Pulmonary EV miRNA profiles identify disease and distinct inflammatory endotypes in COPD

Abstract

Introduction: Chronic obstructive pulmonary disease (COPD) is a heterogeneous condition without effective disease modifying therapies. Identification of novel inflammatory endotype markers such as extracellular vesicles (EVs), which are important intercellular messengers carrying microRNA (miRNA), may enable earlier diagnosis and disease stratification for a targeted treatment approach. Our aim was to identify differentially expressed EV miRNA in the lungs of COPD patients compared with healthy ex-smokers and determine whether they can help define inflammatory COPD endotypes.

Methods: EV miRNA were isolated and sequenced from ex-smoking COPD patients and healthy ex-smoker bronchoalveolar lavage fluid. Results were validated with RT-qPCR and compared to differential inflammatory cell counts.

Results: Expression analysis identified five upregulated miRNA in COPD (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p and miR-625-3p) and three downregulated miRNA (miR-138-5p, miR-338-3p and miR-204-5p), all with a log2 fold change of >1/-1, FDR < 0.05. These miRNAs correlated with disease defining characteristics such as FEF 25-75% (a small airways disease measure) and DLCO% (a surrogate measure of emphysema). Receiver operator curve analysis demonstrated miR-2110, miR-223-3p, and miR-182-5p showed excellent combinatory predictive ability (AUC 0.91, p < 0.0001) in differentiating between health and mild COPD. Furthermore, miR-223-3p and miR-338-3p correlated with airway eosinophilia and were able to distinguish "pure eosinophilic" COPD from other airway inflammatory subtypes (AUC 0.94 and 0.85, respectively).

Discussion: This is the first study to identify differentially expressed miRNA in COPD bronchoalveolar lavage fluid EVs. These findings suggest specific lung derived EV miRNA are a strong predictor of disease presence even in mild COPD. Furthermore, specific miRNA correlated with inflammatory cell numbers in COPD, and may have a role in defining inflammatory endotypes for future treatment stratification.

Keywords: COPD; early diagnostics; extracellular vesicles; inflammatory endotypes; microRNA.

FIGURE 1

Subject enrolment and tests performed in the study to assess extracellular vesicle (EV) microRNA (miRNA) expression. Subjects included in the grey, hashed boxes were recruited later and therefore only underwent testing for EV miRNA via RT-qPCR. *All subjects were ex-smokers having given up smoking at least 6 months prior to study enrolment and with at least a 10-pack year smoking history.

FIGURE 2

Volcano plot showing relationship between P-values and expression data for differentially expressed miRNA validated by RT-qPCR. Red dots show miRNA with P-values < 0.05 after false discovery rate (FDR) correction for multiple testing. Blue dotted line represents zero Log2FC, data points to the right are upregulated in chronic obstructive pulmonary disease (COPD), and data points to the left are downregulated in COPD. FC, fold change; miRNA, microRNA; RT-qPCR, real-time quantitative PCR.

FIGURE 3

Receiver operator curve analysis for miR-2110, miR-223-3p, and miR-182-5p and the combination of miR-2110, miR-223-3p, and miR-182-5p.

FIGURE 4

Venn diagram to describe the inflammatory endotypes in the chronic obstructive pulmonary disease (COPD) subjects based on pre-defined cut-offs.