Air-liquid Interface Cultures Research Articles

Spatiotemporal Clusters of ERK Activity Coordinate Cytokine-induced Inflammatory Responses in Human Airway Epithelial Cells.

Spatially coordinated ERK signaling events ("SPREADs") transmit radially from a central point to adjacent cells via secreted ligands for EGFR and other receptors. SPREADs maintain homeostasis in non-pulmonary epithelia, but it is unknown whether they play a role in the airway epithelium or are dysregulated in inflammatory disease. To address these questions, we measured SPREAD activity with live-cell ERK biosensors in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial (pHBE) cells, in both submerged and biphasic Air-Liquid Interface (ALI) culture conditions (i.e., differentiated cells). Airway epithelial cells were exposed to pro-inflammatory cytokines relevant to asthma and chronic obstructive pulmonary disease (COPD). Type 1 pro-inflammatory cytokines significantly increased the frequency of SPREADs, which coincided with epithelial barrier breakdown in differentiated pHBE cells. Furthermore, SPREADs correlated with IL-6 peptide secretion and the appearance of localized clusters of phospho-STAT3 immunofluorescence. To probe the mechanism of SPREADs, cells were co-treated with pharmacological treatments (gefitinib, tocilizumab, hydrocortisone) or metabolic modulators (insulin, 2-deoxyglucose). Hydrocortisone, inhibitors of receptor signaling, and suppression of metabolic function decreased SPREAD occurrence, implying that pro-inflammatory cytokines and glucose metabolism modulate SPREADs in human airway epithelial cells via secreted EGFR and IL6R ligands. We conclude that spatiotemporal ERK signaling plays a role in barrier homeostasis and dysfunction during inflammation of the airway epithelium. This novel signaling mechanism could be exploited clinically to supplement corticosteroid treatment for asthma and COPD.

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

BackgroundHypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context.MethodsWe used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively.ResultsBy screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs.ConclusionORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation.Graphical

Common Cold Coronavirus 229E Induces Higher Interferon Stimulating Gene Responses in Human Nasal Epithelial Cells from Patients with Chronic Rhinosinusitis with Polyposis.

Viral infections have long been implicated in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Given widespread exposure to the common cold coronavirus 229E (HCoV229E), we sought to investigate how HCoV-229E is cleared and stimulates interferon pathways in air-liquid interface (ALI) cultures from patients with CRSwNP. The objective of this study was to identify whether viral clearance and ISG expression is different in ALI cultures from donors with CRSwNP compared with controls. Plaque assays were used to quantify infectious virus released by infected air-liquid interface (ALI) cultures derived from patients with CRSwNP compared to patients without CRS (controls). Additionally, mock and induced levels of Interferon Stimulated Genes (ISGs) mRNA following HCoV-229E infection were quantified by RT-qPCR. Quantification of infectious virus by plaque assay reveals that CRSwNP ALI cultures were equally susceptible to HCoV-229E infection, and surprisingly viral titers dropped significantly faster than in the control ALI cultures. We further demonstrate that this accelerated viral clearance correlates with increased mRNA expression of at least 4 ISGs following viral infection in the CRSwNP ALIs compared to the control ALIs. This study paradoxically demonstrates that ALI cultures from patients with CRSwNP are more efficient at clearing the common cold HCoV-229E virus compared to controls. We also demonstrate significantly increased ISG mRNA expression following HCoV-229E infection in CRSwNP. These findings call for further investigation into the effect of unimpaired interferon signaling on the type 2 inflammatory environment in patients with CRSwNP.

Activation of STAT3-mediated ciliated cell survival protects against severe infection by respiratory syncytial virus.

Respiratory syncytial virus (RSV) selectively targets ciliated cells in human bronchial epithelium and can cause bronchiolitis and pneumonia, mostly in infants. To identify molecular targets of intervention during RSV infection in infants, we investigated how age regulates RSV interaction with the bronchial epithelium barrier. Employing precision-cut lung slices and air-liquid interface cultures generated from infant and adult human donors, we found robust RSV virus spread and extensive apoptotic cell death only in infant bronchial epithelium. In contrast, adult bronchial epithelium showed no barrier damage and limited RSV infection. Single nuclear RNA-Seq revealed age-related insufficiency of an antiapoptotic STAT3 activation response to RSV infection in infant ciliated cells, which was exploited to facilitate virus spread via the extruded apoptotic ciliated cells carrying RSV. Activation of STAT3 and blockade of apoptosis rendered protection against severe RSV infection in infant bronchial epithelium. Lastly, apoptotic inhibitor treatment of a neonatal mouse model of RSV infection mitigated infection and inflammation in the lung. Taken together, our findings identify a STAT3-mediated antiapoptosis pathway as a target to battle severe RSV disease in infants.

Maternal stress and the early embryonic microenvironment: investigating long-term cortisol effects on bovine oviductal epithelial cells using air–liquid interface culture

The oviduct epithelium is the initial maternal contact site for embryos after fertilization, offering the microenvironment before implantation. This early gestation period is particularly sensitive to stress, which can cause reduced fertility and reproductive disorders in mammals. Nevertheless, the local impact of elevated stress hormones on the oviduct epithelium has received limited attention to date, except for a few reports on polyovulatory species like mice and pigs. In this study, we focused on the effects of chronic maternal stress on cattle, given its association with infertility issues in this monoovulatory species. Bovine oviduct epithelial cells (BOEC) differentiated at the air–liquid interface (ALI) were stimulated with 250 nmol/L cortisol for 1 or 3 weeks. Subsequently, they were assessed for morphology, bioelectrical properties, and gene expression related to oviduct function, glucocorticoid pathway, cortisol metabolism, inflammation, and apoptosis. Results revealed adverse effects of cortisol on epithelium structure, featured by deciliation, vacuole formation, and multilayering. Additionally, cortisol exposure led to an increase in transepithelial potential difference, downregulated mRNA expression of the major glucocorticoid receptor (NR3C1), upregulated the expression of cortisol-responsive genes (FKBP5, TSC22D3), and significant downregulation of oviductal glycoprotein 1 (OVGP1) and steroid receptors PGR and ESR1. The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells, indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine. The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2, an enzyme controlling the cellular capacity to metabolise cortisol. These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.

Single-cell RNA sequencing reveals heterogeneity of ALI model and epithelial cell alterations after exposure to electronic cigarette aerosol

Electronic cigarettes (e-cigarettes) have been advertised as a healthier alternative to traditional cigarettes; however, their exact effects on the bronchial epithelium are poorly understood. Air-liquid interface culture human bronchial epithelium (ALI-HBE) contains various cell types, including basal cell, ciliated cell and secretory cell, providing an in vitro model that simulates the biological characteristics of normal bronchial epithelium. Multiplex single-cell RNA sequencing of ALI-HBE was used to reveal previously unrecognized transcriptional heterogeneity within the human bronchial epithelium and cell type–specific responses to acute exposure to e-cigarette aerosol (e-aerosol) containing distinct components (nicotine and/or flavoring). The findings of our study show that nicotine-containing e-aerosol affected gene expression related to transformed basal cells into secretory cells after acute exposure; inhibition of secretory cell function by down-regulating genes related to epithelial cell differentiation, calcium ion binding, extracellular exosomes, and secreted proteins; and enhanced interaction between secretory cells and other cells. On the other hand, flavoring may alter the growth pattern of epithelial cells and make basal cells more susceptible to SARS-CoV infection. Besides, the data also indicate factors that may promote SARS-CoV-2 infection and suggest therapeutic targets for restoring normal bronchial epithelium function after e-cigarette use. In summary, the current study offered fresh perspectives on alterations in the cellular landscape and cell type–specific responses in human bronchial epithelium that are brought about by e-cigarette use.

Human Mesofluidic Intestinal Model for Studying Transport of Drug Carriers and Bacteria Through a Live Mucosal Barrier.

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 mm to 50 μm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 hr after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

A Microphysiological System with an Anaerobic Air-Liquid Interface and Functional Mucus Layer for Coculture of Intestinal Bacteria and Primary Human Colonic Epithelium.

Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host-colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell-derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air-liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 μm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon.

Dynamic monitoring of a 3D-printed airway tissue model using an organic electrochemical transistor

Assessing the transepithelial resistance to ion flow in the presence of an electric field enables the evaluation of the integrity of the epithelial cell layer. In this study, we introduce an organic electrochemical transistor (OECT) interfaced with a 3D living tissue, designed to monitor the electrical resistance of cellular barriers in real-time. We have developed a non-invasive, tissue-sensing platform by integrating an inkjet-printed large-area OECT with a 3D-bioprinted multilayered airway tissue. This unique configuration enables the evaluation of epithelial barrier integrity through the dynamic response capabilities of the OECT. Our system effectively tracks the formation and integrity of 3D-printed airway tissues in both liquid-liquid and air-liquid interface culture environments. Furthermore, we successfully quantified the degradation of barrier function due to influenza A (H1N1) viral infection and the dose-dependent efficacy of oseltamivir (Tamiflu®) in mitigating this degradation. The tissue-electronic platform offers a non-invasive and label-free method for real-time monitoring of 3D artificial tissue barriers, without disturbing the cellular biology. It holds the potential for further applications in monitoring the structures and functions of 3D tissues and organs, significantly contributing to the advancement of personalized medicine.

Lentiviral vector gene therapy and CFTR modulators show comparable effectiveness in cystic fibrosis rat airway models

Mutation-agnostic treatments such as airway gene therapy have the potential to treat any individual with cystic fibrosis (CF), irrespective of their CF transmembrane conductance regulator (CFTR) gene variants. The aim of this study was to employ two CF rat models, Phe508del and CFTR knockout (KO), to assess the comparative effectiveness of CFTR modulators and lentiviral (LV) vector-mediated gene therapy. Cells were isolated from the tracheas of rats and used to establish air-liquid interface (ALI) cultures. Phe508del rat ALIs were treated with the modulator combination, elexacaftor-tezacaftor-ivacaftor (ETI), and separate groups of Phe508del and KO tracheal epithelial cells were treated with LV-CFTR followed by differentiation at ALI. Ussing chamber measurements were performed to assess CFTR function. ETI-treated Phe508del ALI cultures demonstrated CFTR function that was 59% of wild-type level, while gene-addition therapy restored Phe508del to 68% and KO to 47% of wild-type level, respectively. Our findings show that rat Phe508del-CFTR protein can be successfully rescued with ETI treatment, and that CFTR gene-addition therapy provides significant CFTR correction in Phe508del and KO ALI cultures to levels that were comparable to ETI. These findings highlight the potential of an LV vector-based gene therapy for the treatment of CF lung disease.

Proton pump inhibitors modulate esophageal epithelial barrier function and crosstalk with eosinophils.

Eosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal dysfunction, type-2 inflammation, and esophageal eosinophilic infiltrate. While proton pump inhibitor (PPI) therapy is commonly used for EoE management, the underlying mechanism of action remains unclear. Air-liquid interface culture of esophageal epithelial cells was employed to investigate the impact of the PPI omeprazole on barrier integrity in IL-13-treated cultures. Epithelial chemokine secretion was assessed following stimulation with IL-13 and omeprazole, and the migration of eosinophils from healthy human donors was evaluated using 3 μm pore-sized transwells. A co-culture system of epithelial cells and eosinophils was employed to study chemokine secretion and eosinophil adhesion and activation markers. Omeprazole treatment in the IL-13-treated air-liquid interface (ALI) model resulted in 186 differentially expressed genes and restored barrier integrity compared to ALI treated with IL-13 alone. Omeprazole treatment reduced STAT6 phosphorylation, downregulated calpain 14, and upregulated desmoglein-1 in the IL-13-treated air-liquid interface samples. IL-13-induced upregulation of Eotaxin-3, CXCL10, and periostin, but this was downregulated by omeprazole. Further, the expression of CD11b, CD18, and CD69 was lower on eosinophils from omeprazole-treated epithelial-eosinophil co-cultures, which also had lower levels of eotaxin-3, CXCL10, CCL2, and CCL4. Omeprazole reduced the effects of IL-13 in both the epithelial air-liquid interface model and eosinophil-epithelial co-cultures, reducing barrier dysfunction, chemokine expression, and upregulation of eosinophil adhesion markers.

Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Metabolic dysfunction mediated by HIF-1α contributes to epithelial differentiation defects in eosinophilic esophagitis

Investigating the contributory role that epithelial cell metabolism plays in allergic inflammation is a key factor to understanding what influences dysfunction and the pathogenesis of the allergic disease eosinophilic esophagitis (EoE). We previously highlighted the absence of hypoxia signaling through HIF-1α in EoE contributes to esophageal epithelial dysfunction. However, metabolic regulation by HIF-1α has not been explored in esophageal allergy. Herein, we sought to define the role of HIF-1α-mediated metabolic dysfunction in esophageal epithelial differentiation processes and barrier function in EoE. In RNA-sequencing derived from EoE patient biopsies, we observed the expression pattern of key genes involved in mitochondrial metabolism/oxidative phosphorylation (OXPHOS) and glycolysis. Bioenergetics analysis using Seahorse was performed on EPC2-hTERT cells to decipher the metabolic processes involved in epithelial differentiation processes. In addition, air-liquid interface cultures were employed to delineate metabolic dependency mechanisms required for epithelial differentiation. Transcriptomic analysis identified an increase in genes associated with OXPHOS in patients with EoE. Epithelial origin of this signature was confirmed by complex V immunofluorescence of patient biopsies. Bioenergetic analysis in vitro revealed that differentiated epithelium was less reliant on OXPHOS compared with undifferentiated epithelium. Increased OXPHOS potential and reduced glycolytic capacity was mirrored in HIF1A-knockdown EPC2-hTERT cells which portray a significant absence of terminal markers of epithelial differentiation, including involucrin. Pharmacological glucose transport inhibition phenocopied this, while rescue of the HIF-1α-deficient phenotype using the pan-prolyl hydroxylase inhibitor DMOG resulted in restored expression of epithelial differentiation markers. An OXPHOS-dominated metabolic pattern in EoE patients, brought about largely by the absence of HIF-1α-mediated glycolysis, is linked with the deficit in esophageal epithelial differentiation.

Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines.

Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

An in vitro study of the impact of IL-17A and IL-22 on ciliogenesis in nasal polyps epithelium via the Hippo-YAP pathway

Cilia loss and impaired motile ciliary functions are among the typical pathological features of chronic rhinosinusitis with nasal polyps (CRSwNP). IL17A and IL22 are the canonical cytokines of type 3 inflammation, exhibiting similar functional effects on epithelial cells. In this study, we sought to examine the effects of IL17A and IL22 on ciliated cells and investigate the potential involvement of Hippo-YAP signaling in their influence on ciliogenesis. We assessed both the mRNA and protein expression levels of IL17A and IL22 in nasal tissues obtained from patients with CRSwNP and compared them to those from healthy controls. To further explore the impact of IL17A and IL22, we established a primary human nasal epithelial cell model using different concentrations (2 ng/mL, 10 ng/mL, 50 ng/mL) for a duration of 28 days in an air-liquid interface culture. Additionally, we employed the inhibitor verteporfin to investigate whether IL17A and IL22 exert their effects on ciliated cells via the Hippo-YAP pathway. The mRNA and protein levels of IL17A and IL22 in CRSwNP were significantly higher than those in healthy controls, revealing a robust correlation between IL17A and IL22. YAP was highly expressed in the nucleus of ciliated cells in CRSwNP and displayed a positive correlation with clinical symptoms. Both IL17A and IL22 were found to reduce the number of ciliated cells. IL17A, but not IL22, suppressed ciliogenesis by disrupting the proper development and docking of the basal body of ciliated cells, resulting in motile ciliary dysfunctions. Furthermore, the expression of YAP within the nucleus of ciliated cells gradually declined as these cells reached the final stage of differentiation. However, this process was obstructed by IL17A only. YAP inhibitors, such as verteporfin, markedly reversed the effects of IL17A by increasing the proportion of ciliated cells, suppressing nuclear YAP expression in these cells, and enhancing ciliary beating frequency. Both IL17A and IL22 are overexpressed in nasal epithelium of CRSwNP, which is associated with the impairment of epithelial cell differentiation. Furthermore, IL17A has been shown to exert a disruptive effect on morphogenesis of motile cilia via activation of YAP.

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (>21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells.

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1β and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1β- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1β- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1β may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.

Development of robust antiviral assays using relevant apical-out human airway organoids.

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput (~10-fold) and fewer cells (~100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

A range of 30-62% of functioning multiciliated airway cells is sufficient to maintain ciliary airway clearance.

Primary ciliary dyskinesia is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently leads to recurrent airway infections and bronchiectasis. We aimed to determine: how many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? To answer this question we exploited the molecular defects of the X-linked recessive primary ciliary dyskinesia variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterised by immotile cilia in affected males. We carefully analysed the clinical phenotype and molecular defect (using immunofluorescence and transmission electron microscopy) and performed in vitro studies (particle tracking in air-liquid interface cultures) and in vivo studies (radiolabelled tracer studies) to assess ciliary clearance of respiratory cells from female individuals with heterozygous and male individuals with hemizygous pathogenic DNAAF6 variants. Primary ciliary dyskinesia male individuals with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe primary ciliary dyskinesia symptoms. Owing to random or skewed X-chromosome inactivation in six female carriers with heterozygous pathogenic DNAAF6 variants, 54.3±10% (range 38-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. Our findings indicate that having 30-62% of multiciliated respiratory cells functioning can generate either normal or slightly reduced ciliary clearance. Because heterozygous female carriers displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine could improve ciliary airway clearance in individuals with primary ciliary dyskinesia, as well as clinical symptoms.