Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

Abstract

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Neutrophil Extracellular Traps Do Not Induce Injury and Inflammation in Well-Differentiated RSV-Infected Airway Epithelium

“…This could possibly be explained by the low concentration of NETs added in this study as compared to previous studies [ 15 , 16 , 18 ]. Literature reporting on the effect of NETs for the airway epithelium is heterogeneous in the reported methods for neutrophil stimulation as well as the cell culture models used [ 15 , 16 , 17 , 18 ]. Monolayer cultures are submerged by design, and this allows for the apical addition of large volumes of NET-containing fluid to the cell cultures.…”

“…However, these NETs were obtained by stimulating neutrophils with high dosages of phorbol myristate acetate (PMA, 500 nM), followed by the scraping of neutrophils from the culture plate and vigorous mixing [ 18 ]. In our study, we chose to use a ten-fold lower dosage of PMA for neutrophil stimulation as compared to Hudock et al [ 18 ], using PMA dosages for NET harvesting comparable to the literature [ 15 , 16 , 17 , 26 ]. At the dose of 50 nM, PMA induces NET formation rather than other forms of neutrophilic death [ 15 , 38 , 39 ], and we have previously shown that NETs collected following our method induce a significant increase in airway mucus viscoelasticity [ 26 ].…”

“…In vitro airway epithelial monolayer studies have previously shown that NETs are cytotoxic, induce epithelial cell death and cause a pro-inflammatory response via IL-6 and CXCL8 (IL-8) release [ 15 , 16 ]. In addition, in one study the exposure of RSV-infected Hep2 cells to different NET concentrations was associated with a dose-dependent decrease of syncytia formation in cells when using a low multiplicity of RSV infection (MOI 0.5), but not for higher MOI [ 17 ]. However, in this study the interpretation of the results is somewhat clouded by the detachment of cells exposed to high concentrations of NETs [ 17 ].…”

“…In addition, in one study the exposure of RSV-infected Hep2 cells to different NET concentrations was associated with a dose-dependent decrease of syncytia formation in cells when using a low multiplicity of RSV infection (MOI 0.5), but not for higher MOI [ 17 ]. However, in this study the interpretation of the results is somewhat clouded by the detachment of cells exposed to high concentrations of NETs [ 17 ]. A recent study, using well-differentiated human airway epithelium (HAE) cultures, showed that NETs lead to an inflammatory response via increased IL-8 and IL-1α release [ 18 ].…”