Rémi Safi1, Romy Kallas2, Tara Bardawil3, Carl Joe Mehanna4, Ossama Abbas3, Rola Hamam4, Imad Uthman2, Abdul-Ghani Kibbi3, Dany Nassar5. 1. Department of Anatomy, Cell Biology and Physiological Science, American University of Beirut, Beirut, Lebanon. 2. Division of Rheumatology, Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon. 3. Department of Dermatology, American University of Beirut Medical Center, Beirut, Lebanon. 4. Division of Ophthalmology, Department of Surgery, American University of Beirut Medical Center, Beirut, Lebanon. 5. Department of Anatomy, Cell Biology and Physiological Science, American University of Beirut, Beirut, Lebanon; Department of Dermatology, American University of Beirut Medical Center, Beirut, Lebanon; Hôpital Cochin Tarnier, Département de Dermatologie, Université Paris Descartes, Paris, France. Electronic address: dn18@aub.edu.lb.
Abstract
BACKGROUND AND OBJECTIVES: Behçet'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.
BACKGROUND AND OBJECTIVES: Behçet'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 BDpatients 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 BDpatients was examined using immunolabeling of NETs associated proteins. RESULTS: Circulating Neutrophils from BDpatients 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 BDpatients 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 BDpatients 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 BDpatients. CONCLUSION: Our results provide evidence on the implication of NETosis in the pathophysiology of BD especially in inducing vasculitis.
Authors: Jocelyn Dupuis; Martin G Sirois; Eric Rhéaume; Quang T Nguyen; Marie-Élaine Clavet-Lanthier; Genevieve Brand; Teodora Mihalache-Avram; Gabriel Théberge-Julien; Daniel Charpentier; David Rhainds; Paul-Eduard Neagoe; Jean-Claude Tardif Journal: PLoS One Date: 2020-12-02 Impact factor: 3.240