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When inhaled,ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung lining fluid,generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols,epoxycholesterol-α and β (α-EpCh,β-EpCh) and Secosterol A and B (Seco A,SecoB),in cell lysates and apical washes. Similarly,bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected,O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly,expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1),which is regulated by activation of the liver X receptor (LXR),was suppressed in epithelial cells exposed to O3. Additionally,exposure of LXR knockout mice to O3 enhanced pro-inflammatory cytokine production in the lung,suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols,our data demonstrate adduction of LXR with Seco A. Similarly,supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared to stimulation with T09 alone,indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall,these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR,thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects. View Publication

While all forms of tobacco exposure have negative health effects,the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here,we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM,GCLC,GPX2,NQO1 and HO-1. Vaporization of,and/or the presence of nicotine in,eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE,and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A,140,126,374A,26A-2,147B,941 and 589) for further study. We validated increased expression of multiple miRNAs,including miR126,following eCig exposure. We also found significant reduction in the expression of two miR126 target genes,MYC and MRGPRX3,following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells,some of which are epigenetically programmed at the level of miRNA regulation. View Publication

Human coronaviruses (HCoVs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine 2 (TMPRSS2). We previously reported that clinical isolates of HCoV-229E preferred cell-surface TMPRSS2 to endosomal cathepsin for cell entry,and that they acquired the ability to use cathepsin L by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. Here,we show that clinical isolates of HCoV-OC43 and -HKU1 preferred the cell-surface TMRRSS2 to endosomal cathepsins for cell entry,similar to HCoV-229E. In addition,the cell-culture-adapted HCoV-OC43 lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. These results suggest that circulating HCoVs in the field generally use cell-surface TMPRSS2 for cell entry,not endosomal cathepsins,in human airway epithelial cells. View Publication

Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis,we previously found three immunological clusters in asthma: Th2-high,Th17-high,and Th2/17-low. Although new therapies are emerging for Th2-high disease,identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity,but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma,suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary,CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways. View Publication

BACKGROUND In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However,undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study,we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients. METHODS Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated,mucus-secreting and basal cells,and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally,epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay. RESULTS Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also,immunofluorescence analysis revealed the presence of ciliated,mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight. CONCLUSION In summary,primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated,mucus-producing and basal cells,which adequately reflect the in vivo properties of the human respiratory epithelium. View Publication

The extracellular matrix (ECM) is an important contributor to the asthmatic phenotype. Recent studies investigating airway inflammation have demonstrated an association between hyaluronan (HA) accumulation and inflammatory cell infiltration of the airways. The ECM proteoglycan versican interacts with HA and is important in the recruitment and activation of leukocytes during inflammation. We investigated the role of versican in the pathogenesis of asthmatic airway inflammation. Using cockroach antigen (CRA)-sensitized murine models of allergic asthma,we demonstrate increased subepithelial versican in the airways of CRA-treated mice that parallels subepithelial increases in HA and leukocyte infiltration. During the acute phase,CRA-treated mice displayed increased gene expression of the four major versican isoforms,as well as increased expression of HA synthases. Furthermore,in a murine model that examines both acute and chronic CRA exposure,versican staining peaked 8 days following CRA challenge and preceded subepithelial leukocyte infiltration. We also assessed versican and HA expression in differentiated primary human airway epithelial cells from asthmatic and healthy children. Increases in the expression of versican isoforms and HA synthases in these epithelial cells were similar to those of the murine model. These data indicate an important role for versican in the establishment of airway inflammation in asthma. View Publication

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways,however,remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture,showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread,including localisation to epithelial cell junctions. We extended those observations here,characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation,barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally,M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology,consistent with contrasting virulence in experimentally infected mice. In summary,this study reports pericellular invasion,NHBE cytoskeletal reorganisation,and tissue remodelling with persistent infection in a human airway epithelium model,providing clear insight into the likely route for extrapulmonary spread. View Publication

The embryonic mouse lung is a widely used substitute for human lung development. For example,attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology,gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover,we identify mouse-human differences,including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. View Publication

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar theratype"; however a standardized approach confirming efficacy in these cohorts has not been reported. Here we demonstrate that patients bearing the rare mutation: c.3700 A>G causing protein misprocessing and altered channel function-similar to ΔF508-CFTR are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor respectively this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound effective in increasing the levels of immature CFTR protein augmented the Orkambi® response. Importantly this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue will facilitate therapy development for patients with rare CF mutations. View Publication

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However,limited understanding of human airway patterning has made this goal challenging. Here,we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach,we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases. View Publication

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples,while requiring low cell numbers. To date,a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative,EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris,doublets and dead cells from the analysis. For validation,the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers,HBEC recovered from BAL (2.3% of live cells),BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed,validated,and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. View Publication

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response,and observed a significant increase in secretion of IL-8,GRO-α,IL-1β,and GM-CSF. Interestingly,secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes. View Publication