparative Super-Resolution Mapping of Basal Feet Reveals a Modular but Distinct Architecture in Primary and Motile Cilia.

In situ molecular architecture analysis of organelles and protein assemblies is essential to understanding the role of individual components and their cellular function, and to engineering new molecular functionalities. Through a super-resolution-driven approach, here we characterize the organization of the ciliary basal foot, an appendage of basal bodies whose main role is to provide a point of anchoring to the microtubule cytoskeleton. Quantitative image analysis shows that the basal foot is organized into three main regions linked by elongated coiled-coil proteins, revealing a conserved modular architecture in primary and motile cilia, but showing distinct features reflecting its specialized functions.

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiX technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements.

Cystic Fibrosis Lung Transplant Recipients Have Suppressed Airway Interferon Responses during Infection.

Lung transplantation can be lifesaving in end-stage cystic fibrosis (CF), but long-term survival is limited by chronic lung allograft dysfunction (CLAD). Persistent upper airway (PsA) colonization can seed the allograft. While PsA infection is associated with CLAD in non-CF recipients, this association is less clear for CF recipients experiencing PsA recolonization.

Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants.

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC down to ~300 nM following initial structure-activity studies.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection.

Na+/H+ Exchangers Are Required for the Development and Function of Vertebrate Mucociliary Epithelia.

Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients.

Combination potentiator ('co-potentiator') therapy for CF caused by CFTR mutants, including N1303K, that are poorly responsive to single potentiators.

Background: Current modulator therapies for some cystic fibrosis-causing CFTR mutants, including N1303K, have limited efficacy. We provide evidence here to support combination potentiator (co-potentiator) therapy for mutant CFTRs that are poorly responsive to single potentiators.

Methods: Functional synergy screens done on N1303K and W1282X CFTR, in which small molecules were tested with VX-770, identified arylsulfonamide-pyrrolopyridine, phenoxy-benzimidazole and flavone co-potentiators.

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter.

The most common cystic fibrosis-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1.

Corticosteroid and long-acting ß-agonist therapy reduces epithelial goblet cell metaplasia.

Background: Bronchial epithelial goblet cell metaplasia (GCM) with hyperplasia is a prominent feature of asthma, but the effects of treatment with corticosteroids alone or in combination with a long-acting β -adrenergic receptor agonist (LABA) on GCM in the bronchial epithelium are unknown.

Objectives: To determine whether corticosteroid alone or in combination with a LABA alters protein and gene expression pathways associated with IL-13-induced goblet cell metaplasia.

Results: We evaluated the effects of fluticasone propionate (FP) and of salmeterol (SM), on the response of well-differentiated cultured bronchial epithelial cells to interleukin-13 (IL-13).

In vivo and in vitro ivacaftor response in cystic fibrosis patients with residual CFTR function: N-of-1 studies.

Rationale: Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non-human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product.

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR activity.

Cigarette Smoke Mediates Nuclear to Cytoplasmic Trafficking of Transcriptional Inhibitor Kaiso through MUC1 and P120-Catenin.

Lung cancer is the leading cause of cancer-related death, and 87% of these deaths are directly attributable to smoking. Using three-dimensional cultures of primary human bronchial epithelial cells, we demonstrated that loss of adherens junction protein, epithelial cadherin, and the aberrant interaction of its adherens junction binding partner, p120-catenin (p120ctn), with the cytoplasmic tail of apical mucin-1 (MUC1-CT) represent initiating steps in the epithelial-to-mesenchymal transition. Smoke provoked the rapid nuclear entry of p120ctn in complex with MUC1-CT that was inhibited using the MUC1-CT inhibitory peptides, PMIP and GO-201.

Correlation of Prenatal Diagnosis and Pathology Findings Following Dilation and Evacuation for Fetal Anomalies.

Context: -Despite increased use of dilation and evacuation in the setting of fetuses with developmental anomalies, the pathology examination of fragmented specimens obtained by this technique has been understudied.

Objectives: -To correlate pathologic findings in second-trimester fetal dilation and evacuation specimens with prenatal diagnoses established through ultrasound and/or chromosome studies to determine the value of pathology examination for supplementing or correcting clinical diagnoses.

Design: -In this retrospective study, clinical and pathology findings were correlated in 448 dilation and evacuation specimens performed for second-trimester termination of pregnancy for fetal anomalies discovered on ultrasound examination (278 cases) or chromosome analysis (170 cases).

Epithelial tethering of MUC5AC-rich mucus impairs mucociliary transport in asthma.

The development of pathologic mucus, which is not readily cleared from the airways, is an important contributor to the morbidity and mortality associated with asthma. It is not clear how the major airway mucins MUC5AC and MUC5B are organized within the mucus gel or how this gel contributes to airway obstruction in asthma. Here, we demonstrated that mucus plugs from individuals with fatal asthma are heterogeneous gels with distinct MUC5AC- and MUC5B-containing domains.

Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.

Pendrin (SLC26A4) is a Cl(-)/anion exchanger expressed in the epithelium of inflamed airways where it is thought to facilitate Cl(-) absorption and HCO3 (-) secretion. Studies using pendrin knockout mice and airway epithelial cells from hearing-impaired subjects with pendrin loss of function suggest involvement of pendrin in inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation of airway surface liquid (ASL) volume. Here we identified small-molecule pendrin inhibitors and demonstrated their efficacy in increasing ASL volume.

Potentiators of Defective ΔF508-CFTR Gating that Do Not Interfere with Corrector Action.

Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action.

Some gating potentiators, including VX-770, diminish ΔF508-CFTR functional expression.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit. However, most CF patients carry the ΔF508 mutation, which impairs CFTR folding, processing, function, and stability.