Neutrophils contribute to vasculitis by increased release of neutrophil extracellular traps in Behcet's disease

Background and objectives: Behcet's disease (BD) is a multi-system inflammatory disorder that can cause vasculitis. Here we questioned whether Neutrophils in BD cause vasculitis via releasing Neutrophil Extracellular Traps (NETs), a process called NETosis. Methods: Circulating neutrophils were isolated from a cohort of Middle Eastern BD patients with an active disease and healthy volunteers. The percentage of NETs release was monitored in neutrophils stimulated or not with BD serum, and treated or not with Colchicine, Dexamethasone, Cl-amidine or N-Acetyl Cysteine (NAC). The mRNA expression levels of PAD4 (a key enzyme in NETosis) was also assessed. The effect of NETs on the proliferation and cell death of endothelial cells was investigated using an in vitro co-culture model. The presence of NETs in skin tissues of BD patients was examined using immunolabeling of NETs associated proteins. Results: Circulating Neutrophils from BD patients were more prone to release NETs in vitro and expressed higher levels of PAD4 compared to healthy volunteers. Spontaneous NETs formation in BD neutrophils was inhibited by Colchicine and Dexamethasone, two drugs used to treat BD. NETs formation was also inhibited by Cl-amidine, a specific PAD4 inhibitor, and by NAC, a ROS inhibitor. Interestingly, serum from BD patients stimulated circulating neutrophils from healthy volunteers to release more NETs and increased their mRNA PAD4 expression. Moreover, endothelial cells cultured in the presence of NETs from BD patients showed a decrease in proliferation and an increase in apoptosis and cell death. Finally, NETosis was predominantly identified around affected blood vessels in biopsies of vasculitis from BD patients. Conclusion: Our results provide evidence on the implication of NETosis in the pathophysiology of BD especially in inducing vasculitis. (C) 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.