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Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense

Mehmet Kesimer^*, Margaret Scull, Brian Brighton, Genevieve DeMaria^*, Kimberlie Burns, Wanda O'Neal, Raymond J. Pickles and John K. Sheehan^*,1

^* Department of Biochemistry and Biophysics,

Department of Microbiology and Immunology, and

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

¹Correspondence: 4021 Thurston Bowles Bldg. CB#7248, Chapel Hill, NC 27599-7248, USA. E-mail: kesimer{at}med.unc.edu

Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30–100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12–1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles’ structure. Notably, -2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.— Kesimer, M., Scull, M., Brighton, B., DeMaria, G., Burns, K., O’Neal, W., Pickles, R. J., Sheehan, J. K. Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense.

Key Words: mucins • influenza virus • mucus • glycocalyx • sialic acid