RSV infection in a macrophage-cell line activates the non-canonical NF-κB pathway and induces pro-inflammatory cytokine expression.

Respiratory syncytial virus (RSV) is a highly prevalent infectious agent that causes severe respiratory tract illnesses in infants and children worldwide. Children who have suffered severe RSV infections during infancy are prone to develop recurrent episodes of wheezing and asthma that may be associated with viral persistence. RSV infections in humans and animal models are characterized by extensive inflammatory responses. Epithelial cell lines acutely infected by RSV have shown activation of the NF-κB signaling through two independent pathways: the canonical pathway, mediated by RelA and p50 subunits, and the non-canonical pathway, mediated by the subunits RelB and p52. Herein, we investigated the state of activation of the canonical and non-canonical NF-κB signaling pathways in macrophages either acutely or persistently infected by RSV and examined the expression of pro-inflammatory mediators. Activation of NF-κB subunits was analyzed through Western blot assays using acutely RSV-infected epithelial cells as a control. The expression levels of two pro-inflammatory cytokines and a chemokine were determined by quantitative RT-PCR and through immunobead assays. The results showed that p52 was abundant during acute and persistent RSV infection, indicating that macrophages predominantly activate the non-canonical pathway. We also observed activation of IL-1ß, TNF-α and CCL5/RANTES transcription, though at higher levels in persistently infected macrophages than in acutely infected macrophages. In contrast, the protein levels of these cytokines/chemokine did not correlate with their mRNA transcription, as quantitation displayed higher levels during acute infection than in persistent infection, suggesting post-transcriptional regulation by RSV persistence.

In vitro cell fusion between CD4(+) and HIV-1 Env(+) T cells generates a diversity of syncytia varying in total number, size and cellular content.

Syncytia formation in HIV infections is driven by the virus fusion-active molecules (Env) interacting with membrane components of hosts cells. HIV-syncytia are usually interpreted as pathogenic entities and although they may potentially vary in size, numbers and types of constituent cells, little is known about the extent and significance of their diversity. Here, we describe numerically the cell population dynamics and the diversity of syncytia produced in the in vitro cell-fusion between two Jurkat T cell lines, one CD4(+) and the other Env(+). Cell-fusion partners were differentially stained with the lipophilic DiI and DiO, or with the cytoplasmic CMFDA and CMTMR tracers and syncytia showing double fluorescence were counted in a flow cytometer. The total number of syncytia formed, their size, cellular complexity and ratio of CD4(+)/Env(+) cells recruited, varied significantly in relation with time of reaction and initial proportions of fusion partners. The considerable structural diversity of syncytia formed, in so limited an in vitro cell fusion reaction, suggests that a greater heterogeneity may be formed in the natural course of disease. Identification of the main determinants of syncytia diversity allows for a detailed study of the relation between the syncytia structure and function.