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

The incidence and sites of mucus accumulation, and molecular regulation of mucin gene expression, in COVID-19 lung disease have not been reported.Characterize incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease.Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by AB-PAS and immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. SARS-CoV-2-infected human bronchial epithelial (HBE) cultures were utilized to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures.MUC5B and variably MUC5AC RNA levels were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the sub-acute/chronic disease phase following SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of COVID-19 subjects in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days post inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days post inoculation. SARS-CoV-2 infection of HBE cultures induced expression of EGFR ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways, or dexamethasone administration, reduced SARS-CoV-2-induced mucin expression.SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation post SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Mutations in the FOXF1 gene, encoding the mesenchymal Forkhead Box (FOX) transcription factor, are linked to Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. While proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we utilized murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in ACDMPV patients) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into AEC1s. Alveolar organoids containing Foxf1WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1WT/S52F mice was associated with decreased expression of non-canonical WNT5A ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 cell expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in ACDMPV patients.

Common genetic variants have been associated with idiopathic pulmonary fibrosis (IPF).To determine functional relevance of the 10 IPF-associated common genetic variants we previously identified.We performed expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) mapping, followed by co-localization of eQTL and mQTL with genetic association signals and functional validation by luciferase reporter assays. Illumina MEGA genotyping arrays, mRNA sequencing, and Illumina 850k methylation arrays were performed on lung tissue of participants with IPF (234 RNA and 345 DNA samples) and non-diseased controls (188 RNA and 202 DNA samples).Focusing on genetic variants within 10 IPF-associated genetic loci, we identified 27 eQTLs in controls and 24 eQTLs in cases (false-discovery-rate-adjusted p<0.05). Among these signals, we identified associations of lead variants rs35705950 with expression of MUC5B and rs2076295 with expression of DSP in both cases and controls. mQTL analysis identified CpGs in gene bodies of MUC5B (cg17589883) and DSP (cg08964675) associated with the lead variants in these two loci. We also demonstrated strong co-localization of eQTL/mQTL and genetic signal in MUC5B (rs35705950) and DSP (rs2076295). Functional validation of the mQTL in MUC5B using luciferase reporter assays demonstrates that the CpG resides within a putative internal repressor element.We have established a relationship of the common IPF genetic risk variants rs35705950 and rs2076295 with respective changes in MUC5B and DSP expression and methylation. These results provide additional evidence that both MUC5B and DSP are involved in the etiology of IPF.

Pulmonary fibrosis is an interstitial lung disease caused by various factors such as exposure to workplace environmental contaminants, drugs, or X-rays. Epithelial cells are among the driving factors of pulmonary fibrosis. Immunoglobulin A (IgA), traditionally thought to be secreted by B cells, is an important immune factor involved in respiratory mucosal immunity. In the current study, we found that lung epithelial cells are involved in IgA secretion, which, in turn, promotes pulmonary fibrosis. Spatial transcriptomics and single-cell sequencing suggest that Igha transcripts were highly expressed in the fibrotic lesion areas of lungs from silica-treated mice. Reconstruction of B-cell receptor (BCR) sequences revealed a new cluster of AT2-like epithelial cells with a shared BCR and high expression of genes related to IgA production. Furthermore, the secretion of IgA by AT2-like cells was trapped by the extracellular matrix and aggravated pulmonary fibrosis by activating fibroblasts. Targeted blockade of IgA secretion by pulmonary epithelial cells may be a potential strategy for treating pulmonary fibrosis.

Pulmonary fibrosis is an interstitial lung disease caused by various factors such as exposure to workplace environmental contaminants, drugs, or X-rays. Epithelial cells are among the driving factors of pulmonary fibrosis. Immunoglobulin A (IgA), traditionally thought to be secreted by B cells, is an important immune factor involved in COVID-19 infection and vaccination. In current study, we found lung epithelial cells were involved in IgA secretion which, in turn, promoted pulmonary fibrosis. The spatial transcriptomics and single-cell sequencing suggests that Igha transcripts were highly expressed in the fibrotic lesion areas of lungs from silica-treated mice. Reconstruction of B-cell receptor (BCR) sequences revealed a new cluster of AT2-like epithelial cells with a shared BCR and high expression of genes related to IgA production. Furthermore, the secretion of IgA by AT2-like cells were trapped by extracellular matrix and aggravated pulmonary fibrosis by activating fibroblasts. Targeted blockade of IgA secretion by pulmonary epithelial cells may be a potential strategy for treating pulmonary fibrosis.

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.

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.

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 (