Comparison of Nasal Epithelial Smoking-Induced Gene Expression on Affymetrix Exon 1.0 and Gene 1.0 ST Arrays

We have previously defined the impact of tobacco smoking on nasal epithelium gene expression using Affymetrix Exon 1.0 ST arrays. In this paper, we compared the performance of the Affymetrix GeneChip Human Gene 1.0 ST array with the Human Exon 1.0 ST array for detecting nasal smoking-related gene expression changes. RNA collected from the nasal epithelium of five current smokers and five never smokers was hybridized to both arrays. While the intersample correlation within each array platform was relatively higher in the Gene array than that in the Exon array, the majority of the genes most changed by smoking were tightly correlated between platforms. Although neither array dataset was powered to detect differentially expressed genes (DEGs) at a false discovery rate (FDR) <0.05, we identified more DEGs than expected by chance using the Gene ST array. These findings suggest that while both platforms show a high degree of correlation for detecting smoking-induced differential gene expression changes, the Gene ST array may be a more cost-effective platform in a clinical setting for gene-level genomewide expression profiling and an effective tool for exploring the host response to cigarette smoking and other inhaled toxins

Reversible and permanent effects of tobacco smoke exposure on airway epithelial gene expression

Oligonucleotide microarray analysis revealed 175 genes that are differentially expressed in large airway epithelial cells of people who currently smoke compared with those who never smoked, with 28 classified as irreversible, 6 as slowly reversible, and 139 as rapidly reversible

Smoking-induced gene expression changes in the bronchial airway are reflected in nasal and buccal epithelium

<p>Abstract</p> <p>Background</p> <p>Cigarette smoking is a leading cause of preventable death and a significant cause of lung cancer and chronic obstructive pulmonary disease. Prior studies have demonstrated that smoking creates a field of molecular injury throughout the airway epithelium exposed to cigarette smoke. We have previously characterized gene expression in the bronchial epithelium of never smokers and identified the gene expression changes that occur in the mainstem bronchus in response to smoking. In this study, we explored relationships in whole-genome gene expression between extrathorcic (buccal and nasal) and intrathoracic (bronchial) epithelium in healthy current and never smokers.</p> <p>Results</p> <p>Using genes that have been previously defined as being expressed in the bronchial airway of never smokers (the "normal airway transcriptome"), we found that bronchial and nasal epithelium from non-smokers were most similar in gene expression when compared to other epithelial and nonepithelial tissues, with several antioxidant, detoxification, and structural genes being highly expressed in both the bronchus and nose. Principle component analysis of previously defined smoking-induced genes from the bronchus suggested that smoking had a similar effect on gene expression in nasal epithelium. Gene set enrichment analysis demonstrated that this set of genes was also highly enriched among the genes most altered by smoking in both nasal and buccal epithelial samples. The expression of several detoxification genes was commonly altered by smoking in all three respiratory epithelial tissues, suggesting a common airway-wide response to tobacco exposure.</p> <p>Conclusion</p> <p>Our findings support a relationship between gene expression in extra- and intrathoracic airway epithelial cells and extend the concept of a smoking-induced field of injury to epithelial cells that line the mouth and nose. This relationship could potentially be utilized to develop a non-invasive biomarker for tobacco exposure as well as a non-invasive screening or diagnostic tool providing information about individual susceptibility to smoking-induced lung diseases.</p

Single-Cell Characterization of Pulmonary Nodules Implicates Suppression of Immunosurveillance across Early Stages of Lung Adenocarcinoma

UNLABELLED: A greater understanding of molecular, cellular, and immunological changes during the early stages of lung adenocarcinoma development could improve diagnostic and therapeutic approaches in patients with pulmonary nodules at risk for lung cancer. To elucidate the immunopathogenesis of early lung tumorigenesis, we evaluated surgically resected pulmonary nodules representing the spectrum of early lung adenocarcinoma as well as associated normal lung tissues using single-cell RNA sequencing and validated the results by flow cytometry and multiplex immunofluorescence (MIF). Single-cell transcriptomics revealed a significant decrease in gene expression associated with cytolytic activities of tumor-infiltrating natural killer and natural killer T cells. This was accompanied by a reduction in effector T cells and an increase of CD4+ regulatory T cells (Treg) in subsolid nodules. An independent set of resected pulmonary nodules consisting of both adenocarcinomas and associated premalignant lesions corroborated the early increment of Tregs in premalignant lesions compared with the associated normal lung tissues by MIF. Gene expression analysis indicated that cancer-associated alveolar type 2 cells and fibroblasts may contribute to the deregulation of the extracellular matrix, potentially affecting immune infiltration in subsolid nodules through ligand-receptor interactions. These findings suggest that there is a suppression of immune surveillance across the spectrum of early-stage lung adenocarcinoma. SIGNIFICANCE: Analysis of a spectrum of subsolid pulmonary nodules by single-cell RNA sequencing provides insights into the immune regulation and cell-cell interactions in the tumor microenvironment during early lung tumor development

Identifying a nasal gene expression signature associated with hyperinflation and treatment response in severe COPD

Hyperinflation contributes to dyspnea intensity in COPD. Little is known about the molecular mechanisms underlying hyperinflation and how inhaled corticosteroids (ICS) affect this important aspect of COPD pathophysiology. To investigate the effect of ICS/long-acting β2-agonist (LABA) treatment on both lung function measures of hyperinflation, and the nasal epithelial gene-expression profile in severe COPD. 117 patients were screened and 60 COPD patients entered a 1-month run-in period on low-dose ICS/LABA budesonide/formoterol (BUD/F) 200/6 one inhalation b.i.d. Patients were then randomly assigned to 3-month treatment with either a high dose BDP/F 100/6 two inhalations b.i.d. (n = 31) or BUD/F 200/6 two inhalations b.i.d. (n = 29). Lung function measurements and nasal epithelial gene-expression were assessed before and after 3-month treatment and validated in independent datasets. After 3-month ICS/LABA treatment, residual volume (RV)/total lung capacity (TLC)% predicted was reduced compared to baseline (p < 0.05). We identified a nasal gene-expression signature at screening that associated with higher RV/TLC% predicted values. This signature, decreased by ICS/LABA treatment was enriched for genes associated with increased p53 mediated apoptosis was replicated in bronchial biopsies of COPD patients. Finally, this signature was increased in COPD patients compared to controls in nasal, bronchial and small airways brushings. Short-term ICS/LABA treatment improves RV/TLC% predicted in severe COPD. Furthermore, it decreases the expression of genes involved in the signal transduction by the p53 class mediator, which is a replicable COPD gene expression signature in the upper and lower airways.Trial registration: ClinicalTrials.gov registration number NCT01351792 (registration date May 11, 2011), ClinicalTrials.gov registration number NCT00848406 (registration date February 20, 2009), ClinicalTrials.gov registration number NCT00158847 (registration date September 12, 2005)

Comparison of Proteomic and Transcriptomic Profiles in the Bronchial Airway Epithelium of Current and Never Smokers

Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear.Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in <or=5% of airway samples from a previously published dataset.1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure