Culture with apically applied healthy or disease sputum alters the airway surface liquid proteome and ion transport across human bronchial epithelial cells.

Airway secretions contain many signalling molecules and peptides/proteins that are not found in airway surface liquid (ASL) generated by normal human bronchial epithelial cells (NHBE) in vitro. These play a key role in innate defence and mediate communication between the epithelium, immune cells and the external environment. We investigated how culture of NHBE with apically applied secretions from healthy or disease (Cystic Fibrosis, CF) lungs affected epithelial function with a view to providing better in vitro models of the in vivo environment. NHBE from 6-8 different donors were cultured at air-liquid interface (ALI), with apically applied sputum from normal healthy donors (NLS) or CF donors (CFS) for 2-4 hours, 48 hours or with sputum reapplied over 48 hours. Proteomic analysis was carried out on the sputa and on NHBE ASL before and after culture with sputa. Transepithelial electrical resistance (TEER), short circuit current (Isc) and changes to ASL height were measured. There were 71 proteins common to both sputa but not ASL. The protease:protease inhibitor balance was increased in CFS compared to NLS and ASL. Culture of NHBE with sputa for 48 hours identified additional factors not present in NLS, CFS or ASL alone. Culture with either NLS or CFS for 48 hours increased CFTR activity, calcium activated chloride channel (CaCC) activity and changed ASL height. These data indicate that culture with healthy or disease sputum changes the proteomic profile of ASL and ion transport properties of NHBE and this may increase physiological relevance when using in vitro airway models.

Naringenin Regulates Lipopolysaccharide-Induced Abnormal Airway Surface Liquid Secretion

Airway surface liquid (ASL) is one of the key factors affecting the respiratory system's physiological function. Abnormal ASL secretion can increase the incidence of various respiratory diseases. Lipopolysaccharide (LPS) stimulation can damage the airway epithelial barrier, affect the concentration of ASL contents, and down-regulate ion channel expression, which in turn causes abnormal ASL secretion. Naringenin, which exists in many Citrus foods, has the ability to promote airway surface liquid secretion. This work is designed to investigate the regulatory mechanism of naringenin on LPS-induced abnormal ASL secretion. The effects of naringenin and LPS on the viability of Calu-3 cells were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). ASL secretion volume was measured by a micropipette on air–liquid interface cultured cells. The concentration of Cl−, Na+, lysozyme, and total protein in ASL were respectively measured by assay kits. The mRNA expressions were determined by quantitative real-time polymerase chain reaction, and proteins were measured by enzyme-linked immunosorbent assay. The results indicated that LPS could affect ASL secretion and regulate cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 1 (AQP1) and aquaporin 5 (AQP5) expression. Naringenin had the ability to regulate the ASL secretion by increasing secretion volume, and Cl− and Na+ concentrations, reducing lysozyme and total protein content, and regulating CFTR, AQP1, and AQP5 expression. This study indicated that naringenin had regulating effects to attenuate LPS-induced abnormal ASL secretion.

Ion concentrations in nasal airway surface liquid: a prediction model for the identification of cystic fibrosis carriers

Background: Cystic fibrosis (CF) carriers seem to have a higher risk to develop chronic rhino-sinusitis (CRS), although the full underlying mechanisms are unknown. Ion concentrations in nasal airway surface liquid (ASL) may be influenced by the heterozygosity for CF gene mutation, with possible impacts on the development of CRS. Methods: A cheap and feasible standardized technique was designed to measure the ion levels in nasal ASL. With this purpose we collected, under basal conditions, samples from the nasal cavity of 165 adults: 14 homozygous for CF, 83 carriers and 68 healthy controls. Sodium (Na) and Chlorine (Cl) concentrations were then evaluated among different groups. Results: Statistical analysis revealed a significant difference of Na and Cl values between controls and carriers and between controls and homozygotes. Receiver operating characteristic (ROC) curves and derived indicators (Youden‘s index and Area Under the Curve, AUC) were used to further evaluate the diagnostic capability of Na and Cl concentrations to differentiate heterozygotes from controls. ROC curves demonstrated that the optimal diagnostic cut-off value of Na is at 124, and the optimal cut-off value of Cl is at 103,2. Conclusion: ASL sampling can be considered a new diagnostic tool for providing quantitative information on nasal ion composition. According to our findings, Na and Cl concentrations of nasal ASL could represent a useful tool to assess heterozygotes and healthy controls.

Low pH and temperature of airway surface liquid are key determinants that potentiate SARS-CoV-2 infectivity

: Overwhelming responses are seen at preclinical and clinical levels to understand and combat coronavirus disease 2019 [COVID-19] pandemic that is caused by severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]. Encouraging successes are achieved in view of diagnostic, therapeutic and preventive measures including vaccines development. In fact, structural information of SARS-CoV-2 and molecular steps that help this virus to target AECs are appreciably studied. Furthermore, the heterogeneous and complex nature of COVID-19 is extensively revealed at molecular, genetic, and epigenetic and microenvironment levels. In spite of these developments in COVID-19 pathogenesis, reasons behind the targeted infection by SARS-CoV-2 to AECs are poorly understood. In this mini-review, we highlight the roles of pH and temperature of airway surface liquid [ASL] as a key determining factor that may contribute towards enhanced targeted infection by SARS-CoV-2 leading to COVID-19.

The Application of Bicarbonate Recovers the Chemical-Physical Properties of Airway Surface Liquid in Cystic Fibrosis Epithelia Models

Cystic fibrosis (CF) is a genetic disease associated with the defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes obstructive disease and chronic bacterial infections in airway epithelia. Deletion of phenylalanine at position 508, p.F508del, the most frequent mutation among CF patients, causes a folding and traffic defect, resulting in a dramatic reduction in the CFTR expression. To investigate whether the direct application of bicarbonate could modify the properties of the airway surface liquid (ASL), we measured the micro-viscosity, fluid transport and pH of human bronchial epithelial cells monolayers. We have demonstrated that the treatment of a CF-epithelia with an iso-osmotic solution containing bicarbonate is capable of reducing both, the ASL viscosity and the apical fluid re-absorption. We suggest the possibility of design a supportive treatment based on topical application of bicarbonate, or any other alkaline buffer.

Airway Surface Liquid pH Regulation in Airway Epithelium Current Understandings and Gaps in Knowledge

Knowledge on the mechanisms of acid and base secretion in airways has progressed recently. The aim of this review is to summarize the known mechanisms of airway surface liquid (ASL) pH regulation and their implication in lung diseases. Normal ASL is slightly acidic relative to the interstitium, and defects in ASL pH regulation are associated with various respiratory diseases, such as cystic fibrosis. Basolateral bicarbonate (HCO3−) entry occurs via the electrogenic, coupled transport of sodium (Na+) and HCO3−, and, together with carbonic anhydrase enzymatic activity, provides HCO3− for apical secretion. The latter mainly involves CFTR, the apical chloride/bicarbonate exchanger pendrin and paracellular transport. Proton (H+) secretion into ASL is crucial to maintain its relative acidity compared to the blood. This is enabled by H+ apical secretion, mainly involving H+/K+ ATPase and vacuolar H+-ATPase that carry H+ against the electrochemical potential gradient. Paracellular HCO3− transport, the direction of which depends on the ASL pH value, acts as an ASL protective buffering mechanism. How the transepithelial transport of H+ and HCO3− is coordinated to tightly regulate ASL pH remains poorly understood, and should be the focus of new studies.