Estudo dos mecanismos de necrose programada induzida pela infecção com vírus sincicial respiratório em macrófagos e seu papel na patogênese da doença

Abstract

Respiratory syncytial virus (RSV) is considered the main cause of respiratory infections during childhood. The secretion of pro-inflammatory cytokines may contribute to the damage of airways caused by RSV infection. Although macrophages have a sentinel role against inhaled pathogens, it seems that they also play a crucial role in the development of severe disease during airway infection. Previous studies have shown that necrosis may be regulated through the activation of signaling pathways mediated by TNF when apoptosis is inhibited. Therefore, programmed necrosis (or necroptosis), is a regulated process induced by the activation of death receptors and it depends on the activation of a complex consisting of RIPK1, RIPK3 and MLKL proteins. Therefore, the hypothesis of this study is that during RSV infection, the virus stimulates TNF secretion by macrophages and that, in an autocrine manner, TNF induces programmed necrosis by macrophages via RIPK1, RIPK3 and MLKL signaling. Here we showed that RSV is capable of inducing death by necrosis of alveolar and peritoneal macrophages. This effect is dependent on virus concentration and time of infection, as well as replicative activity of RSV. RSV stimulates TNF production by macrophages and TNF secretion is necessary for the effect of RSV on cell death. Treatment of macrophages with neutralizing anti-TNF antibody significantly reduced necrosis induced by RSV. Likewise, TNFR1 KO macrophages were resistant to RSV-induced necrosis. Treatment of cells with the selective necroptosis inhibitors NEC-1, GW-42X and NSA, suppressed RSV effect on macrophage necrosis, indicating that RSV triggers macrophage necroptosis. In addition, depletion of alveolar macrophages decreased weight loss and viral load in the lung of animals infected with RSV. Also, we measured TNF in the nasal wash of pediatric patients hospitalized for RSV bronchiolitis. We observed that RSV-positive patients presented higher levels of TNF in their nasal washes when compared to those infected by other respiratory viruses. With these data, we demonstrate that RSV-induced macrophage necroptosis was mediated by autocrine TNF and was dependent on the activation of RIPK1, RIPK3 and MLKL. Moreover, macrophage necroptosis plays a detrimental role during RSV infection in vivo.