Induction of Epithelial-Mesenchymal Transition (EMT) by Neutrophil Extracellular Traps (NETs) as Possible Molecular Mechanism in CLAD

Abstract

Purpose Chronic Lung Allograft Dysfunction (CLAD) is the main limitation to long term post lung transplant survival and is characterized by interstitial or airway fibrosis. Recruitment and activation of Neutrophils in the graft has been described in CLAD. Neutrophils are an essential component of the innate immune system which can undergo different activation processes, among which NETosis. NETs can drive epithelial cell injury and induce endothelial mesenchymal transition or epithelial mesenchymal transition (EMT) of breast cancer cells. We here aim to investigate whether NETs can induce EMT on a human alveolar basal epithelial cell line (A549) as possible mechanism of fibrosis in CLAD. Methods NETosis was induced on healthy donor-derived neutrophils (Neu) with PMA. EMT was studied on A549 cells in three conditions (PMA-activated Neu (PMA-Neu), purified NETs from PMA-Neu and TGF-β). To study the induction of EMT, we evaluate the expression of two main proteins representative of EMT mechanism (α-SMA and E-cadherin) by western blot of cell lysates. Since it is known that neutrophils activated against NETosis can induce cell damage, we decided to evaluate cell toxicity after treatments with PMA-Neu or NETs by flow cytometry. Results Confocal microscope images confirmed that PMA treatment leads to release NETs by neutrophils. NETs changed the typical epithelial morphology of A549 cells into a mesenchymal phenotype. These morphological changes are also visible after treatment with TGF-β, the best known EMT effector. Morphological changes were confirmed by western blot analysis of EMT markers expression. Both PMA-Neu and purified NETs induced EMT in A549 after 24h, increasing α-SMA (1.21±0.025 and 1.34±0.025, respectively) and decreasing E-Cadherin expression (0.245±0.10 and 0.08±0.002, respectively). Moreover we observed that NETs activate EMT without toxic effects rather than PMA-Neu which induced 33.70 ± 4.37 % cell death compared to control cells. Conclusion This study highlights the contribution NETs as trigger of lung fibrosis, and suggest this mechanism might contribute to CLAD pathogenesis.