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Genetic characterization of macrophages from induced sputum of asthma and COPD patients

ABSTRACT: Background: Macrophages are important cells in pathogenesis of obstructive lung diseases including asthma and chronic obstructive pulmonary disease (COPD). The aim of the study was a multivariate, genetic, comparative analysis of macrophages from patients with asthma and COPD. Methods: Macrophages were isolated from induced sputum (IS) by magnetic bead separation. Transcriptomic measurements were carried out using Affymetrix Human Gene 2.1 ST ArrayStrip in 17 samples: 8 asthma (4 ICS naive , 4 ICS treated), 4 COPD and 5 control samples. The expression of the most significantly differentiating genes was evaluated by real time PCR in 34 samples (15 patients with asthma, 11 patients with COPD and 17 controls). Results: USP53 was the only gene differentially expressed (p adjusted clue =0.09) in the comparison between ICS naïve and ICS treated asthma. The PCR verification showed increased expression of BTF3, CDS2, DNAJC13, DDX5, GNAI2, SCGB1A1, SIRPB1, TRAF3IP, USP53, WDR49 in the asthma compared to COPD group. The changed gene expression profile of macrophages were characterized mostly by gene ontology terms linked with cell motility, cilium function, cell junction and adhesion organization. Conclusions: Gene expression profiling of sputum macrophages revealed distinct molecular capacity in asthma and COPD. The role of sputum macrophages in the pathophysiology of obstructive lung diseases is probably connected with biological processes associated with their motility, cilium dysfunction and cell junction organization.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112260 | GEO | 2018/08/09

REPOSITORIES: GEO

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Project description:Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary diseases (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8-day storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV200) >30% and an individual yield >50ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV200 higher than 30%. 70% of the samples had a yield >50ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.

Project description:Chronic obstructive pulmonary disease (COPD) is one of the most prevalent lung diseases, and involves persistent airflow limitation and incorporates both emphysema and chronic bronchitis. Cigarette smoking has been identified as the main risk factor for disease development and progression. In a basic model of COPD, the disease is initiated when the physiologic response mechanisms to cigarette smoke exposure are overwhelmed; for example, because of long-term exposure effects or other aging-related changes. In this parallel-group case-controlled clinical study we asked to what extent the different transitions in a chronic-exposure-to-disease model are reflected in the proteome and cellular transcriptome of induced sputum samples from the lung. For this, we selected 60 age- and gender-matched individuals for each of four study groups: current healthy smokers, current-smoker COPD patients, former smokers, and never smokers (a total of 240 individuals). Induced sputum was collected, the cell-free supernatant was analyzed by quantitative proteomics (isobaric-tag based), and the cellular mRNA fraction was analyzed by microarray-based expression profiling. The sputum proteome of current smokers (healthy or COPD patients) clearly reflected the common physiological responses to smoke exposure, including alterations in mucin/trefoil proteins (e.g., MUC5AC and TFF1/3up-regulation), peptidase regulators (e.g., TIMP1 up-regulation), and a prominent xenobiotic/oxidative stress response (e.g., NQO1 and ALDH3A1 up-regulation). The latter response also was observed in the sputum transcriptome, which additionally demonstrated an immune-related polarization change (toward a M2 signature). The (long-term) former smoker group showed nearly complete reversal of the observable biological effects. Thirteen differentially abundant proteins between the COPD and healthy smoker groups were identified. These abundant proteins included previously reported COPD-associated proteins (e.g., TIMP1 (up-regulation) and APOA1 (down-regulation)) and novel proteins such as C6orf58 and BPIFB1 (LPLUNC1) (both up-regulated in the COPD group compared with the healthy smokers). In summary, our study demonstrates that sputum proteomics/transcriptomics can capture the complex and reversible physiological response to cigarette smoke exposure, which appears to be only slightly modulated in early-stage COPD patients. The study has been registered on ClinicalTrials.gov with identifier NCT01780298.

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Genetic characterization of macrophages from induced sputum of asthma and COPD patients

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