Analysis of BAL extracellular vesicles unveils NF-kB activation at the onset of chronic lung allograft dysfunction

The onset of chronic lung allograft dysfunction (CLAD) represents the greatest long-term challenge in lung transplantation (LT). Here we aimed to identify early molecular signals of CLAD by analyzing the effects of bronchoalveolar lavage (BAL)-derived extracellular vesicles (EVs) on airway cells and validating these findings in patient lung tissues. In our BAL biobank we identified thirteen LT patients with a BAL sample at CLAD diagnosis, and 13 patients with a stable graft function and a BAL sample obtained at least 12 post LT (Ctrl). All patients were then followed for at least 18 EVs were isolated, immunophenotyped, and co-cultured with airway cells. The cells transcriptome and proteome were profiled. Selected targets were validated by immunohistochemistry. Logistic regression and survival analyses were performed for prediction of CLAD progression. During follow-up, 7 CLAD patients experienced allograft dysfunction aggravation and one control developed CLAD. CLAD patients showed more EVs originating from epithelial cells and leukocytes than stable LT recipients. Exposure of airway cells to CLAD-EVs led to the upregulation of p70S6K and canonical NF-kB signaling, altering their intracellular and extracellular proteome. Activation of NF-kB was also detected at the onset of CLAD in transbronchial biopsies and BAL cytology, and it persisted throughout the progression to end-stage CLAD. RelA overexpression was associated with poorer graft performance and worse outcomes. RelA-driven NF-kB activation is a key factor in the development of CLAD by promoting persistent inflammation. This pathway may be a promising therapeutic target to improve long-term graft survival after LT.