Introduction:Azithromycin improves symptomology in various chronic airway diseases exacerbated by viral infections. However, the mechanisms underlying the apparent antiviral effects of azithromycin remain unclear. Methods:Airway epithelial cells from healthy children were cultured, expanded and differentiated into air-liquid interface cultures. Submerged and differentiated primary cultures were treated with 10 µM of AZM for 24 h and subsequently infected with human rhinovirus (HRV)-1b for 24 h. Virus receptor expression, replication, progeny release and inflammatory cytokines (IL-1β, -6, -8 and IP-10) were then measured. Barrier integrity was determined via qPCR, in-cell western (ICW), immunofluorescence confocal microscopy, confocal microscopy, transepithelial electrical resistance (RT) measurement and an apparent permeability (P app ) assay. Results:Treatment with AZM for 24 h at the concentrations of 0.1, 1 and 10 µM did not have any significant impact on either cellular viability or cytotoxicity in un-infected cells. No significant effect on viral receptor, cytokine expression was observed in non-infected cells treated with 10 µM AZM. Similarly, there was no significant change in both occludin and ZO-1 expression in non-infected cells. However, claudin-1 gene expression was significantly reduced but corresponding protein expression was significantly increased following 10 µM AZM. Although RT was significantly lower, this was not corroborated by any significant change in epithelial permeability after 10 µM AZM treatment. Subsequent to HRV-1b infection, 10 µM AZM treatment significantly reduced cytotoxicity induced by infection. Viral receptor expression were not affected with AZM pre-treatment but a significant decrease in viral replication was observed. Except for IP-10, expression of IL-1β, -6, and -8 was significantly reduced. Gene and protein expression of key epithelial junctions were significantly higher in treated, infected cells, which were concomitant with epithelial barrier function. Discussions:This study identified that AZM can protect against HRV-1b-induced epithelial damage. Our data, demonstrating the antiviral, anti-inflammatory, and barrier-protective effects in vitro are strongly indicative of pleiotropic mechanisms of AZM for mitigating viral infection and its consequences. These effects are likely to contribute to the benefits observed in clinical trials of AZM in a number of chronic respiratory diseases.

BACKGROUND:The airway epithelium is the primary structural and functional airway barrier and orchestrates innate immunity. Some children may have underlying epithelial vulnerabilities that contribute to the pathogenesis of acute wheeze and asthma (AWA). OBJECTIVE:To investigate the pediatric AWA epithelial environment and responses at time of exacerbation to elucidate cellular vulnerabilities; thus, a differentiated in vitro model with differentiated cell types is required. METHODS:Here, feasibility of establishing a differentiated model of primary nasal epithelial cells from children presenting to hospital with AWA was assessed. This was followed by examination of mucociliary differentiation, barrier integrity and function, viral receptor expression, and appropriate pro-inflammatory and antiviral cytokines compared with nasal epithelial cells derived from children who were non-wheezing non-atopic and non-wheezing with positive atopy. RESULTS:A differentiated pediatric AWA model was established with interparticipant heterogeneity reflected appropriately. The AWA model was of epithelial lineage and had mucociliary differentiation, barrier integrity and function, viral receptor expression, and the production of innate immune cytokines. However, the AWA epithelium had a more permeable paracellular barrier, higher expression of rhinovirus receptor ICAM1, intrinsically higher levels of pro-inflammatory and antiviral cytokines IL-6, IL-8, CCL5, CXCL10, and IFNL1, and potentially fewer cilia. Relationships between demographic and clinical data and AWA epithelial parameters were also identified. CONCLUSION:Collectively, a differentiated epithelial model of pediatric AWA was established, and results indicated underlying epithelial vulnerabilities in these children. This platform may be used to investigate the role of the AWA epithelium in pathogen infections, disease etiology, and therapeutic pipelines.

The airway mucosal epithelium is the main gateway of entry for numerous human respiratory viruses, including human influenza virus, respiratory syncytial virus (RSV), coronavirus, and rhinoviruses (RV). For respiratory viruses to perpetuate infection, they must be able to traverse the airway mucosal epithelium and then spread into distal sites of the respiratory tract and lung parenchyma. However, this cellular interface has evolved well-developed apical junctional complexes (AJCs), including tight and adherens junctions, that bridge adjacent epithelial cells together. The resulting structure not only provides a strong physical barrier but also plays an active role in preventing and/or limiting the spread and dissemination of viral pathogens. Respiratory viruses, such as RVs, have been shown to target various components of these AJCs, either directly or indirectly, thus facilitating paracellular viral penetration, resulting in airway epithelial barrier dysfunction. Disruption of these AJCs may also result in unintended contact with hidden viral receptors, further enabling viral infection of the airway epithelium. Here we describe the various models of RV-infected airway epithelial cells and the methods used for the characterization and analysis of AJCs following RV infection.

The nasal epithelium is the primary point of contact for inhaled respiratory viruses such as rhinovirus, respiratory syncytial virus, influenza, and coronavirus, among others. In order to establish infection, these viruses must engage their respective receptors located on host epithelial cells and begin replication. However, the nasal epithelium is also a pivotal orchestrator of both structural and innate immune defenses against these pathogens and thus mounts a broad antiviral response to halt the progression of the infection into the lower airways. Of note, the most common virus found in the airways of children presenting to the hospital emergency department with acute wheezing and asthma is rhinovirus C (RV-C), followed by rhinovirus A (RV-A). Here, we illustrate infection of a preclinical differentiated nasal epithelial model with clinical isolates of RV-A and -C, in conjunction with several methods utilized for characterization of epithelial responses post-infection in vitro.

Background:Early childhood wheeze is a major risk factor for asthma. However, not all children who wheeze will develop the disease. The airway epithelium has been shown to be involved in asthma pathogenesis. Despite this, the airway epithelium of children with acute wheeze remains poorly characterized. Methods:Upper airway epithelial cells (AEC) from children with acute wheeze and non-wheeze controls were cultured and expanded. Markers of epithelial lineage (Cytokeratin (KRT)-5, -19) and vimentin were assessed via qPCR and immunocytochemistry. Inflammatory cytokines (Interleukin (IL)-1β, -6, and -8) were measured using ELISA. Tight junction (TJ) protein expression and barrier integrity were determined via In-Cell Western and paracellular permeability assays, respectively. Results:Upper AECs from children with acute wheeze had significantly higher KRT19 and lower vimentin gene expression compared to non-wheeze controls but similar KRT5 levels. Similar staining intensities of KRT5 and KRT19 proteins were observed in both cohorts. IL-6 and IL-8 levels were not significantly different, but IL-1β was increased in cultures from children with acute wheeze compared to controls. Tight junction protein expression of claudin-1, occludin and ZO-1 were significantly lower in acute wheeze cohorts, concomitant with increased paracellular permeability. Conclusion:Airway epithelium of children experiencing acute wheeze appears abnormal, primarily with compromised epithelial barrier integrity.

Background: Emerging evidence suggests significant transcriptomic overlap between upper and lower airways, and prior work has shown a dysregulated response to injury in the lower airway of children with wheeze. However, there is little data that directly compares functionality between these two regions. Objective: Here, we hypothesized that dysregulated epithelial repair is observed in upper and lower airways of children with and without wheeze. Methods: Upper airway epithelial cell (AEC) cultures were established from children with asymptomatic recurrent wheeze (n=22), acute wheeze exacerbations (n=29) or without wheeze (n=29). Of these, matched upper and lower AEC cultures were established from children with and without wheeze (n≥6). Scratch wound and cell migration assays were performed, and therapeutic efficacy of celecoxib and dimethyl-celecoxib to enhance repair was determined. Results: Matched upper and lower AEC from children with asymptomatic wheeze displayed reduced repair response compared to non-wheeze counterparts. Upper AEC from children with acute wheeze showed a variable repair response where children with a poor repair "vulnerable epithelium" endotype had a shorter time to next exacerbation and higher number of exacerbations relative to full repair counterparts. Defective upper AEC repair associated with rhinitis and sibling asthma. Aberrant cell migration was observed in upper AEC from children with wheeze. Celecoxib and dimethyl-celecoxib enhanced upper AEC repair and migration. Conclusion: This study provides evidence of functional surrogacy, specifically airway repair responses, and highlights a novel vulnerable epithelium endotype in children with wheeze that is therapeutically targetable.

Introduction: Poor lung health in children born prematurely is associated with severe viral infection during early life. An altered response of the airway epithelium, which acts as the frontline defence against foreign pathogens, may underpin severe infection. This study aimed to determine if nasal epithelial cells from preterm infants display a defective viral response compared to term infants. Methods: Primary epithelial cultures were established and infected with human rhinovirus 1b (hRV1b) and respiratory syncytial virus (RSV). Barrier integrity was measured via transepithelial electrical resistance (TEER) and fluorescent permeability assays. Collected supernatant and RNA was analysed via ELISA and qPCR, for characterisation of caspase genes, viral load, viral receptors, and inflammatory mediators. Results: Successful differentiation occurred in 90% of term samples (final n=9, 2.75±0.55 years) but only 52% of preterm samples (final n=12, 1.38±0.09 years). Preterm ALI cultures were more permeable at baseline (4.3cm/sec vs 9.9cm/sec, p<0.05), however TEER did not differ between cohorts (p>0.05). Interestingly, baseline cytokine concentrations were elevated in preterm cultures (IL-8: 5908pg/mL vs 4242pg/mL, p<0.05; CCL5: 37.04pg/mL vs 32.00pg/mL, p<0.01). Following infection, no difference was detected in cell lysis, apoptosis, viral receptor expression, inflammation, viral replication, permeability or TEER between term and preterm samples under tested conditions. Conclusion: Preliminary data suggests certain viral-specific responses do not differ between term and preterm epithelial cells. However, underlying differences in inflammation and permeability may alter infection outcomes in those born preterm.

Introduction: Paediatric acute wheezing illness (AWI) airway models have not yet been established or characterised for intrinsic epithelial vulnerabilities, due to poor yield and culture survival. Aim: To establish and characterise a paediatric airway epithelial culture model of AWI. Methods: Nasal epithelial cells (NECs) sampled from children with AWI (n=6; 3 males; age 10.7±2.8) and community controls (n=6; 3 males; age 6.6±2.1) were cultured at air-liquid interface. Epithelial lineage markers (Cytokeratin (Ck)19, Ck5), vimentin, β-actin, β-tubulin, zonula occludens (ZO1) and rhinoviral receptors (ICAM1, LDLR, CDHR3) were assessed via qPCR. Barrier integrity and function was determined via trans-epithelial resistance and permeability assays. Basal inflammatory cytokine production (Interleukin (IL)6, -8) was measured via ELISA. Results: Similar gene expression was observed in both cohorts (AWI vs control (X ̅±SD) for Ck19 (2.88±1.21 vs 3.16±0.91), Ck5 (3.31±1.49 vs 3.51±1.78), vimentin (0.12±0.09 vs 0.15±0.10), β-actin (0.29±0.06 vs 0.28±0.06), ZO1 (0.0059±0.00089 vs 0.0054±0.0029), LDLR (0.69±0.32 vs 0.72±0.32), ICAM1 (0.23±0.24 vs 0.20±0.35) and CDHR3 (0.96±0.27 vs 1.61±0.94). β-tubulin was significantly lower the AWI cohort (7.17±2.56×10-6 vs 2.27±1.37×10-5; p<0.05). Similar trans-epithelial resistance (Ω.cm2) (274.87±76.20 vs 302.60±135.65; n=5), permeability (cm/sec) (14.58±9.62×10-4 vs 72.25±116.48×10-4; n=5) and basal inflammatory cytokines (pg/ml/µg protein) (IL6: 1.57±1.85, n=4 vs 0.52±0.21, n=3; IL8: 7.33±3.42; n=4 vs 4.05±1.49; n=3) were observed. Conclusion: We successfully established a well-differentiated culture model of children with AWI that can be used for future studies.

Rationale: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter airway epithelial cells (AECs). Objective: To determine what factors are associated with ACE2 expression particularly in patients with asthma and chronic obstructive pulmonary disease (COPD). Methods: We obtained upper and lower AECs from 145 people from two independent cohorts, aged 2-89, Newcastle (n=115), and from Perth (n= 30) Australia. The Newcastle cohort was enriched with people with asthma (n=37) and COPD (n=38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR, protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AECs. Results: Increased gene expression of ACE2 was associated with older age (p=0.02) and male sex (p=0.03), but not pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma (p=0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in asthma (p=0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface was increased (p=0.02). ACE2 protein levels were lower in endobronchial biopsies from asthma patients. Conclusions: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

Evidence from animal models demonstrate that intrauterine growth restriction (IUGR) alters airway structure and function which may affect susceptibility to disease. Airway inflammation and dysregulated epithelial barrier properties are features of asthma which have not been examined in the context of IUGR. This study used a maternal hypoxia-induced IUGR mouse model to assess lung-specific and systemic inflammation and airway epithelial tight junctions (TJs) protein expression. Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O-2) from gestational day (GD) 11 to 17.5 (IUGR group; term, GD 21). Following hypoxic exposure, mice were returned to a normoxic environment (21% O-2). A Control group was housed under normoxic conditions throughout pregnancy. Offspring weights were recorded at 2 and 8 weeks of age and euthanized for bronchoalveolar lavage (BAL) and peritoneal cavity fluid collection for inflammatory cells counts. From a separate group of mice, right lungs were collected for Western blotting of TJs proteins. IUGR offspring had greater inflammatory cells in the BAL fluid but not in peritoneal fluid compared with Controls. At 8 weeks of age, interleukin (IL)-2, IL-13, and eotaxin concentrations were higher in male IUGR compared with male Control offspring but not in females. IUGR had no effect on TJs protein expression. Maternal hypoxia-induced IUGR increases inflammatory cells in the BAL fluid of IUGR offspring with no difference in TJs protein expression. Increased cytokine release, specific to the lungs of IUGR male offspring, indicates that both IUGR and sex can influence susceptibility to airway disease.

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Introduction/Aim. Early childhood wheeze is a major risk factor for asthma. However, not all children who wheeze will develop the disease. The airway epithelium (including altered epithelial barrier function) has been shown to be involved in asthma pathogenesis (Looi et al, CEA 2018, 48:513). Despite this, the airway epithelium of children with acute wheeze remains poorly characterized. This study aimed to characterize the airway epithelium in children with acute wheeze. Methods. Nasal epithelial cells (NECs) from children with acute wheeze (n = 11; 6 males; mean age 3.7 _ 1.3 years old) and community controls (n = 8; 4 males; mean age 2.7 _ 0.3 years old), were expanded (Martinovich et al, Sci Rep 2017, 7:17971) andalso differentiated into airliquid interface (ALI) cultures. Markers of epithelial lineage (Cytokeratin (Ck)-5, -19) and vimentin was assessed via qPCR and immunocytochemistry. Inflammatory cytokines (Interleukin (IL)-6, -8 and -1β) were measured using ELISA. Barrier integrity was determined via In-Cell Western assay for tight junction (TJ) expression and transepithelial electrical resistance (RT) measurement and permeability assay for epithelial function. Results. NECs from children with acute wheeze had significantly higher Ck-19 and lower vimentin gene expression compared to community controls (P < 0.05) but Ck-5 was not significantly different. Similar staining intensities of Ck-5 and -19 proteins were observed in both acute wheeze and controls. IL-6 and -8 levels were not significantly different, but IL-1β was increased in NEC cultures of acute wheeze when compared to controls. TJ protein expression of claudin-1, occludin and ZO-1 were significantly lower in acute wheeze cohorts and was concomitant with decreased epithelial RT and increased permeability. Conclusion. Airway epithelium of children experiencing acute wheeze appears abnormal primarily with compromised epithelial barrier integrity and function. Collectively, these data infer potentially exaggerated epithelial responses following pathogen exposure. The mechanisms underlying this compromised epithelial remains unknown and warrants further assessment.

The epithelium is integral to the protection of many different biological systems and for the maintenance of biochemical homeostasis. Emerging evidence suggests that particular children have epithelial vulnerabilities leading to dysregulated barrier function and integrity, that resultantly contributes to disease pathogenesis. These epithelial vulnerabilities likely develop in utero or in early life due to various genetic, epigenetic and environmental factors. Although various epithelia are uniquely structured with specific function, prevalent allergic-type epithelial diseases in children potentially have common or parallel disease processes. These include inflammation and immune response dysregulation stemming from atypical epithelial barrier function and integrity. Two diseases where aetiology and pathogenesis are potentially linked to epithelial vulnerabilities include Paediatric Asthma and Eosinophilic Oesophagitis (EoE). For example, rhinovirus C (RV-C) is a known risk factor for paediatric asthma development and is known to disrupt respiratory epithelial barrier function causing acute inflammation. In addition, EoE, a prevalent atopic condition of the oesophageal epithelium, is characterised by similar innate immune and epithelial responses to viral injury. This review examines the current literature and identifies the gaps in the field defining viral-induced effects on a vulnerable respiratory epithelium and resulting chronic inflammation, drawing from knowledge generated in acute wheezing illness, paediatric asthma and EoE. Besides highlighting the importance of epithelial structure and barrier function in allergic disease pathogenesis regardless of specific epithelial sub-types, this review focuses on the importance of examining other parallel allergic-type disease processes that may uncover commonalities driving disease pathogenesis. This in turn may be beneficial in the development of common therapeutics for current clinical management and disease prevention in the future.

The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.

BACKGROUND:Dysregulated airway epithelial repair following injury is a proposed mechanism driving posttransplant bronchiolitis obliterans (BO), and its clinical correlate bronchiolitis obliterans syndrome (BOS). This study compared gene and cellular characteristics of injury and repair in large (LAEC) and small (SAEC) airway epithelial cells of transplant patients.METHODS:Subjects were recruited at the time of routine bronchoscopy posttransplantation and included patients with and without BOS. Airway epithelial cells were obtained from bronchial and bronchiolar brushing performed under radiological guidance from these patients. In addition, bronchial brushings were also obtained from healthy control subjects comprising of adolescents admitted for elective surgery for nonrespiratory-related conditions. Primary cultures were established, monolayers wounded, and repair assessed (±) azithromycin (1 µg/mL). In addition, proliferative capacity as well as markers of injury and dysregulated repair were also assessed.RESULTS:SAEC had a significantly dysregulated repair process postinjury, despite having a higher proliferative capacity than large airway epithelial cells. Addition of azithromycin significantly induced repair in these cells; however, full restitution was not achieved. Expression of several genes associated with epithelial barrier repair (matrix metalloproteinase 7, matrix metalloproteinase 3, the integrins β6 and β8, and β-catenin) were significantly different in epithelial cells obtained from patients with BOS compared to transplant patients without BOS and controls, suggesting an intrinsic defect.CONCLUSIONS:Chronic airway injury and dysregulated repair programs are evident in airway epithelium obtained from patients with BOS, particularly with SAEC. We also show that azithromycin partially mitigates this pathology.

Background: Aberrant responses by the cystic fibrosis airway epithelium during viral infection may underly the clinical observations. Whether CFTR modulators affect antiviral responses by CF epithelia is presently unknown. We tested the hypothesis that treatment of CF epithelial cells with ivacaftor (Iva) or ivacaftor/lumacaftor (Iva/Lum) would improve control of rhinovirus infection. Methods: Nineteen CF epithelial cultures (10 homozygous for p.Phe508del as CFTR Class 2, 9 p.Phe508del/p.Gly551Asp as Class 3) were infected with rhinovirus 1B at multiplicity of infection 12 for 24 h. Culture RNA and supernatants were harvested to assess gene and protein expression respectively. Results: RNA-seq analysis comparing rhinovirus infected cultures to control identified 796 and 629 differentially expressed genes for Class 2 and Class 3, respectively. This gene response was highly conserved when cells were treated with CFTR modulators and were predicted to be driven by the same interferonpathway transcriptional regulators (IFNA, IFNL1, IFNG, IRF7, STAT1). Direct comparisons between treated and untreated infected cultures did not yield any differentially expressed genes for Class 3 and only 68 genes for Class 2. Changes were predominantly related to regulators of lipid metabolism and inflammation, aspects of epithelial biology known to be dysregulated in CF. In addition, CFTR modulators did not affect viral copy number, or levels of pro-inflammatory cytokines produced post-infection. Conclusions: Though long-term clinical data is not yet available, results presented here suggest that first generation CFTR modulators do not interfere with core airway epithelial responses to rhinovirus infection. Future work should investigate the latest triple modulation therapies. (C) 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.