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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

© 2020 American Academy of Allergy, Asthma & Immunology Background: Emerging evidence suggests that disease vulnerability is expressed throughout the airways, the so-called unified airway hypothesis, but the evidence to support this is predominantly indirect. Objectives: We sought to establish the transcriptomic profiles of the upper and lower airways and determine their level of similarity irrespective of airway symptoms (wheeze) and allergy. Methods: We performed RNA sequencing on upper and lower airway epithelial cells from 63 children with or without wheeze and accompanying atopy, using differential gene expression and gene coexpression analyses to determine transcriptional similarity. Results: We observed approximately 91% homology in the expressed genes between the 2 sites. When coexpressed genes were grouped into modules relating to biological functions, all were found to be conserved between the 2 regions, resulting in a consensus network containing 16 modules associated with ribosomal function, metabolism, gene expression, mitochondrial activity, and antiviral responses through IFN activity. Although symptom-associated gene expression changes were more prominent in the lower airway, they were reflected in nasal epithelium and included IL-1 receptor like 1, prostaglandin-endoperoxide synthase 1, CCL26, and periostin. Through network analysis we identified a cluster of coexpressed genes associated with atopic wheeze in the lower airway, which could equally distinguish atopic and nonatopic phenotypes in upper airway samples. Conclusions: We show that the upper and lower airways are significantly conserved in their transcriptional composition, and that variations associated with disease are present in both nasal and tracheal epithelium. Findings from this study supporting a unified airway imply that clinical insight regarding the lower airway in health and disease can be gained from studying the nasal epithelium.

© 2018 Elsevier Ltd. Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With

Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction

Background: Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. Objectives: To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Methods: Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT) and permeability to fluorescent dextran. Results: Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. Conclusion: This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it co

© 2017 The Author(s). Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Copyright © 2016 by the American Thoracic Society. Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epitheliumis prevented byuninhibitedNE.However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aimof this studywas to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (a1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAEC non-CF and pAEC CF , respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAEC CF wound repair was also assessed.We report that viability after 48 hours was significantly decreased by 100 nM NE in pAEC non-CF and pAEC CF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAEC CF , but this effect was reversed by the addition of a1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases inthe airway contribute directly toCF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail.

© 2016 Taylor & Francis. Rationale: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. Aim of the Study: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Materials and Methods: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. Results: HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. Conclusion: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased ep

© 2016 John Wiley & Sons Ltd Background: The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. Objective: To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. Methods: Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-ß production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. Results: Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-ß production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-ß restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. Conclusions: Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children

© 2016 Asian Pacific Society of Respirology Background and objective: Evidence into the role of TGF-ß1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-ß1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells. Methods: Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-ß1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins avß6, avß8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-ß1 during wound repair. Results: TGF-ß1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-ß1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-ß1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-ß1 activators of epithelial cells, integrin avß6 and avß8 was also measured and there was no difference in avß6 gene expression between the two cohorts. Although integrin avß8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the avß8 mediated TGF-ß1 activation was significantly downregulated. Conclusion: Our data has highlighted the importance of TGF-ß1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells.

Background: Cystic fibrosis (CF) remains the most common life-shortening genetic disease affecting children. The defective protein product of the affected gene, cystic fibrosis transmembrane conductance regulator (CFTR), blocks Cl - and water absorption into the airway epithelium resulting in a reduction of the air surface liquid (ASL). Reduced ASL impedes ciliary activity and reduces clearance of airway secretions contributing to an abnormal airway microenvironment that is pro-inflammatory and promotes infection. Thus, an intervention to address the basic CFTR defect has the potential to be disease modifying. One possibility may be via the utilization of stem cells. We have previously demonstrated the potential of stem cell based therapy using hAECs in lung fibrosis (Moodley Y, et al. Am J Respir Crit Care Med. 2010;182:643-51). Hence, the aim of this study was to assess the potential of hAECs to restore ion transport function to CF airway epithelial in vitro. Methods: Primary AECs were obtained from children with CF (homozygous for F508del) when attending the Princess Margaret Hospital for Children for their annual bronchoscopy and bronchoalveolar lavage and cultures established (Sutanto EN, et al. Am J Respir Cell Mol Biol. 2011;44:761-7). Co-cultures were established at ratios of 1:1, 1:10 and 1:100 of primary AEC and hAEC respectively (Cell Applications CA, USA). Air liquid interface (ALI) cultures were also set up with hAECs and primary AECs, differentiation confirmed and resulting ASL height determined. Finally, Ussing chamber and yellow fluorescent protein (YFP) halide reporter assays were used to measure resulting CFTR function. Results: Co-cultures were successfully established at ratios of 1:1, 1:10 and 1:100 of primary AEC and hAEC respectively. ALI cultures established with hAEC at a ratio of 1:1 had a significantly greater ASL height compared to matched primary AEC ALI cultures (9.5 ± 1.0μm vs 6.0 ± 0.5μm, n=6). Ussing chamber results identified that c

© ERS 2015. Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis. Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis. A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis. Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease.