Airway Epithelial Gene Research Articles

Deep multiomic profiling reveals molecular signatures that underpin preschool wheeze and asthma

Wheezing in childhood is prevalent, with over one-half of all children experiencing at least 1 episode by age 6. The pathophysiology of wheeze, especially why some children develop asthma while others do not, remains unclear. This study addresses the knowledge gap by investigating the transition from preschool wheeze to asthma using multiomic profiling. Unsupervised, group-agnostic integrative multiomic factor analysis was performed using host/bacterial (meta)transcriptomic and bacterial shotgun metagenomic datasets from bronchial brush samples paired with metabolomic/lipidomic data from bronchoalveolar lavage samples acquired from children 1-17 years old. Two multiomic factors were identified: one characterizing preschool-aged recurrent wheeze and another capturing an inferred trajectory from health to wheeze and school-aged asthma. Recurrent wheeze was driven by type 1-immune signatures, coupled with upregulation of immune-related and neutrophil-associated lipids and metabolites. Comparatively, progression toward asthma from ages 1 to 18 was dominated by changes related to airway epithelial cell gene expression, type 2-immune responses, and constituents of the airway microbiome, such as increased Haemophilus influenzae. These factors highlighted distinctions between an inflammation-related phenotype in preschool wheeze, and the predominance of airway epithelial-related changes linked with the inferred trajectory toward asthma. These findings provide insights into the differential mechanisms driving the progression from wheeze to asthma and may inform targeted therapeutic strategies.

Long-Term PM2.5 Exposure and Upregulation of CLCA1 Expression in Nasal Epithelium from Youth with Asthma.

Little is known about long-term PM2.5 exposure and airway epithelial gene expression. To test for association between long-term PM2.5 exposure and nasal epithelial gene expression in youth with asthma. Transcriptome-wide association study (TWAS) of long-term PM2.5 in nasal epithelium from youth aged 6-20 years in the: 1) Epigenetic Variation and Childhood Asthma in Puerto Ricans study (EVA-PR, n=182); 2) Vitamin D Kids Asthma Study (VDKA, n=58); and 3) Stress and Treatment Response in Puerto Rican and African American Children with Asthma study (STAR, n=81). Satellite hybrid models were used to estimate PM2.5 exposure in the prior year at each participant's residence. Multivariable negative binomial regression was used for each TWAS, adjusting for age, sex, and other covariates. A meta-analysis of all TWAS results was then conducted using an inverse variance-weighted average approach. Most participants (~95%) in the meta-analysis of TWAS for PM2.5 exposure identified as Puerto Rican or Black. Long-term PM2.5 was associated with: 1) upregulated expression of CLCA1 (calcium-activated chloride channel regulator 1, false discovery rate-adjusted P [FDR-P]=0.008), SYCP2 (synaptonemal complex protein 2, FDR-P=0.01), and CYP2A6 (cytochrome p450 family 2 subfamily A member 6, FDR-P=0.02), and 2) downregulated expression of EDAR (ectodysplasin A receptor, FDR-P=0.01). In a meta-analysis, CLCA1 upregulation was associated with ≥1 positive allergen-specific IgE (FDR-P <0.001) and increased blood eosinophils (FDR-P <0.001) and total IgE (FDR-P <0.001). In a meta-analysis of TWASs in predominantly Puerto Rican and Black youth with asthma, long-term PM2.5 exposure was associated with upregulated airway epithelial CLCA1 expression, in turn linked to biomarkers of T2-high immunity.

Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway

BackgroundAirway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma.MethodsThe datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor.ResultsBoth bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-β1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression.ConclusionsTaken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

Dysfunctional neutrophil type 1 interferon responses in preschool children with recurrent wheezing and IL-4–mediated aeroallergen sensitization

BackgroundThe innate mechanisms associated with viral exacerbations in preschool children with recurrent wheezing are not understood. ObjectiveWe assessed differential gene expression in blood neutrophils from preschool children with recurrent wheezing, stratified by aeroallergen sensitization, at baseline and after exposure to polyinosinic-polycytidylic acid (poly(I:C)). We also examined whether poly(I:C)-stimulated blood neutrophils influenced airway epithelial gene expression. MethodsBlood neutrophils were purified and cultured overnight with poly(I:C) and underwent next-generation sequencing with Reactome pathway analysis. Primary human small airway epithelial cells were treated with poly(I:C)-treated neutrophil culture supernatants and were analyzed for Type 1 interferon gene expression with a targeted array. Symptoms and exacerbations were assessed in participants over 12 months. Results436 genes were differently expressed in neutrophils from children with versus without aeroallergen sensitization at baseline, with significant downregulation of Type 1 interferons. These Type 1 interferons were significantly upregulated in sensitized children after poly(I:C) stimulation. Confirmatory experiments demonstrated similar upregulation of Type 1 interferons in IL-4 treated neutrophils stimulated with poly(I:C). Poly(I:C)-treated neutrophil supernatants from children with aeroallergen sensitization also induced a Type 1 interferon response in epithelial cells. Children with aeroallergen sensitization also had higher symptom scores during exacerbations and these symptom differences persisted for three days after prednisolone treatment. ConclusionsType 1 interferon responses are dysregulated in preschool children with aeroallergen sensitization, which is in turn associated with exacerbation severity. Given the importance of Type 1 interferon signaling in viral resolution, additional studies of neutrophil Type 1 interferon responses are needed in this population.

Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

Nepalese indoor cookstove smoke extracts alter human airway epithelial gene expression, DNA methylation and hydroxymethylation

Household air pollution caused by inefficient cooking practices causes 4 million deaths a year worldwide. In Nepal, 86% of the rural population use solid fuels for cooking. Over 25% of premature deaths associated with air pollution are respiratory in nature. Here we aimed to identify molecular signatures of different cookstove and fuel type exposures in human airway epithelial cells, to understand the mechanisms mediating cook stove smoke induced lung disease. Primary human airway epithelial cells in submerged culture were exposed to traditional cook stove (TCS), improved cook stove (ICS) and liquefied petroleum gas (LPG) stove smoke extracts. Changes to gene expression, DNA methylation and hydroxymethylation were measured by bulk RNA sequencing and HumanMethylationEPIC BeadChip following oxidative bisulphite conversion, respectively. TCS smoke extract alone reproducibly caused changes in the expression of 52 genes enriched for oxidative stress pathways. TCS, ICS and LPG smoke extract exposures were associated with distinct changes to DNA methylation and hydroxymethylation. A subset of TCS induced genes were associated with differentially methylated and/or hydroxymethylated CpGs sites, and enriched for the ferroptosis pathway and the upstream regulator NFE2L2. DNA methylation and hydroxymethylation changes not associated with a concurrent change in gene expression, were linked to biological processes and molecular pathways important to airway health, including neutrophil function, transforming growth factor beta signalling, GTPase activity, and cell junction organisation. Our data identified differential impacts of TCS, ICS and LPG cook stove smoke on the human airway epithelium transcriptome, DNA methylome and hydroxymethylome and provide further insight into the association between indoor air pollution exposure and chronic lung disease mechanisms.

Cis- and trans-eQTM analysis reveals novel epigenetic and transcriptomic immune markers of atopic asthma in airway epithelium

Expression quantitative trait methylation (eQTM) analyses uncover associations between DNA methylation markers and gene expression. Most eQTM analyses of complex diseases have focused on cis-eQTM pairs (within 1 megabase). This study sought to identify cis- and trans-methylation markers associated with gene expression in airway epithelium from youth with and without atopic asthma. In this study, the investigators conducted both cis- and trans-eQTM analyses in nasal (airway) epithelial samples from 158 Puerto Rican youth with atopic asthma and 100 control subjects without atopy or asthma. The investigators then attempted to replicate their findings in nasal epithelial samples from 2 studies of children, while also examining whether their results in nasal epithelium overlap with those from an eQTM analysis in white blood cells from the Puerto Rican subjects. This study identified 9,108 cis-eQTM pairs and 2,131,500 trans-eQTM pairs. Trans-associations were significantly enriched for transcription factor and microRNA target genes. Furthermore, significant cytosine-phosphate-guanine sites (CpGs) were differentially methylated in atopic asthma and significant genes were enriched for genes differentially expressed in atopic asthma. In this study, 50.7% to 62.6% of cis- and trans-eQTM pairs identified in Puerto Rican youth were replicated in 2 smaller cohorts at false discovery rate-adjusted P< .1. Replicated genes in the trans-eQTM analysis included biologically plausible asthma-susceptibility genes (eg, HDC, NLRP3, ITGAE, CDH26, and CST1) and are enriched in immune pathways. Studying both cis- and trans-epigenetic regulation of airway epithelial gene expression can identify potential causal and regulatory pathways or networks for childhood asthma. Trans-eQTM CpGs may regulate gene expression in airway epithelium through effects on transcription factor and microRNA target genes.

WS18.06 The impact of cytomegalovirus on airway epithelial gene expression

Introduction: The pathogenicity of cytomegalovirus (CMV) has predominantly been recognized in immunosuppressed populations & newborns. However, a recent study in persons with CF observed that CMV seropositive individuals died from lung disease 8 years sooner than those who were seronegative. Further, CMV DNA was identified in those individuals’ sputum (Parkins 2019, ERJ). This observation aligns with increasing evidence associating CMV infection with worse outcomes in many inflammatory conditions, and a higher overall mortality. To date, models of CMV infection of human bronchial epithelial (HBE) cells have not been established, limiting our ability to study its impact in CF airways.

Longitudinal Effects of 1-Year Smoking Cessation on Human Bronchial Epithelial Transcriptome

Longitudinal Effects of 1-Year Smoking Cessation on Human Bronchial Epithelial Transcriptome

Potentiation of long-acting \u03b22-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP

IntroductionOver 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50–80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35–50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).HypothesisIncreasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.MethodsExpression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.ResultsUsing archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.ConclusionModulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

579: Use of transthoracic electroporation for airway epithelial cell gene delivery in mice

579: Use of transthoracic electroporation for airway epithelial cell gene delivery in mice

Integrative Analysis Reveals a miRNA-mRNA Regulatory Network and Potential Causative Agents in the Asthmatic Airway Epithelium.

BackgroundDuring asthma progression, the intricate molecular networks, including microRNA (miRNA) transcriptional regulation in airway epithelium, remain largely undefined. The abnormal expression of miRNAs in asthmatic airway epithelium is a recent and fast-growing area in developing diagnostic and therapeutic targets for asthma.Material and MethodsAnalyses were conducted to compare airway epithelial miRNAs and gene expression between patients with asthma and healthy subjects from three datasets (two for miRNAs expression profiles and one for gene expression profile). The interactions network between differentially expressed (DE)-miRNAs and mRNAs was further identified for functional analysis. To verify the involvement and functions of all the identified miRNAs in asthma, we constructed two cellular models of asthma. The most promising causal miRNA candidate, miR-1246, was examined in an in vitro system to explore its targets and roles in asthma pathophysiology.ResultsThrough integrative analysis, we obtained six miRNAs with 31 validated target genes in airway epithelium associated with asthma. Next, we confirmed that these miRNAs were all associated with asthma progression by in vitro functional experiments. They may participate in eosinophilic inflammation (miR-92b-3p, miR-1246, miR-197-3p, and miR-124-5p) or remodeling (miR-197-3p, miR-193a-5p, miR-1246, and miR-92b-3p). Additionally, some other non-screened valuable miRNAs were also examined and identified (miR-21-5p and miR-19b-3p), and some detected in blood correlated with the disease status. Furthermore, we found that miR-1246 could directly target POSTN and influence epithelial-to-mesenchymal transition and fibrosis in airway epithelial cells.ConclusionWe constructed a preliminary epithelial regulatory network in asthma based on six identified miRNAs and their valuable target genes. Candidate factors in the biological miRNA-mRNA network in airway epithelium may provide further information on the pathogenesis of asthma. Strikingly, among all screened miRNAs, miR-1246, which could interact with POSTN may have multifunctional effects in the course of asthma and be a promising agent for asthma treatment and molecular subtyping.

Improving lung cancer risk stratification leveraging whole transcriptome RNA sequencing and machine learning across multiple cohorts

BackgroundBronchoscopy for suspected lung cancer has low diagnostic sensitivity, rendering many inconclusive results. The Bronchial Genomic Classifier (BGC) was developed to help with patient management by identifying those with low risk of lung cancer when bronchoscopy is inconclusive. The BGC was trained and validated on patients in the Airway Epithelial Gene Expression in the Diagnosis of Lung Cancer (AEGIS) trials. A modern patient cohort, the BGC Registry, showed differences in key clinical factors from the AEGIS cohorts, with less smoking history, smaller nodules and older age. Additionally, we discovered interfering factors (inhaled medication and sample collection timing) that impacted gene expressions and potentially disguised genomic cancer signals.MethodsIn this study, we leveraged multiple cohorts and next generation sequencing technology to develop a robust Genomic Sequencing Classifier (GSC). To address demographic composition shift and interfering factors, we synergized three algorithmic strategies: 1) ensemble of clinical dominant and genomic dominant models; 2) development of hierarchical regression models where the main effects from clinical variables were regressed out prior to the genomic impact being fitted in the model; and 3) targeted placement of genomic and clinical interaction terms to stabilize the effect of interfering factors. The final GSC model uses 1232 genes and four clinical covariates – age, pack-years, inhaled medication use, and specimen collection timing.ResultsIn the validation set (N = 412), the GSC down-classified low and intermediate pre-test risk subjects to very low and low post-test risk with a specificity of 45% (95% CI 37–53%) and a sensitivity of 91% (95%CI 81–97%), resulting in a negative predictive value of 95% (95% CI 89–98%). Twelve percent of intermediate pre-test risk subjects were up-classified to high post-test risk with a positive predictive value of 65% (95%CI 44–82%), and 27% of high pre-test risk subjects were up-classified to very high post-test risk with a positive predictive value of 91% (95% CI 78–97%).ConclusionsThe GSC overcame the impact of interfering factors and achieved consistent performance across multiple cohorts. It demonstrated diagnostic accuracy in both down- and up-classification of cancer risk, providing physicians actionable information for many patients with inconclusive bronchoscopy.

Ivacaftor or lumacaftor/ivacaftor treatment does not alter the core CF airway epithelial gene response to rhinovirus

BackgroundAberrant 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. MethodsNineteen 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. ResultsRNA-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 interferon-pathway 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. ConclusionsThough 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.

Expression Quantitative Trait Methylation Analysis Reveals Methylomic Associations With Gene Expression in Childhood Asthma

Nasal (airway) epithelial methylation profiles have been associated with asthma, but the effects of such profiles on expression of distant cis-genes are largely unknown. To identify genes whose expression is associated with proximal and distal CpG probes (within 1 Mb), and to assess whether and how such genes are differentially expressed in atopic asthma. Genome-wide expression quantitative trait methylation (eQTM) analysis in nasal epithelium from Puerto Rican subjects (aged 9-20 years) with (n= 219) and without (n= 236) asthma. After the eQTM analysis, a Gene Ontology Enrichment analysis was conducted for the top 500 eQTM genes, and mediation analyses were performed to identify paths from DNA methylation to atopic asthma through gene expression. Asthma was defined as physician-diagnosed asthma and wheeze in the previous year, and atopy was defined as at least one positive IgE to allergens. Atopic asthma was defined as the presence of both atopy and asthma. We identified 16,867 significant methylation-gene expression pairs (false-discovery rate-adjusted P< .01) in nasal epithelium from study participants. Most eQTM methylation probes were distant (average distance, ∼378 kb) from their target genes, and also more likely to be located in enhancer regions of their target genes in lung tissue than control probes. The top 500 eQTM genes were enriched in pathways for immune processes and epithelial integrity and were more likely to have been previously identified as differentially expressed in atopic asthma. In a mediation analysis, we identified 5,934 paths through which methylation markers could affect atopic asthma through gene expression in nasal epithelium. Previous epigenome-wide association studies of asthma have estimated the effects of DNA methylation markers on expression of nearby genes in airway epithelium. Our findings suggest that distant epigenetic regulation of gene expression in airway epithelium plays a role in atopic asthma.

T2 and T17 cytokines alter the cargo and function of airway epithelium-derived extracellular vesicles

BackgroundAsthma is a common and heterogeneous disease that includes subgroups characterized by type 2 (T2) or type 17 (T17) immune responses for which there is a need to identify the underlying mechanisms and biomarkers in order to develop specific therapies. These subgroups can be defined by airway epithelium gene signatures and the airway epithelium has also been implicated to play a significant role in asthma pathology. Extracellular vesicles (EVs) carry functional biomolecules and participate in cell-to-cell communication in both health and disease, properties that are likely to be involved in airway diseases such as asthma. The aim of this study was to identify stimulus-specific proteins and functionality of bronchial epithelium-derived EVs following stimulation with T2 or T17 cytokines.MethodsEVs from cytokine-stimulated (T2: IL-4 + IL-13 or T17: IL-17A + TNFα) human bronchial epithelial cells cultured at air-liquid interface (HBEC-ALI) were isolated by density cushion centrifugation and size exclusion chromatography and characterized with Western blotting and electron microscopy. Transcriptomic (cells) and proteomic (EVs) profiling was also performed.ResultsOur data shows that EVs are secreted and can be isolated from the apical side of HBEC-ALI and that cytokine stimulation increases EV release. Genes upregulated in cells stimulated with T2 or T17 cytokines were increased also on protein level in the EVs. Proteins found in T17-derived EVs were suggested to be involved in pathways related to neutrophil movement which was supported by assessing neutrophil chemotaxis ex vivo.ConclusionsTogether, the results suggest that epithelial EVs are involved in airway inflammation and that the EV proteome may be used for discovery of disease-specific mechanisms and signatures which may enable a precision medicine approach to the treatment of asthma.

Effects of cannabis oil extract on immune response gene expression in human small airway epithelial cells (HSAEpC): implications for chronic obstructive pulmonary disease (COPD)

BackgroundChronic obstructive pulmonary disease (COPD) is commonly associated with both a pro-inflammatory and a T-helper 1 (Th1) immune response. It was hypothesized that cannabis oil extract can alleviate COPD symptoms by eliciting an anti-inflammatory Th2 immune response. Accordingly, the effects of cannabis oil extract on the expression of 84 Th2 and related immune response genes in human small airways epithelial cells (HSAEpC) were investigated.MethodsHSAEpC from a single donor were treated with three dilutions of a standardized cannabis oil extract (1:400, 1:800 and 1:1600) along with a solvent control (0.25% [2.5 ul/ml] ethanol) for 24 h. There were four replicates per treatment dilution, and six for the control. RNA isolated from cells were employed in pathway-focused quantitative polymerase chain reaction (qPCR) microarray assays.ResultsThe extract induced significant (P < 0.05) changes in expression of 37 tested genes. Six genes (CSF2, IL1RL1, IL4, IL13RA2, IL17A and PPARG) were up-regulated at all three dilutions. Another two (CCL22 and TSLP) were up-regulated while six (CLCA1, CMA1, EPX, LTB4R, MAF and PMCH) were down-regulated at the 1:400 and 1:800 dilutions. The relationship of differentially-expressed genes of interest to biologic pathways was explored using the Database for Annotation, Visualization and Integrated Discovery (DAVID).ConclusionsThis exploratory investigation indicates that cannabis oil extract may affect expression of specific airway epithelial cell genes that could modulate pro-inflammatory or Th1 processes in COPD. These results provide a basis for further investigations and have prompted in vivo studies of the effects of cannabis oil extract on pulmonary function.Trial registrationNONE (all in vitro experiments).

Characterization of an immortalized human small airway basal stem/progenitor cell line with airway region-specific differentiation capacity

BackgroundThe pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line.MethodsSmall airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF.ResultsThe hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that “cilium” and “immunity” were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2–1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF.ConclusionsThe immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Gene Expression Alterations in the Bronchial Epithelium of e-Cigarette Users

Although e-cigarette (ECIG) use has increased in the United States, their potential health effects remain uncertain. Understanding the effects of tobacco cigarette (TCIG) smoke on bronchial airway epithelial gene expression have previously provided insights into tobacco-related disease pathogenesis. Identifying the impact of ECIGs on airway gene expression could provide insights into their potential long-term health effects. We sought to compare the bronchial airway gene-expression profiles of former TCIG smokers now using ECIGs with the profiles of former and current TCIG smokers. We performed gene-expression profiling of bronchial epithelial cells collected from current TCIG smokers (n= 9), current ECIG users who are former TCIG smokers (n= 15), and former TCIG smokers (n= 21). We then compared our findings with previous studies of the effects of TCIG use on bronchial epithelium, as well an invitro model of ECIG exposure. Among 3,165 genes whose expression varied between the three study groups (q< 0.05), we identified 468 genes altered in ECIG users relative to former smokers (P< .05). Seventy-nine of these genes were up- or down-regulated concordantly among ECIG and TCIG users. We did not detect ECIG-associated gene-expression changes in known pathways associated with TCIG usage. Genes downregulated in ECIG users are enriched among the genes most downregulated by exposure of airway epithelium to ECIG vapor invitro. ECIGs induce both distinct and shared patterns of gene expression relative to TCIGs in the bronchial airway epithelium. The concordance of the genes altered in ECIG users and in the invitro study suggests that genes altered in ECIG users are likely to be changed as the direct effect of ECIG exposure.

EPITHELIAL CELL GENE NETWORKS UPREGULATED IN OBESE ASTHMATIC CHILDREN

Introduction Obesity is a major pediatric health concern affecting almost 20% of American children and adolescents. Obesity is a risk factor for the development of asthma and is associated with increased asthma severity. Children with obesity and asthma have reduced responsiveness to inhaled corticosteroids, increased use of systemic corticosteroids during exacerbations, and a reduced FEV1/FVC ratio. The mechanisms underlying the influence of obesity on asthma pathogenesis remain poorly understood. Methods Children (ages 6-17 years) with a priori defined exacerbation-prone asthma and blood eosinophils ≥150/mm3 had repeated nasal lavage sampling during a one-week period following reported URI symptoms. Nasal cell differentials were determined by cytospin and nasal gene expression assessed by RNA-sequencing. Differential gene expression was assessed by cell deconvolution and modular analysis. Results 154 URI events from 106 participants were captured and 47 of those events resulted in asthma exacerbation. 42/106 participants were obese defined by a BMI ≥95th percentile. Children with obesity had higher expression levels of 7 geneset modules when compared to those without obesity. These modules were associated with the airway epithelium and represent molecular functions including keratinization, tissue kallikrein induction, extracellular matrix production, and EGFR signaling. Measured effect sizes ranged from 1.24-2.05 fold higher. Conclusions We identified several airway epithelial associated gene networks upregulated in children with obesity and asthma. Previous studies of obesity and asthma have focused largely on metabolic and inflammatory alterations. Our results highlight the relevance of epithelial response pathways, which may serve as novel targets for biomarkers or therapies for obesity associated asthma.