The respiratory tract possesses a highly regulated innate defense system that includes cilia-mediated mucociliary clearance (MCC). Efficient MCC relies on appropriate hydration of airway surfaces, which is controlled by a blend of transepithelial sodium and liquid absorption, as well as anion and liquid secretion. The latter is mediated primarily by the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Succinate is derived from parasites, microorganisms, and inflammatory cells, and its concentration increases in the airway surface liquid during infections, activating the G protein-coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Because MCC is tightly regulated by second messengers, we tested the hypothesis that succinate signaling was linked to CFTR activity. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport, and induced tracheal constriction in mouse airways. In the CftrΔF508/ΔF508 mouse, increased mucus transport and tracheal constriction were not observed, whereas succinate-induced electrogenic anion secretion remained unaffected. Stimulation of normal human bronchial epithelial cells with succinate activated CFTR-dependent anion secretion and increased airway surface liquid height. Moreover, human bronchial epithelial cells derived from ΔF508-CF individuals that lacked succinate-induced anion secretion, unless incubated with elexacaftor-tezacaftor-ivacaftor, which restored succinate-induced anion secretion, confirmed the tight relationship between SUCNR1 signaling and CFTR function. We have identified a novel mechanism for regulating CFTR/MCC activation that is defective in cystic fibrosis airways. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways.

The respiratory tract possesses a highly regulated innate defense system which includes efficient cilia-mediated mucus transport or mucociliary clearance (MCC). This essential process relies on appropriate hydration of airway surfaces which is controlled by a blend of transepithelial sodium and liquid absorption via the epithelial sodium channel (ENaC), and anion and liquid secretion, primarily regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. MCC is tightly regulated by second messenger signalling pathways. Succinate is derived from parasites, microorganisms and inflammatory cells, and its concentration increases in the airway surface liquid (ASL) during infections. Increases in ASL succinate activates the G-protein coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Here, we tested the hypothesis that succinate signalling was linked to CFTR activity, ASL hydration and increased MCC. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport and induced bronchoconstriction in mouse airways. In parallel, stimulation of human bronchial epithelial cells (HBEC) with succinate activated anion secretion and increased ASL height. All functions activated by succinate/SUCNR1 were impeded when working with tissues and cells isolated from animal models or individuals affected cystic fibrosis (CF) or when CFTR was inhibited. Moreover, when HBECs derived from ΔF508 individuals were incubated with the triple drug combination of elexacaftor/tezacaftor/ivacaftor (ETI), succinate-induced anion secretion was restored, confirming the tight relationship between SUCNR1 signalling and CFTR function. Our results identify a novel activation pathway for CFTR that participates in the defence response of the airways, which is defective in CF. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways. ### Competing Interest Statement The authors have declared no competing interest.

AbstractIntroductionOtitis media is an umbrella term for middle ear inflammation; ranging from acute infection to chronic mucosal disease. It is a leading cause of antimicrobial therapy prescriptions and surgery in children. Despite this, treatments have changed little in over 50 years. Research has been limited by the lack of physiological models of middle ear epithelium.MethodsWe develop a novel human middle ear epithelial culture using an air‐liquid interface (ALI) system; akin to the healthy ventilated middle ear in vivo. We validate this using immunohistochemistry, immunofluorescence, scanning and transmission electron microscopy, and membrane conductance studies. We also utilize this model to perform a pilot challenge of middle ear epithelial cells with SARS‐CoV‐2.ResultsWe demonstrate that human middle ear epithelial cells cultured at an ALI undergo mucociliary differentiation to produce diverse epithelial subtypes including basal (p63+), goblet (MUC5AC+, MUC5B+), and ciliated (FOXJ1+) cells. Mature ciliagenesis is visualized and tight junction formation is shown with electron microscopy, and confirmed by membrane conductance. Together, these demonstrate this model reflects the complex epithelial cell types which exist in vivo. Following SARS‐CoV‐2 challenge, human middle ear epithelium shows positive viral uptake, as measured by polymerase chain reaction and immunohistochemistry.ConclusionWe describe a novel physiological system to study the human middle ear. This can be utilized for translational research into middle ear diseases. We also demonstrate, for the first time under controlled conditions, that human middle ear epithelium is susceptible to SARS‐CoV‐2 infection, which has important clinical implications for safe otological surgery.Level of EvidenceNA.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator ( CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly, inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.

Objective Translational research into subglottic disease is restricted by the availability of primary human tissue originating from this subsite. Primary epithelial cells are also limited by their inability to survive beyond several divisions in culture outside of the body. Specific subglottic cell lines, useful for in vitro studies, have not yet been described. We therefore demonstrate what we believe to be the first immortalized subglottic epithelial cell line. Methods Subglottic tissue was derived from a single adult patient's neoplasia-free human subglottic brushing specimen. Cells were immortalized using a lentiviral vector expressing simian virus 40 T antigen. Karyotyping was performed on the transformed cells using single nucleotide polymorphism array comparative genomic hybridization. Transformed cells were phenotypically characterized by light microscopy, immunohistochemistry, and electrophysiology studies. Results The immortalized subglottic cell line (SG01) was able to divide successfully beyond 20 passages. Karyotyping demonstrated no significant genomic imbalance after immortalization. The cells demonstrated normal epithelial morphology and cytokeratin expression throughout. SG01 cells were also successfully cultured at air-liquid interface (ALI). At ALI cells demonstrated cilia, mucus production, and relevant ion channel expression. Conclusion The novel SG01 subglottic epithelial cell line has been established. This cell line provides a unique resource for researchers to investigate subglottic diseases, such as subglottic stenosis. Level of Evidence NA. Laryngoscope, 129:2640-2645, 2019

Objective: Gastroesophageal reflux is thought to be a risk factor for laryngotracheal stenosis. Bile acids are a component of gastric refluxate and have previously been implicated in the development of fibrosis in other airway subsites. There is clear evidence that bile acids reflux into the upper airway. We therefore investigated the potential role of bile acids in the pathophysiology of laryngotracheal fibrosis and stenosis, specifically investigating the highly conserved process of epithelial-mesenchymal transition (EMT). Study Design: Translational research study. Methods: Human primary tracheal epithelial cells (PTECs) were challenged with the four most common digestive bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic). EMT markers transforming growth factor (TGF)-beta 1, Matrix metalloproteinase (MMP)-9, and procollagen proteins were measured in the supernatant at 48 hours via enzyme-linked immunosorbent assay. Real-time polymerase chain reaction was also used to measure E-cadherin and fibronectin expression. Results: Significantly greater concentrations of TGF-beta 1 and MMP-9 were measured in the culture supernatants of cells treated with each bile acid at 10 mu mol/L. Lithocholic acid and deoxycholic acid induced significantly increased expression of procollagen protein. Upregulation of fibronectin and downregulation of E-cadherin were observed with all bile acids, except for deoxycholic acid. Conclusion: This is the first proof of principle demonstration that physiologically relevant bile acid challenge induces EMT mechanisms in PTECs. This implies a potential role for bile acids in laryngotracheal scarring and airway remodeling of potential translational significance in laryngotracheal stenosis.

Background: Gastro-oesophageal reflux disease (GORD) frequently affects bronchiectasis patients and has been associated with increased disease severity and mortality. Methods: We prospectively assessed reflux and microaspiration in sequential, unselected patients with HRCT-confirmed bronchiectasis from 2 centres comparing findings to age, sex, and BMI-matched chronic bronchitis patients and healthy volunteers. Reflux was assessed by questionnaire, high resolution manometry and 24h pH-impedance, and micro-aspiration by measurement of pepsin and bile acids from BAL or sputum. Bronchiectasis severity was assessed by the BSI. Quality of life (QoL) was measured by SGRQ and QOL-B. Results: 93 bronchiectasis, 25 chronic bronchitis patients and 13 healthy volunteers were enrolled. Bronchiectatics had significantly higher oesophageal acid exposure, total, acid, weakly acid and proximal events on pH-impedance, a higher prevalence of manometric dysmotility, and higher pepsin, bile acids and inflammatory mediators compared to chronic bronchitis patients and healthy controls. Significant correlations of pepsin with IL-8, VEGF, SGRQ, total, distal and proximal reflux and acid exposure time were noted. Bronchiectatics with reflux had more frequent exacerbations [OR (95% CI) 1.21 (0.78-1.65); p<0.01], worse radiological involvement (p<0.01), higher MRC dyspnoea scores (p<0.01), higher colonisation rates (p=0.03), higher BSI [OR 2.58 (0.92-4.24); p<0.01] and worse QOL (p<0.001) than bronchiectatics without reflux. Conclusion: Recognition of the high disease burden of GORD and microaspiration in bronchiectasis with increased exacerbations and severity and poorer QoL despite common proton pump inhibitor use is needed.

RATIONALE:Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP.OBJECTIVES:To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth.METHODS:Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses.MEASUREMENTS AND MAIN RESULTS:Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium.CONCLUSIONS:Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

OBJECTIVE:Gastro-oesophageal reflux disease is thought to be a risk factor for head and neck malignancies. Bile acids are one of the principle components of gastric refluxate and have previously been implicated in the development of oesophageal and bowel malignancies. There is clear evidence that bile acids reflux into the laryngopharynx. Despite this, the carcinogenic properties of bile acids in this area are yet to be fully identified. We therefore investigated the potential role of bile acids in pharyngeal malignancy, through the highly conserved process of epithelial-mesenchymal transition (EMT). EMT occurs in invasion and metastasis and is a central process in the development of epithelial carcinoma.DESIGN:Translational research study.METHODS:Human hypopharyngeal squamous carcinoma FaDu cells were challenged with primary (cholic or chenodeoxycholic) and secondary (deoxycholic or lithocholic) bile acids. EMT-relevant proteins TGF-β1 and MMP-9 were measured in the cell culture supernates at 48 h via ELISA. Cell viability was confirmed >95% via CellTiter-Blue assay.RESULTS:Significantly greater concentrations of TGF-β1 were measured in the culture supernates of cells treated with cholic acid, deoxycholic acid and chenodeoxycholic acid. MMP-9 levels were increased in deoxycholic acid and lithocolic acid stimulations when compared to control (P < 0.05).CONCLUSION:This is the first demonstration that bile acids induce TGF-β1 and MMP-9 in pharyngeal cells. TGF-β1 is considered a master switch for EMT, while MMP-9 is a part of the EMT proteome which degrades basement membranes. This implies a potential role for bile acids in pharyngeal carcinogenesis through the mechanism of EMT and suggests potential novel therapeutic targets.

Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B). The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte−macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L−1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L−1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo. This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anionand fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine- and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2-induced reduction in anion secretion was not due to a decrease in HCO3- transport given that neither a change in CFTR-dependent HCO3- efflux nor Na+/HCO3- cotransporter-dependent HCO3- influx were CO2-sensitive. Hypercapnia also reduced the volume of forskolin-stimulated fluid secretion over 24h, yet had no effect on the HCO3- content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR-dependent, electrogenic Cl- and fluid secretion, but not CFTR-dependent HCO3- secretion, which highlights a differential sensitivity of Cl- and HCO3- transporters to raised CO2 in Calu-3 cells. Hypercapnia also reduced forskolin-stimulated CFTR-dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs.