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

Netsanet A Negewo,1 Peter G Gibson,2– 4 Jodie L Simpson,1 Vanessa M McDonald,2– 5 Katherine J Baines1 1Immune Health Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia; 2Centre of Excellence in Treatable Traits, University of Newcastle, New Lambton Heights, NSW, Australia; 3Department of Respiratory and Sleep Medicine, John Hunter Hospital, Newcastle, NSW, Australia; 4Asthma and Breathing Research Centre, Hunter Medical Research Centre, New Lambton Heights, NSW, Australia; 5School of Nursing and Midwifery, The University of Newcastle, Callaghan, NSW, AustraliaCorrespondence: Katherine J Baines, Hunter Medical Research Institute, Level 2 East Wing, Locked Bag 1000, New Lambton Heights, NSW, 2305, Australia, Tel +61 2 40420090, Fax +61 2 40420046, Email katherine.baines@newcastle.edu.auPurpose: 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