Abstract
Exosomes are small membrane vesicles that regulate intracellular function and cell-to-cell communication. Our report has shown circulating exosomes are induced during lung allograft rejection (Am J Transplant, 2017; 17:474-484). Rab27, a member of the Rab subfamily of GTPases, is essential for exosome secretion. The aim of this study is to determine the role of Rab27A signaling in human bronchial epithelial cells (BEAS-2B) stimulated with exosomes from human lung transplant recipients (LTxRs) diagnosed with acute rejection, bronchiolitis obliterans syndrome (BOS), or stable.
Exosomes were isolated using ultracentrifugation and 100µg of exosomes were added to BEAS-2B and incubated for 24hrs. Supernatants collected were analyzed for IL-6 using luminex platform. Epithelial mesenchymal transformation was determined using vimentin and α-SMA by Western blot. Protein band in western blot was normalized with β-Actin and densitometry was performed using Image-J software.
Supernatants of BEAS-2B cells, stimulated with BOS-exosome, showed significantly higher production of IL-6 (2927, 4592 MFI p=<0.01) as compared to stable-exosome. Rab27A siRNA knockdown decreased the production of IL-6 (2998 MFI vs 1307 MFI p=<0.01) in supernatants. Further, Rab27A knockdown significantly reduced fibronectin protein in BOS (50% reduction) exosome treated BEAS-2B cells compared to stable (p=<0.01). Vimentin and α-SMA were significantly higher (p=<0.01) in BOS-exosome treated BEAS-2B cells compared to stable-exosome.
Rab27A signaling plays an important role in the production of pro-inflammatory cytokine IL-6 by airway epithelial cells following incubation with exosomes isolated from LTxRs with rejection. In addition, Rab27A signaling plays a role in exosome mediated upregulation of fibronectin protein production resulting in epithelia mesenchymal transition of the airway epithelial cells.