Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in young children and babies, being mainly associated with bronchiolitis. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an immune response is triggered and neutrophils are recruited. In this study, we investigated the mechanisms underlying NET production induced by RSV. We show that RSV induced the classical ROS-dependent NETosis in human neutrophils and that RSV was trapped in DNA lattices coated with NE and MPO. NETosis induction by RSV was dependent on signaling by PI3K/AKT, ERK and p38 MAPK and required histone citrullination by PAD-4. In addition, RIPK1, RIPK3 and MLKL were essential to RSV-induced NETosis. MLKL was also necessary to neutrophil necrosis triggered by the virus, likely promoting membrane-disrupting pores, leading to neutrophil lysis and NET extrusion. Finally, we found that RSV infection of alveolar epithelial cells or lung fibroblasts triggers NET-DNA release by neutrophils, indicating that neutrophils can identify RSV-infected cells and respond to them by releasing NETs. The identification of the mechanisms responsible to mediate RSV-induced NETosis may prove valuable to the design of new therapeutic approaches to treat the inflammatory consequences of RSV bronchiolitis in young children.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report that RSV infection triggers necroptosis in primary mouse macrophages and human monocytes in a RIPK1-, RIPK3- and MLKL-dependent manner. Moreover, necroptosis pathways are harmful to RSV clearance from alveolar macrophages. Additionally,Ripk3^−/−mice were protected from RSV-induced weight loss and presented with reduced viral loads in the lungs.Alveolar macrophage depletion also protected mice from weight loss and decreased lung RSV virus load. Importantly, alveolar macrophage depletion abolished the upregulation ofRipk3andMlklgene expression induced by RSV infection in the lung tissue.Autocrine tumor necrosis factor (TNF)-mediated RSV-triggered macrophage necroptosis and necroptosis pathways were also involved in TNF secretion even when macrophages were committed to cell death, which can worsen lung injury during RSV infection. In line,Tnfr1^−/−mice had a marked decrease inRipk3andMlklgene expression and a sharp reduction in the numbers of necrotic alveolar macrophages in the lungs. Finally, we provide evidence that elevated nasal levels of TNF are associated with disease severity in infants with RSV bronchiolitis.We propose that targeting TNF and/or the necroptotic machinery may be valuable therapeutic approaches to reduce the respiratory morbidity caused by RSV infection in young children.

The virome constitutes the viral component of the microbiome and it consists of the genomes of all the viruses that inhabit a particular region of the human body, including those that cause acute, persistent or latent infection, and retroviral elements integrated to host chromosomes. The human virome is composed by eukaryotic viruses, bacteriophages and archaeal viruses. The understanding of the virome composition and role on human health has been delayed by the absence of specific tools and techniques to accurately characterize viruses. However, more recently, advanced methods for viral diagnostics, such as deep sequencing and metagenomics, have allowed a better understanding of the diverse viral species present in the human body. Previous studies have shown that the respiratory virome modulates the host immunity and that, since childhood, the human lung is populated by viruses for whom there is no disease association. Whether these viruses are potentially pathogenic and the reason for their persistence remain elusive. Increased respiratory viral load can cause exacerbation of chronic pulmonary diseases, including COPD, cystic fibrosis, and asthma. Moreover, the presence of resident viral populations may contribute to the pathogenesis of community-acquired respiratory virus infections. In this mini review, I will discuss the recent progress on our understanding of the human lung virome and summarize the up-to-date knowledge on the relationships among community-acquired respiratory viruses, the lung virome and the immune response to better understand disease pathophysiology and the factors that may lead to viral persistence.

Background.Respiratory syncytial virus (RSV) infection is the major cause of respiratory disease in lower respiratory tract in infants and young children. Attempts to develop effective vaccines or pharmacological treatments to inhibit RSV infection without undesired effects on human health have been unsuccessful. However, RSV infection has been reported to be affected by flavonoids. The mechanisms underlying viral inhibition induced by these compounds are largely unknown, making the development of new drugs difficult.Methods.To understand the mechanisms induced by flavonoids to inhibit RSV infection, a systems pharmacology-based study was performed using microarray data from primary culture of human bronchial cells infected by RSV, together with compound-proteomic interaction data available forHomo sapiens.Results.After an initial evaluation of 26 flavonoids, 5 compounds (resveratrol, quercetin, myricetin, apigenin, and tricetin) were identified through topological analysis of a major chemical-protein (CP) and protein-protein interacting (PPI) network. In a nonclustered form, these flavonoids regulate directly the activity of two protein bottlenecks involved in inflammation and apoptosis.Conclusions.Our findings may potentially help uncovering mechanisms of action of early RSV infection and provide chemical backbones and their protein targets in the difficult quest to develop new effective drugs.