View Abstract

A3095 - Increased Peribronchiolar and Perivascular Cysteine Protease Activity Is Associated with Bronchiolitis Obliterans Syndrome
Author Block: C. Morrone1, N. F. Smirnova2, N. Kneidinger3, M. Mann4, H. B. Schiller5, O. Eickelberg6, A. O. Yildirim7; 1ILBD/CPC, Helmholtz Zentrum, Munich, Germany, 2Comprehensive Pneumology Center, Munich, Germany, 3Univ of Giessen Lung Ctr, Giessen, Germany, 4Proteomics and Signal Transduction, Max Planck Institute of biochemistry, Munich, Germany, 5MPIB, Martinsried, Germany, 6Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States, 7Institute of Lung Biology and Disease (iLBD),, Comprehensive Pneumology Center (CPC), Neuherberg, Germany.
After lung transplantation (LTx), 50% of the patients do not survive the first 5 years due to chronic lung allograft dysfunction (CLAD). Bronchiolitis obliterans syndrome (BOS) is the most common CLAD phenotype, characterized by mononuclear infiltration and fibrotic tissue deposition around the bronchioles, which is associated with a progressive obstruction. The mechanism underlying BOS development remains to be defined. Here, we aimed to investigate factors associated with the tissue remodeling during the inflammatory response in BOS that affect the survival of BOS patients. Human BALF, and lung tissue from LTx patients with and without signs of BOS were analyzed by quantitative proteomics, Western blot (WB), protease activity measurements, ELISA, and immunostaining. Orthotopic left lung transplanted mice were used as model of no CLAD (syngeneic group) and CLAD (allogeneic group). BALF and lung tissue were analyzed in time course experiments until two months after LTx by WB and staining. Quantitative proteomics of human BALF showed the downregulation of cystatins (CSTs), members of cysteine protease inhibitor superfamily, in particular cystatin C (p = 0.0001) and cystatin S (p = 0.0188), that negatively regulate cysteine-proteases. Cysteine proteases have been shown to play a role in the activation of profibrotic factors and in inflammatory responses. Using WB, and protease activity assays we confirmed increased activity and amount of cysteine protease in human BALF from BOS patients. Histological analysis of lungs from BOS proved the accumulation of these proteases in the peribronchiolar and perivascular compartment that were affected by mononuclear infiltration and fibrosis. We confirmed these findings in an orthotopic lung transplantation model for CLAD in mice. In agreement with human outcomes, histological evidence from the CLAD mouse model confirmed the expression of cysteine proteases in the peribronchiolar area starting from seven days after LTx, with its peak at one month, and slightly reduced levels after two months. This finding suggests a potential involvement of cysteine proteases in the rejection of bronchioles. We are currently using the CLAD mouse model to study the functional role of this pathway in BOS pathogenesis. In this study we showed that BOS is associated with an increase in the amount and activity of cysteine proteases. Their accumulation in the peribronchiolar and perivascular area, that clearly shows signs of fibrosis, justifies further investigation into the role of these proteases in the pathogenesis of BOS.