NETosis in Broncho-Alveolar Lavage-Fluid (BAL-f) from Bronchiolitis Obliterans Syndrome (BOS) Patients

Abstract

Purpose Bronchiolitis obliterans Syndrome (BOS) is a major long term complication after lung transplant. The role of Neutrophil (Neu) activation in BOS pathogenesis has been repeatedly suggested but needs to be fully clarified. Human Neutrophil Elastase (HNE) as well as complexes of alpha-1-antitrypsin (AAT)/HNE are more frequently detected in BAL-f from BOS patients thus suggesting chronic Neu activation BOS. NETosis is a mechanism of Neu response to pathogens which can drive severe epithelial injury. Here, we aim to analyze possible role of NETosis in BOS by assessing number of Neu and related markers in BAL-f. Methods A total of 16 BAL samples from BOS (n° 8, 3 BOS1, 2 BOS2 and 2 BOS3) and from stable lung recipients (n°8) were included. Patients with sarcoidosis (Sarco) were used as control (n°3). The following measures were performed: BAL Neu counts; HNE, IL1beta and IL8 (ELISA); NETs (measuring HNE-DNA complexes by ELISA); AAT/HNE complexes (WB). Results NETs were measurable in all BAL samples from Tx recipients, with a not-significant trend towards higher values among BOS patients (p=0.275). Interestingly NETs levels did not correlate with Neu counts (r=0.575; p=0.143) but, limited to BOS patients, significantly correlated with IL-8 (r=0.786; p=0.028), HNE (r=0.736; p=0.028), and AAT/HNE complexes (r=0.73; p=0.05).No correlation with IL1beta levels was found. Conclusion This is the first preliminary evidence of the possibility to detect NETs in BAL-f of lung transplant recipients and, interestingly points out that NETs levels do not seem to correlate simply with Neu counts but rather with inflammation and specific Neu activation. Thus, they could potentially represent a marker of higher inflammatory state. In addition, given the evidence that NETs drive epithelial cell injury and cause EMT their significant pathogenic role in BOS can be suggested and future therapeutic strategies can be hypothesized.