Dynamic regulation of airway surface liquid pH by TMEM16A and SLC26A4 in cystic fibrosis nasal epithelia with rare mutations.

In cystic fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel lead to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy. However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation.

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis.

The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA.

Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay.

We present a protocol to generate organoids from air-liquid-interface (ALI)-differentiated nasal epithelia. We detail their application as cystic fibrosis (CF) disease model in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent forskolin-induced swelling (FIS) assay. We describe steps for isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells, and their differentiation in ALI cultures.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion.

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia.

Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability.

Recurrent Respiratory Syncytial Virus Infection in a CD14-Deficient Patient.

Background: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection.

Methods: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection.

CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.

Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE.

High-resolution 3D imaging of fixed and cleared organoids.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells.

Contribution of Host Defence Proteins and Peptides to Host-Microbiota Interactions in Chronic Inflammatory Lung Diseases.

The respiratory tract harbours a variety of microorganisms, collectively called the respiratory microbiota. Over the past few years, alterations in respiratory and gut microbiota composition have been associated with chronic inflammatory diseases of the lungs. How these changes influence disease development and progression is an active field of investigation.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke.

Proinflammatory Cytokines Impair Vitamin D-Induced Host Defense in Cultured Airway Epithelial Cells.

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action.

Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke.

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown.

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells.

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding.

ADAM17 and EGFR regulate IL-6 receptor and amphiregulin mRNA expression and release in cigarette smoke-exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD).

Aberrant activity of a disintegrin and metalloprotease 17 (ADAM17), also known as TACE, and epidermal growth factor receptor (EGFR) has been suggested to contribute to chronic obstructive pulmonary disease (COPD) development and progression. The aim of this study was to investigate the role of these proteins in activation of primary bronchial epithelial cells differentiated at the air-liquid interface (ALI-PBEC) by whole cigarette smoke (CS), comparing cells from COPD patients with non-COPD CS exposure of ALI-PBEC enhanced ADAM17-mediated shedding of the IL-6 receptor (IL6R) and the EGFR agonist amphiregulin (AREG) toward the basolateral compartment, which was more pronounced in cells from COPD patients than in non-COPD controls. CS transiently increased IL6R and AREG mRNA in ALI-PBEC to a similar extent in cultures from both groups, suggesting that posttranslational events determine differential shedding between COPD and non-COPD cultures.

TNF-α and IL-1β-activated human mesenchymal stromal cells increase airway epithelial wound healing in vitro via activation of the epidermal growth factor receptor.

Background: Mesenchymal stromal cells (MSCs) are investigated for their potential to reduce inflammation and to repair damaged tissue. Inflammation and tissue damage are hallmarks of chronic obstructive pulmonary disease (COPD) and MSC infusion is a promising new treatment for COPD. Inflammatory mediators attract MSCs to sites of inflammation and affect their immune-modulatory properties, but little is known about their effect on regenerative properties of MSCs.

Antimicrobial Peptides and Innate Lung Defenses: Role in Infectious and Noninfectious Lung Diseases and Therapeutic Applications.

Respiratory infections are a major clinical problem, and treatment is increasingly complicated by the emergence of microbial antibiotic resistance. Development of new antibiotics is notoriously costly and slow; therefore, alternative strategies are needed. Antimicrobial peptides, central effector molecules of the immune system, are being considered as alternatives to conventional antibiotics.

Basal cells contribute to innate immunity of the airway epithelium through production of the antimicrobial protein RNase 7.

Basal cells play a critical role in the response of the airway epithelium to injury and are recently recognized to also contribute to epithelial immunity. Antimicrobial proteins and peptides are essential effector molecules in this airway epithelial innate immunity. However, little is known about the specific role of basal cells in antimicrobial protein and peptide production and about the regulation of the ubiquitous antimicrobial protein RNase 7.

Polymeric immunoglobulin receptor down-regulation in chronic obstructive pulmonary disease. Persistence in the cultured epithelium and role of transforming growth factor-β.

Rationale: The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown.

Objectives: To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium.

Role of activin-A in cigarette smoke-induced inflammation and COPD.

Activin-A is a pleiotropic cytokine belonging to the transforming growth factor-β superfamily and has been implicated in asthma and pulmonary fibrosis. However, the role of activin-A and its endogenous inhibitor, follistatin, in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. We first quantified activin-A and follistatin in the lungs of air- or cigarette smoke-exposed mice and in the lungs of patients with COPD by immunohistochemistry, ELISA and quantitative real-time PCR.