Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

Nina Höglund; Ninna Koho; Heini Rossi; Jenni Karttunen; Anne-Mari Mustonen; Petteri Nieminen; Kirsi Rilla; Sanna Oikari; Anna Mykkänen

doi:10.3389/fvets.2022.894189

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

Front Vet Sci. 2022 Jun 21:9:894189. doi: 10.3389/fvets.2022.894189. eCollection 2022.

Authors

Nina Höglund¹, Ninna Koho¹, Heini Rossi¹, Jenni Karttunen¹, Anne-Mari Mustonen^{2

3}, Petteri Nieminen², Kirsi Rilla², Sanna Oikari², Anna Mykkänen¹

Affiliations

¹ Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
² Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
³ Department of Environmental and Biological Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu, Finland.

Abstract

Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell-cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 10¹² ± 1.42 × 10¹² particles/mL in the EA horses and 1.33 × 10¹² ± 1.07 × 10¹² particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.

Keywords: airway disease; bronchoalveolar lavage fluid (BALF); equine asthma; extracellular vesicle (EV); inflammation; size-exclusion chromatography.