Abstract
Lung transplantation (LTx) is a treatment option for end-stage pulmonary diseases. Despite improvements in surgical techniques and post-transplant (Tx) immunosuppression, LTx continues to have one of the poorest long-term outcomes compared to other solid organ Tx, with five year survival of about 50%.
Using an orthotopic single LTx model of B6D2F1/J to DBA/2J strain combination, we demonstrate that >80% of transplanted animals developed histological signs of chronic rejection by day 30 of the transplanted lung but not native. We collected blood at days 7, 14, 28, and 30 and serum was tested by ELISA for development of antibodies to lung self-antigens (SAgs), Collagen V (Col-V) and K-alpha 1 Tubulin (Kα1T).
Significantly elevated levels of antibodies to Col-V and Kα1T were demonstrable on day 14 when there was no histological evidence for chronic rejection of the transplanted lungs. Circulating exosomes were isolated from sera collected on days 7 and 14 which demonstrated increased levels of lung SAgs by western blots in the exosomes isolated from day 14 sera. These results clearly demonstrate that not only antibodies to lung SAgs are present in sera from day 14, but also circulating exosomes with Col-V and Ka1T are easily demonstrable in sera collected on day-14. Semi-quantitation of western blot by densitometry further demonstrated significantly increased levels of exosomes with lung SAgs starting from day 14 until sacrifice on day 30. Splenocytes isolated following sacrifice demonstrated specific reactivity to lung SAgs with significantly high frequency of IFN-γ (Col-V 109±2.5, Kα1T 101±5.5) and TNF-α (Col-V 39±1.5, Kα1T 53±5.5) secreting cells by ELISPOT.
Taken together our results demonstrate that circulating exosomes with lung SAgs and de novo development of antibodies to lung SAgs occurs prior to histological evidence of chronic rejection in this murine model of chronic rejection following orthotopic single LTx suggesting that circulating exosomes with lung SAgs can contribute in the pathogenesis of chronic rejection following LTx. Further, our data suggests that circulating exosomes with lung SAgs could be a potential biomarker for patients at risk for developing chronic rejection following human LTx.