Detection and Characterization of Extracellular Vesicles in Sputum Samples of COPD Patients

Abstract

Background: Only one study has reported the presence of extracellular vesicles (EVs) in COPD patients' sputum. Thus, we aimed to isolate and characterize EVs from COPD and healthy individuals' sputum.

Methods: A total of 20 spontaneous sputum samples from COPD patients (m/f: 19/1) and induced sputum samples from healthy controls (m/f: 8/2) were used for EV isolation. The sputum supernatants were resuspended in PBS, precleared by centrifugation at 800× g for 10 min at 4 °C, and passed through a 0.22 μm filter (Millipore, Burlington, MA, USA). EVs were isolated by a standard membrane affinity spin column method (exoEasy maxi kit, Qiagen, Hilden, Germany). The EVs were then characterized by assessing their morphology and size using Transmission Electron Microscopy (TEM) and determining the CD9 and CD81 EV-markers with Western blot analysis.

Results: The EVs had a spherical shape and their mean diameter in the COPD patients was significantly greater than in the controls. Enrichment of the EV markers, CD9 and CD81, were detected in both the healthy and COPD individuals. Total EV-associated protein was significantly increased in the COPD patients compared to the controls. ROC analysis showed that total EV-associated protein in the sputum could be used to differentiate between the controls and COPD patients, with a sensitivity of 80% and a specificity of 70% at a cut-off point of 55.59 μg/mL (AUC = 0.8150).

Conclusions: EVs were detectable in both the COPD and healthy individuals' sputum. The ratio of EVs in the 150-200 nm range was twice as high in the COPD patients than in the controls. The COPD patients' sputum contained increased total EV-associated protein as compared to controls, highlighting their value as a new source of specific exoproteins.

Keywords: COPD; biomarkers; exosomes; extracellular vesicles; lung; sputum.

Figure 1

Transmission electron microscopic image of sputum-derived extracellular vesicles from (A) Healthy Control and (B) COPD patient. Extracellular vesicles (EVs) with round shape morphology with up to 200 nm size are present (left and right, bar: 200 nm). (C) Western blot analysis of exosomes collected from sputum samples of healthy controls (HC) and COPD patients. The presence of the exosome specific tetraspanins, CD81 and CD9, as well as the non-exosomal protein marker calnexin was assessed.

Figure 2

Scattergrams present EV-associated total protein in the sputum of COPD patients compared to healthy controls. (A) Total EV-associated protein isolated from sputum samples of COPD patients (n = 20) compared to healthy controls (n = 10). (B) Diagnostic performance of sputum EV-associated total protein according to ROC Analysis. Sputum EV-associated protein was an accurate to test to differentiate between COPD and healthy individuals with a sensitivity of 80% and a specificity of 70% at a cut-off point of 55.59 μg/mL (AUC = 0.8150).