Professor Darryl Knight

© 2016 Asian Pacific Society of Respirology.Epithelial cells represent the most important surface of contact in the body and form the first line of defence of the body to external environment. Consequently, epithelia have numerous roles in order to maintain a homeostatic defence barrier. Although the epithelium has been extensively studied over several decades, it remains the focus of new research, indicating a lack of understanding that continues to exist around these cells in specific disease settings. Importantly, evidence is emerging that airway epithelial cells in particular have varied complex functions rather than simple passive roles. One area of current interest is its role following injury. In particular, the epithelial-specific cellular mechanisms regulating their migration during wound repair remain poorly understood and remain an area that requires much needed investigation. A better understanding of the physiological, cellular and molecular wound repair mechanisms could assist in elucidating pathological processes that contribute to airway epithelial pathology. This review attempts to highlight migration-specific and cell-extracellular matrix (ECM) aspects of repair used by epithelial cells under normal and disease settings, in the context of human airways.

© 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.

© 2015 Elsevier Ltd.The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor ß-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the ß-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the ß-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the ß-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFß1 in the presence or absence of the selective small molecule ICG-001 to inhibit ß-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, ß-catenin, fibronectin and ITGß1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFß1 induced EMT, characterized by reduced E-cadherin expression with increased expression of a-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFß1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFß1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGß1 and fibronectin expression. These data support our hypothesis that modulating the ß-catenin/CBP signaling axis plays a key role in epithelial plasticity and function.

© 2015 BMJ Publishing Group Ltd & British Thoracic Society.Background Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the underlying mechanisms of this clinically relevant observation are poorly understood. Objectives To investigate the importance of intact TLR7 signalling in vivo during RV exacerbation using mouse models of house dust mite (HDM)-induced allergic airways disease exacerbated by a superimposed RV infection. Methods Wild-type and TLR7-deficient (Tlr7^-/-) BALB/c mice were intranasally sensitised and challenged with HDM prior to infection with RV1B. In some experiments, mice were administered recombinant IFN or adoptively transferred with plasmacytoid dendritic cells (pDC). Results Allergic Tlr7^-/- mice displayed impaired IFN release upon RV1B infection, increased virus replication and exaggerated eosinophilic inflammation and airways hyper reactivity. Treatment with exogenous IFN or adoptive transfer of TLR7-competent pDCs blocked these exaggerated inflammatory responses and boosted IFN? release in the absence of host TLR7 signalling. TLR7 expression in the lungs was suppressed by allergic inflammation and by interleukin (IL)-5-induced eosinophilia in the absence of allergy. Subjects with moderate-to-severe asthma and eosinophilic but not neutrophilic airways inflammation, despite inhaled steroids, showed reduced TLR7 and IFN?2/3 expression in endobronchial biopsies. Furthermore, TLR7 expression inversely correlated with percentage of sputum eosinophils. Conclusions This implicates IL-5-induced airways eosinophilia as a negative regulator of TLR7 expression and antiviral responses, which provides a molecular mechanism underpinning the effect of eosinophil-targeting treatments for the prevention of asthma exacerbations.

Copyright © 2015 by the American Thoracic Society.Rationale: Chronic obstructive pulmonary disease (COPD) and influenza virus infections are major global health issues. Patients with COPD are more susceptible to infection, which exacerbates their condition and increases morbidity and mortality. The mechanisms of increased susceptibility remain poorly understood, and current preventions and treatments have substantial limitations. Objectives: To characterize the mechanisms of increased susceptibility to influenza virus infection in COPD and the potential for therapeutic targeting. Methods: We used a combination of primary bronchial epithelial cells (pBECs) from COPD and healthy control subjects, a mouse model of cigarette smoke-induced experimental COPD, and influenza infection. The role of the phosphoinositide-3-kinase (PI3K) pathway was characterized using molecular methods, and its potential for targeting assessed using inhibitors. Measurements and Main Results: COPDpBECs were susceptible to increased viral entry and replication. Infected mice with experimental COPD also had more severe infection (increased viral titer and pulmonary inflammation, and compromised lung function). These processes were associated with impaired antiviral immunity, reduced retinoic acid-inducible gene-I, and IFN/cytokine and chemokine responses. Increased PI3K-p110a levels and activity inCOPDpBECs and/or mice were responsible for increased infection and reduced antiviral responses. Global PI3K, specific therapeutic p110a inhibitors, or exogenous IFN-b restored protective antiviral responses, suppressed infection, and improved lung function. Conclusions: The increased susceptibility of individuals with COPD to influenza likely results from impaired antiviral responses, which are mediated by increased PI3K-p110a activity. This pathway may be targeted therapeutically in COPD, or in healthy individuals, during seasonal or pandemic outbreaks to prevent and/or treat influenza.

© 2015 Mahmood et al.Background: Small airway fibrosis is the main contributor in airflow obstruction in chronic obstructive pulmonary disease. Epithelial mesenchymal transition (EMT) has been implicated in this process, and in large airways, is associated with angiogenesis, ie, Type-3, which is clas­sically promalignant. Objective: In this study we have investigated whether EMT biomarkers are expressed in small airways compared to large airways in subjects with chronic airflow limitation (CAL) and what type of EMT is present on the basis of vascularity. Methods: We evaluated epithelial activation, reticular basement membrane fragmentation (core structural EMT marker) and EMT-related mesenchymal biomarkers in small and large airways from resected lung tissue from 18 lung cancer patients with CAL and 9 normal controls. Tissues were immunostained for epidermal growth factor receptor (EGFR; epithelial activation marker), vimentin (mesenchymal marker), and S100A4 (fibroblast epitope). Type-IV collagen was stained to demonstrate vessels. Results: There was increased expression of EMT-related markers in CAL small airways compared to controls: EGFR (P<0.001), vimentin (P<0.001), S100A4 (P<0.001), and fragmentation (P<0.001), but this was less than that in large airways. Notably, there was no hypervascularity in small airway reticular basement membrane as in large airways. Epithelial activation and S100A4 expression were related to airflow obstruction. Conclusion: EMT is active in small airways, but less so than in large airways in CAL, and may be relevant to the key pathologies of chronic obstructive pulmonary disease, small airway fibrosis, and airway cancers.

© 2015 Stefanowicz et al.Background: Epigenetic adjustments of the chromatin architecture through histone modifications are reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific and therefore, it is important to understand its contribution to individual cellular responses in tissues like the airway epithelium which forms the mucosal barrier to the inhaled environment within the lung. The airway epithelium of asthmatics is abnormal with dysregulation of genes such as epidermal growth factor receptor (EGFR), the dN isoform of the transcription factor p63 (dNp63), and signal transducer and activator of transcription 6 (STAT6), integral to differentiation, proliferation, and inflammation. It is important to establish in diseases like asthma how histone modifications affect tissue responses such as proliferation and differentiation. Objectives: To characterize the global histone acetylation and methylation status in the epithelium of asthmatic compared to healthy subjects and to identify the impact of these variations on genes involved in epithelial functions. Methods: Whole lungs were obtained from healthy and asthmatic subjects (n = 6) from which airway epithelial cells (AECs) were isolated and airway sections were taken for analysis of histone lysine acetylation and methylation by immunohistochemistry. AECs were subjected to chromatin immunoprecipitation (ChIP) using anti-H3K18ac and anti-H3K4me2 antibodies followed by RT-PCR targeting dNp63, EGFR, and STAT6. AECs were also treated with TSA and changes in dNp63, EGFR, and STAT6 expression were determined. Results: We identified an increase in the acetylation of lysine 18 on histone 3 (H3K18ac) and trimethylation of lysine 9 on histone 3 (H3K9me3) in the airway epithelium of asthmatic compared to healthy subjects. We found increased association of H3K18ac around the transcription start site of dNp63, EGFR, and STAT6 in AECs of asthmatics. However, we were unable to modify the expression of these genes with the use of the HDAC inhibitor TSA in healthy subjects. Discussion: The airway epithelium from asthmatic subjects displays increased acetylation of H3K18 and association of this mark around the transcription start site of dNp63, EGFR, and STAT6. These findings suggest a complex interaction between histone modifications and gene regulation in asthma.

© 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.

Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. Further, there is now strong evidence that epithelium dysfunction is involved in the development of inflammatory disorders of the lung. Here we review the characteristic immune responses that are orchestrated by the epithelium in response to diverse triggers such as pollutants, cigarette smoke, bacterial peptides, and viruses. We focus in part on the role of epithelium-derived interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), as well as CC family chemokines as critical regulators of the immune response. We cite examples of the function of the epithelium in host defense and the role of epithelium dysfunction in the development of inflammatory diseases. © 2013 Elsevier Inc.

Background and objective Airway epithelial cells represent the first line of defence against inhaled insults, including air pollution. Air pollution can activate innate immune signalling in airway epithelial cells leading to the production of soluble mediators that can influence downstream inflammatory cells. Our objective was to develop and validate a model of dendritic cell exposure to airway epithelial cell-conditioned media. After establishing the model, we explored how soluble mediators released from airway epithelial cells in response to air pollution influenced the phenotype of dendritic cells. Methods Human airway epithelial cells were cultured under control and urban particulate matter (PM10) exposure conditions with or without pharmacological inhibitors of the uric acid pathway. Culture supernatants were collected for conditioned media experiments with peripheral blood mononuclear cell-derived dendritic cells analysed by flow cytometry. Results Monocytes derived from peripheral blood mononuclear cells cultured in interleukin-4 and granulocyte macrophage colony stimulating factor differentiated into immature dendritic cells that phenotypically differentiated into mature dendritic cells in response to conditioned media from phorbol myristate acetate-activated THP-1 monocytes. Exposure of immature dendritic cells to conditioned media from airway epithelial cells exposed to PM10 resulted in dendritic cell maturation that was independent of uric acid. Conclusions We present a conditioned media model useful for interrogating the contribution of soluble mediators produced by airway epithelial cells to dendritic cell phenotype and function. Furthermore, we demonstrate that PM10 exposure induces airway epithelial cell production of soluble mediators that induce maturation of dendritic cells independent of uric acid. We developed and validated a model of airway epithelial cell conditioned media exposure to primary human dendritic cells. Using this model, we then tested the hypothesis that urban particulate matter exposure to airway epithelial cells resulted in production of soluble mediators capable of inducing dendritic cell maturation. © 2014 Asian Pacific Society of Respirology.