Passaging Primary Human Bronchial Epithelia Reduces CFTR-Mediated Fluid Transport and Alters mRNA Expression

Primary human bronchial epithelial cultures (HBECs) are used to study airway physiology, disease, and drug development. HBECs often replicate human airway physiology/pathophysiology. Indeed, in the search for cystic fibrosis (CF) transmembrane conductance regulator (CFTR) therapies, HBECs were seen as the “gold standard” in preclinical studies. However, HBECs are not without their limitations: they are non-immortalized and the requirement for human donors, especially those with rare genetic mutations, can make HBECs expensive and/or difficult to source. For these reasons, researchers may opt to expand HBECs by passaging. This practice is common, but to date, there has not been a robust analysis of the impact of expanding HBECs on their phenotype. Here, we used functional studies of airway surface liquid (ASL) homeostasis, epithelial barrier properties, and RNA-seq and Western blotting to investigate HBEC changes over two passage cycles. We found that passaging impaired CFTR-mediated ASL secretion and led to a reduction in the plasma membrane expression of the epithelial sodium channel (ENaC) and CFTR. Passaging also resulted in an increase in transepithelial resistance and a decrease in epithelial water permeability. We then looked for changes at the mRNA level and found that passaging significantly affected 323 genes, including genes involved in inflammation, cell growth, and extracellular matrix remodeling. Collectively, these data highlight the potential for HBEC expansion to impact research findings

Two Small Molecules Restore Stability to a Sub-population of the Cystic Fibrosis Transmembrane conductance Regulator with the Predominant Disease-causing Mutation

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl(−) channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl(−) currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl(−) channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl(−) currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl(−) channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development

Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an ARDS Model

Rationale: Acute Respiratory Distress Syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca 2+ channel involved in store operated Ca 2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but to date, no Orai1-specific inhibitors exist. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models.  Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly-isolated ARDS patient immune cells. A murine acute lung injury model caused by bacterial pneumonia was then used. Mice were infected with P. aeruginosa, S. aureus, methicillin-resistant S. aureus (MRSA) or multidrug-resistant P. aeruginosa (MDR- Pa) and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with an IC50 of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including MRSA. ELD607 worked as an immunomodulator that reduced cytokine levels, lowered neutrophilia and promoted both macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multi-organ inflammation and treat antibiotic-resistant bacteria. </p