DENV has also been shown

DENV has also been shown to counteract IFN signaling in the infected cells, mainly through the expression of NS5 and NS4B (Munoz-Jordan et al., 2005; Ashour et al., 2009; Morrison et al., 2013). We indeed observed lessened ISG induction in the infected fbpase but not in the uninfected cells in the same virally exposed population. Consistent with these results, we found that although the infection of HLCs was normally self-limiting when a low MOI was used, virtually all cells could be infected if a high (>5) MOI was used in the initial inoculation, as no viral spread was needed in this case to infect all the cells and the IFN response was not able to clear the infection once it had been established. As reduced viral load generally correlates with lessened disease severity (Marques et al., 2015), therapeutic strategies aiming at reducing viral load early may better harness the benefit of host IFN response to thwart disease progression. A “cytokine storm” triggered by DENV is thought to be the major event of DENV pathogenesis (Guzman and Harris, 2015). Several cytokines that have been proposed to be associated with plasma leakage, including TNF, IL-6, IL-8, IL-10, and IL-12 (Guzman and Harris, 2015), were dramatically upregulated in HLCs by DENV infection. Activation of an inflammatory response via the cellular NF-κB pathway by the secreted viral protein NS1 is one underlying mechanism of the cytokine activation (Modhiran et al., 2015; Beatty et al., 2015; Silva et al., 2011). We observed a remarkable upregulation of mRNA levels of TNF and CD137 (TNFRSF9), the major signal and receptor for the canonical NF-κB pathway. The expression of CD40 gene was also induced by DENV infection, but its ligand, CD40LG, was not expressed in these hepatic cells, suggesting that the non-canonical NF-κB pathway is not activated in the HLC system. Because the TNF gene can also be stimulated by type I IFN, its induction by DENV infection may represent the crosstalk before the cellular antiviral response and the inflammatory response at the tissue level. Furthermore, ROS induction by DENV infection can also influence the outcome of the NF-κB pathway activation (Morgan and Liu, 2011; Olagnier et al., 2014). Finally, direct activation of NF-κB pathway by DENV protease has also been reported. In this case, the DENV protease induces the degradation of NF-κB inhibitors IκBα and IκBβ via cleavage (Lin et al., 2014). Regardless of the mechanism of the initial activation, these signal molecules likely elicit an amplified response through both autocrine and paracrine pathways. The nuclear translocation of RELA/p65 was indeed detected in all the cells including the uninfected cells. Unlike the IFN signaling and ISG induction, we did not observe any evidence of DENV proteins interfering with NF-κB signaling in the infected cells. The unabated activation of NF-κB pathway and the release of cytokines from hepatocytes, either infected or exposed to soluble signals (NS1, cytokines) produced by infected blood cells, likely contribute to the inflammatory aspects of dengue pathology. Induction of apoptosis of immune cells following DENV infection is a common feature in vivo. However, it has not been well documented whether DENV causes massive apoptosis of hepatocytes in the same way. We observed rapid cell death and CASP3 activation early in the infection time course. However, a population of the infected cells eventually survived and showed no signs of cleavage of CASP3 or PARP, but only when IFN signaling was not completely blocked. Together, these data point to an anti-apoptotic role of IFN signaling during DENV infection of the HLCs, such as the one reported for IFI6 in DENV-infected endothelial cells (Qi et al., 2015). In addition, viral protein-induced autophagy has been proposed as an additional mechanism for protecting the infected epithelial cells from apoptosis (McLean et al., 2011). In vivo, the infected hepatocytes may also be cleared by antibody-dependent cell-mediated cytotoxicity and T cell-mediated cytotoxicity through DENV antigen (such as NS1) displayed on the cell surface. Despite all of these factors, the strong capacity of regeneration of the liver likely contributes to the rareness of acute liver failure.