Severity of Lung Function Impairment Drives Transcriptional Phenotypes of COPD and Relates to Immune and Metabolic Processes

Abstract

Purpose

This study sought to characterize transcriptional phenotypes of COPD through unsupervised clustering of sputum gene expression profiles, and further investigate mechanisms underlying the characteristics of these clusters.

Patients and methods

Induced sputum samples were collected from patients with stable COPD (n = 72) and healthy controls (n = 15). Induced sputum was collected for inflammatory cell counts, and RNA extracted. Transcriptional profiles were generated (Illumina Humanref-8 V2) and analyzed by GeneSpring GX14.9.1. Unsupervised hierarchical clustering and differential gene expression analysis were performed, and gene alterations validated in the ECLIPSE dataset (GSE22148).

Results

We identified 2 main clusters (Cluster 1 [n = 35] and Cluster 2 [n = 37]), which further divided into 4 sub-clusters (Sub-clusters 1.1 [n = 14], 1.2 [n = 21], 2.1 [n = 20] and 2.2 [n = 17]). Compared with Cluster 1, Cluster 2 was associated with significantly lower lung function (p = 0.014), more severe disease (p = 0.009) and breathlessness (p = 0.035), and increased sputum neutrophils (p = 0.031). Sub-cluster 1.1 had significantly higher proportion of people with comorbid cardiovascular disease compared to the other 3 sub-clusters (92.5% vs 57.1%, 50% and 52.9%, p < 0.013). Through supervised analysis we determined that degree of airflow limitation (GOLD stage) was the predominant factor driving gene expression differences in our transcriptional clusters. There were 452 genes (adjusted p < 0.05 and ≥2 fold) altered in GOLD stage 3 and 4 versus 1 and 2, of which 281 (62%) were also found to be significantly expressed between these GOLD stages in the ECLIPSE data set (GSE22148). Differentially expressed genes were largely downregulated in GOLD stages 3 and 4 and connected in 5 networks relating to lipoprotein and cholesterol metabolism; metabolic processes in oxidation/reduction and mitochondrial function; antigen processing and presentation; regulation of complement activation and innate immune responses; and immune and metabolic processes.

Conclusion

Severity of lung function drives 2 distinct transcriptional phenotypes of COPD and relates to immune and metabolic processes.

Keywords:

• COPD
• gene expression
• inflammation
• sputum

Abbreviations

BMI, body mass index; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; mMRC, modified Medical Research Council; GOLD, Global Initiative for Chronic Obstructive Lung Disease; BODE, body mass index, airflow obstruction, dyspnea, exercise capacity; SGRQ, St George Respiratory Questionnaire; CCI, Charlson Comorbidity Index; HADS, Hospital Anxiety and Depression Scale; ICS, inhaled corticosteroids; CRP, C-reactive protein; SD, standard deviation.

Acknowledgment

The authors would like to acknowledge the technical assistance of Naomi Fibbens, Alan Hsu, Gabrielle Le Brocq, Amber Smith, Bridgette Ridewood, Michelle Gleeson, and Kellie Fakes.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

NA Negewo and JL Simpson have nothing to disclose for this study. KJ Baines reports grants from NHMRC, Lung Foundation of Australia, Hunter Medical Research Institute and John Hunter Hospital Charitable Trust, outside the submitted work. VM McDonald reports grants from NHMRC, Lung Foundation of Australia, grants from Ramaciotti Foundation, during the conduct of the study; grants from Medical Research Futures Fund, NHMRC, personal fees from GSK, personal fees from AstraZeneca, and guideline writer role in COPDX committee, outside the submitted work. PG Gibson reports grants from GSK, personal fees from AstraZeneca, Novartis and GSK, outside the submitted work.