Probes for INS

Background: Orai1 is a plasma membrane Ca2+ channel that is involved in store-operated calcium entry (SOCE). In pulmonary cells, SOCE regulates gene expression and stimulates cytokine, mucin, and protease secretion. Activation of Orai1/SOCE results in recruitment of neutrophils to the lungs. Orai1 activation is also upstream of transcription factors such as nuclear factor of activated T cells, which facilitate onset of inflammation. In cystic fibrosis (CF), the immune response is dysregulated, and the lung is chronically inflamed, but Orai1 expression in the CF lung is poorly understood. Orai1 is a promising target for drug development, so we tested the hypothesis that Orai1 was upregulated in CF lungs. Methods: We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). We imaged sections by confocal and super resolution microscopy and analyzed Orai1 and STIM1 expression, colocalization, and particle size in different pulmonary cell types. Results: Orai1 was broadly expressed throughout the lung, but expression was greatest in immune cells. At messenger RNA and protein levels, there were no consistent trends in expression levels between the three groups. Orai1 must interact with STIM1 to activate SOCE, so we used STIM1/Orai1 colocalization as a marker of Orai1 activity. Using this approach, we found significantly greater colocalization between these proteins in CF and asthma lung epithelia (CF 50%, asthma 54%, normal 15%), interstitia (CF 57%, asthma 49%, normal 16%), and luminal immune cells (CF 66%, asthma 70%, normal 38%). Orai1 also aggregates as part of its interaction process. Using super-resolution microscopy, we found significantly more Orai1 and STIM1 aggregation in immune cells from CF and asthmatic lungs (average Orai1 particle size: CF 52 nm, asthma 63 nm, normal 28 nm; average STIM1 particle size: CF 77 nm, asthma 59 nm, normal 14 nm). We also looked at Orai1 in peripheral blood neutrophils from normal and CF donors (5 per group). All CF subjects took elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), but under baseline conditions, there were significantly bigger puncta in CF neutrophils (CF 10 nm, normal 6 nm), suggesting that these patients continued to have significant inflammation despite taking ELX/TEZ/IVA, and mean percentage predicted forced expiratory volume in 1 second in our CF cohort was 55 ± 22%, indicating that these patients had persistent lung disease. Conclusions: This is the first comprehensive analysis of Orai1 and STIM1 expression in lungs from normal and CF donors. We found evidence that Orai1 was more active in CF than normal lungs. This novel application of super-resolution microscopy has the potential to be used in clinical settings for analysis of ex vivo patient samples and to evaluate inflammation in people with CF. Although traditional biomarkers of inflammation such as serum cytokine levels are useful for rapid detection of systemic inflammation, our technique allows for precise localization of upstream inflammatory signaling biomarkers at the cellular level and in fixed samples. Therefore, these data suggest that Orai1 has a key role in CF lung inflammation and attest to the potential of anti-inflammatory therapeutics that target Orai1.We used LungMAP to analyze single-cell ribonucleic acid (RNA) sequencing data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, CF, and asthma (disease control) donors (4 male/4 female per group). W

Background: Mucociliary clearance (MCC) is a vital innate defense mechanism that is impaired in people with cystic fibrosis (CF) and animal CF models. Dysfunctional MCC contributes to airway inflammation and infection, which hasten lung function decline. Most people with CF benefit from highly effective CF transmembrane conductance regulator (CFTR) modulators, but some mutations are unresponsive to currently available modulators, and even people with CF who benefit from modulator therapy may be unable to clear chronic pulmonary infections. Accordingly, CFTR-independent methods to increase MCC are needed. We previously discovered that the combination of low-dose cholinergic with βadrenergic agonists synergistically increased MCC velocity (MCCV) in ex vivo tracheal preparations from ferrets and newborn piglets. MCC was also significantly greater in tracheas from CF ferrets to a value of approximately 55% of that in wild-type animals. The MCCV increases were produced without inducing airway narrowing [1]. To further our preclinical work, we tested three hypotheses. We hypothesized that synergistic increases in MCCV by the combined agonists involve epithelial sodium channel (ENaC) inhibition, greater secretion of bicarbonate, and additivity with CFTR modulators. Methods: To test these hypotheses, we measured MCCV in excised newborn piglet tracheas with 10 µM formoterol (beta-adrenergic agonist) plus 0.3 µM methacholine (cholinergic agonist) with and without 10 µM benzamil (ENaC inhibitor) using particle tracking. Bicarbonate secretion rates were measured in tracheal mucosa of Yucatan minipigs mounted in Ussing chambers using a pH-stat method with pH electrodes and automated titrators (Metrohm Titrando 902). To assess whether the synergy agonists improved CF tissues exposed to CFTR modulators, we used high-speed digital microscopy to measure the effective diffusivity (Deff in µm2 /msec) of approximately 2-µm fluorescent polystyrene spheres (0.1%, ThermoFisher) added to the apical surface fluid layer of human CF primary nasal cell cultures (F508del homozygote) grown under air-liquid interface conditions with and without elexacaftor/ tezacaftor/ivacaftor (ELX/TEZ/IVA) (3 μM ELX, 3 μM TEZ, 10 μM IVA). Results: Baseline MCCV was 6 times as high with benzamil inhibition of ENaC (0.5 ± 0.7 mm/min to 3.0 ± 0.7 mm/min; p = 0.02, 4 piglets), but when benzamil was present during synergistically stimulated MCCV, no further increase was seen, consistent with the hypothesis that ENaC was already inhibited by the synergy agonists (MCCV: synergy agonists, 13.9 ± 1.6 mm/ min vs. synergy agonists + benzamil, 14.0 ± 1.6 mm/min; p = 0.97, n = 4 piglets, each condition). The synergy agonists increased bicarbonate secretion rates by about 83% (0.6 ± 0.2 µmol/cm2 per hour at baseline vs. 1.1 ± 0.3 µmol/cm2 per hour with synergy agonists, 5 experiments with 3 pig tracheas). Particle diffusivity in CF primary nasal cell cultures showed synergy agonists plus ELX/TEZ/IVA > synergy agonists > ELX/TEZ/IVA > no treatment. Conclusions: Results were consistent with our hypotheses. The combination of beta adrenergic plus low-dose cholinergic agonists produces synergistic increases in MCCV by inhibiting ENaC and increasing bicarbonate secretion and appears to be at least additive to the effects induced by ELX/TEZ/IVA modulator therapy.

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Methods: Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, as well as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. One week later, the medium was changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Results: Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 ± 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 ± 9%, n = 3). Conclusions: In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.

Background: Mucociliary clearance is heavily affected by mucus concentration, with its attendant biophysical properties. Mucus concentration is tightly regulated by luminal mucin secretion and mucus hydration. Although small (distal) airways (