Serine proteases in neutrophil extracellular traps exhibit anti-Respiratory Syncytial Virus activity.

Human respiratory syncytial virus (hRSV) is an infectious agent in infants and young children which there are no vaccines or drugs for treatment. Neutrophils are recruited for airway, where they are stimulated by hRSV to release large amounts of neutrophil extracellular traps (NETs). NETs are compound by DNA and proteins, including microbicidal enzymes. They constitute a large part of the mucus accumulated in the lung of patients, compromising their breathing capacity. In contrast, NETs can capture/inactivate hRSV, but the molecules responsible for this effect are unknown. OBJECTIVES: We selected microbicidal NET enzymes (elastase, myeloperoxidase, cathepsin-G, and proteinase-3) to assess their anti-hRSV role. METHODS AND RESULTS: Through in vitro assays using HEp-2 cells, we observed that elastase, proteinase-3, and cathepsin-G, but not myeloperoxidase, showed virucidal effects even at non-cytotoxic concentrations. Elastase and proteinase-3, but not cathepsin-G, cleaved viral F-protein, which is responsible for viral adhesion and fusion with the target cells. Molecular docking analysis indicated the interaction of these macromolecules in the antigenic regions of F-protein through the active regions of the enzymes. CONCLUSIONS: Serine proteases from NETs interact and inactive hRSV. These results contribute to the understanding the role of NETs in hRSV infection and to designing treatment strategies for the inflammatory process during respiratory infections.

Neutrophil extracellular traps possess anti-human respiratory syncytial virus activity: Possible interaction with the viral F protein.

Human respiratory syncytial virus (hRSV) is one of the main etiological agents of diseases of the lower respiratory tract, and is often responsible for the hospitalization of children and the elderly. To date, treatments are only palliative and there is no vaccine available. The airways of patients infected with hRSV exhibit intense neutrophil infiltration, which is responsible for the release of neutrophil extracellular traps (NETs). These are extracellular structures consisting of DNA associated with intracellular proteins, and are efficient in capturing and eliminating various microorganisms, including some viruses. hRSV induces the release of NETs into the lung tissue of infected individuals; however, the pathophysiological consequences of this event have not been elucidated. The objective of this study was to utilize in vitro and in silico assays to investigate the impact of NETs on hRSV infection. NETs, generated by neutrophils stimulated with phorbol myristate acetate (PMA), displayed long fragments of DNA and an electrophoretic profile suggestive of the presence of proteins that are classically associated with these structures (elastase, cathepsin G, myeloperoxidase, and histones). The presence of NETs (>2 µg/ml) in HEp-2 cell culture medium resulted in cellular cytotoxicity of less than 50%. Pre-incubation (1 h) of viral particles (multiplicity of infection (MOI) values of 0.1, 0.5, and 1.0) with NETs (2-32 µg/ml) resulted in cellular protection from virus-induced death of HEp-2 cells. Concurrently, there was a reduction in the formation of syncytia, which is related to decreased viral spread in infected tissue. Results from western blotting and molecular docking, suggest interactions between F protein of the hRSV viral envelope and BPI (bactericidal permeability-increasing protein), a microbicidal member of NETs. Interactions occurred at sites important for the neutralization and coordination of the hRSV infection/replication process. Our results showed that the presence of NETs decreases hRSV-induced cellular damage, possibly by directly affecting viral particle capture and/or interfering with the fusion activity of the F protein. These findings broaden the understanding of the role of NETs during hRSV infection.