Role of exosomes in rejection following human lung transplantation – an in vitro analysis using human airway epithelial cells

Abstract

Abstract Studies have demonstrated that exosomes expressing donor human leukocyte antigen (HLA) and lung self-antigens (SAg), Collagen V (Col-V) and Kα1 Tubulin (Kα1T), can be detected in the circulation of human lung transplant recipients (LTxR) who developed antibodies (Ab) to donor HLA and diagnosed with rejection However, the mechanisms by which Ab mediate induction of exosomes into the circulation from airway epithelial cells (AEC) remains unclear. Ab ligation to HLA could enhance PERK-mediated pathways involved in endoplasmic reticular (ER) stress-dependent exosome release. The goal of this study is to determine the mechanism by which Ab induce exosomes from AEC. Towards this we employed human AEC, ligated antigens by Ab specific to HLA (HiPRA) and lung SAg (rabbit anti-Col-V or Kα1T) and the role of ER stress signaling molecule in exosomes release were determined. Effect of exosome inhibition by GW4869 was also analyzed. AEC were incubated with HiPRA or Ab to SAg for 24hr in both normoxia and hypoxia (0.1% O2) conditions. AEC incubated with HiPRA or Ab to SAg demonstrated induction of exosomes containing lung SAg, Col-V and Kα1T. Incubation with HiPRA and Ab (20μg/ml) to SAg also demonstrated induction of exosomes containing lung SAg, MHC class II, CD80, CD86. Treatment with GW4869, exosome inhibitor (20μM) reduced the exosomes and SAg. Effect of HiPRA and Ab to SAg on stress kinase in both normoxic and hypoxic conditions and influence of GW4869 in these pathways will be delineated. Identification of stress kinase and exosome inhibition will provide novel results for Ab mediated upregulation of stress leading to exosome release from the transplanted organ resulting in immune responses thus increasing the risk for chronic rejection following LTx.