Abstract
We recently demonstrated increased circulating exosomes with down regulated tumor suppressor gene liver kinase B1 (LKB1) in lung transplant recipients (LTxRs) diagnosed with bronchiolitis obliterans syndrome (BOS). Further, downregulated LKB1 was also noted in transbronchial lung biopsies from BOS LTxRs. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. We hypothesized that LKB1-STRADα pathway downregulation accompanied with increases in circulating exosomes can be a biomarker for chronic rejection following murine lung transplantation.
A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. Both LKB1 and STRADα expression was examined in transplanted and native non-transplanted lungs. Exosome release was measured both in transplanted and non-transplanted mice in a time dependent manner. We also examined the effect of LKB1 knock down using CRISPR-CAS9 on mTOR signaling, and fibrosis using an in vitro culture system with a human airway epithelial cell line, BEAS-2B.
Single lung transplanted from a B6D2F1 mouse into a DBA/2J mouse demonstrated signs of chronic rejection by day 34 post-transplant (increased fibrosis and cellular infiltration) and significantly increased exosome release (p=0.04 at day 14, p=0.02 at day 34). Circulating exosomes isolated on day 34 following chronic rejection also showed significant, downregulation of LKB1 (p=0.02) compared to native non-transplanted lungs. Significant downregulation of LKB1 (p=0.013) and STRADα (p=0.002) expression was found in donor lung compared to recipient lung. CRISPR-CAS9 mediated LKB1 knockdown cells demonstrated significant upregulation of genes resulting in increased levels of fibronectin (p=0.005) and collagen-I (p=0.004) in BEAS-2B cells.
Using a murine model of chronic lung allograft rejection, we demonstrated that downregulation of the LKB1-STRADα axis regulates exosome release and increase fibrosis of transplanted lung suggesting that down regulation of LKB1in circulating exosomes can be a biomarker for chronic lung allograft rejection following human lung transplantation.