Sputum-Derived Extracellular Vesicles as Non-Invasive Biomarkers for Predicting Acute Exacerbations of Chronic Obstructive Pulmonary Disease

Abstract

Background

Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are major drivers of morbidity, mortality, and healthcare costs worldwide. Predicting these events early remains difficult because current systemic biomarkers lack specificity for localized airway pathology. Sputum-derived extracellular vesicles (EVs) originate directly from the respiratory mucosa and carry distinct molecular payloads. We investigated whether the quantitative profile and microRNA (miRNA) cargo of sputum-derived EVs could serve as a reliable, non-invasive liquid biopsy to predict AECOPD risk.  

Methods

In this prospective translational cohort study, induced sputum samples were collected from 160 clinically stable patients with moderate-to-severe COPD (GOLD Stages II–IV) who were subsequently tracked for 12 months. Sputum EVs were isolated using size-exclusion chromatography and verified via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting for classical tetraspanin markers (CD9, CD63, and CD81). Total EV protein concentrations were quantified, and a specific panel of inflammatory and hypoxia-related exosomal microRNAs—specifically miR-21, miR-155, miR-146a, and miR-223—was analyzed via RT-qPCR.

Results

During the 12-month follow-up window, 68 patients experienced at least one documented acute exacerbation ($42.5\%$). At baseline baseline, patients who later went on to develop frequent exacerbations ($\ge 2$ events/year) exhibited a significantly higher mean concentration of sputum EVs in the 150–200 nm size range, alongside a two-fold increase in total EV-associated protein compared to those who remained stable ($p < 0.001$).

Transcriptomic analysis of the EV cargo showed a stark molecular shift in the group that went on to exacerbate: baseline expression of exosomal miR-21 and miR-223 (markers of active neutrophilic inflammation and tissue hypoxia) was significantly upregulated, while miR-155 was markedly suppressed ($p < 0.001$). Receiver operating characteristic (ROC) analysis revealed that combining total sputum EV protein levels (using a predictive cut-off threshold of $55.6\text{ }\mu\text{g/mL}$) with the miR-21/miR-155 ratio yielded excellent prognostic accuracy for predicting a 12-month AECOPD event, delivering an area under the curve (AUC) of $0.86$ ($95\%\text{ CI: } 0.77\text{–}0.94$; Sensitivity: $84\%$, Specificity: $78\%$).

Conclusions

Sputum-derived extracellular vesicles and their distinct microRNA signatures offer a highly accurate, non-invasive snapshot of sub-clinical lower-airway inflammation. Measuring these localized mucosal packages allows clinicians to reliably predict impending acute exacerbations, paving the way for targeted, early preventive strategies in high-risk COPD phenotypes.  

Keywords

Chronic Obstructive Pulmonary Disease (COPD); Acute Exacerbation (AECOPD); Extracellular Vesicles; Sputum Liquid Biopsy; MicroRNA; Translational Biomarkers.