P014 Ebola virus induces a differential cytokine response and NFKB activation in infected cells

Introduction Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response. Macrophages and dendritic cells not only orchestrate innate and adaptive immune responses but are also early target cells of EBOV. Using infected human macrophages, we analyzed the antiviral response to EBOV compared to a promising EBOV vaccine candidate, recombinant vesicular stomatitis virus expressing EBOV-GP (rVSV/EBOV-GP). rVSV/EBOV-GP conferred complete protection against EBOV in a NHP model (Jones et al., 2005). Methods Primary human macrophages or Vero cells were infected with EBOV or rVSV/EBOV-GP. Western blot analysis was performed to detect induction of antiviral signaling. To investigate if EBOV is able to block pro-inflammatory signaling pathways, EBOV-infected cells were treated with LPS and the cytokine/chemokine response was analyzed. Cytokine mRNA expression levels were analyzed by qRT-PCR and supernatants were used to detect secretion using 18-plex Luminex assay. Immunofluorescence analysis was used to analyze p65 translocation. Results rVSV/EBOV-GP infection of macrophages led to the activation of antiviral pathways, including dsRNA-dependent protein kinase R (PKR) activation, induction of apoptosis, degradation of IκBα, and increased expression of cytokines at RNA level with IFNβ and TNFα showing the highest expression levels. However, cytokine levels were not significantly elevated in supernatants of rVSV/EBOV-GP-infected macrophages. The observed lack of cytokine secretion is likely due to the inhibition of the nuclear export of cellular mRNA by VSV (Stojdl et al., 2003). As cytokines were present in supernatants of rVSV/EBOV-GP-infected PBMCs, we currently investigate which cell type secretes the cytokines. EBOV infection did not induce apoptosis in infected cells and activation of PKR was suppressed. Interestingly, EBOV was not able to block apoptosis induced by exogenous stimuli in Vero cells, including dsRNA-mediated apoptosis. Degradation of IκBα and nuclear translocation of p65 was observed in EBOV-infected macrophages, indicating that EBOV activates NFκB signaling. Cytokine expression was upregulated in EBOV-infected macrophages, and most of the cytokines/chemokines tested in the Luminex assay, including IL6 and TNFα, were secreted from EBOV-infected cells at 6 hpi or later. Treatment of EBOV-infected macrophages with LPS revealed an increase in IL6 expression, suggesting that LPS-mediated signaling is not blocked by EBOV. In contrast to non-infected cells treated with LPS, IL10 was not upregulated in EBOV-infected macrophages even after stimulation with LPS. Conclusion Significant antiviral responses were observed after infection of macrophages with rVSV/EBOV-GP whereas multiple antiviral responses including PKR activation and apoptosis signaling are not induced or blocked in EBOV-infection. rVSV/EBOV-GP induced an increase in cytokine gene expression, but secretion of the corresponding proteins from macrophages was not detected. Furthermore, we observed NFκB activation and a differential cytokine response in macrophages infected with EBOV. Our data suggest a complex regulation of cytokine expression by EBOV in infected primary cells. Further analysis are planned to reveal the underlying mechanisms.