br Results br Discussion Human HLCs

Results
Discussion Human HLCs derived from pluripotent stem Angiogenesis Compound Library are permissive to infection by hepatotropic viruses, including HCV and HBV. Interestingly, the SEC14L2 gene, an essential host factor for supporting the replication of wild-type, unadapted HCV genomes, was induced during the hepatic differentiation (data not shown) but not expressed in the hepatoma cell line Huh 7.5 cells (Saeed et al., 2015). This difference may account for the ability of the HLCs to support infection by HCV patient serum (Wu et al., 2012; Saeed et al., 2015). Here we demonstrate that these cells also support productive infection and persistent replication of DENV. The timing of transition to viral permissiveness during the hepatic differentiation differs for the three viruses. For HCV, the transition required the induction of a liver-specific microRNA, miR-122 (Wu et al., 2012; Jopling et al., 2005), while HBV infection of the differentiated HLCs correlated with the induction of the HBV receptor, sodium taurocholate cotransporting peptide (NTCP) (Shlomai et al., 2014; Yan et al., 2012). Similar to the situation with HBV, transition to DENV permissiveness correlated with the induction of DENV entry factors, the TIM-TAM family of proteins and their ligand, for hepatic cells (Meertens et al., 2012). Elucidating gene-expression changes that are associated with the shift to virus susceptibility during differentiation represents a valuable approach with the potential of identifying cellular determinants that control viral infection. This approach is especially attractive in the case of DENV because of the extraordinarily broad tropism it exhibits in cell culture (Acosta et al., 2014). In fact, hPSCs and the early progenies are rare examples of cells that are refractory to DENV infection in vitro. In the liver, hepatocytes are organized as polarized epithelial cell layers to separate sinusoidal blood from the secreted bile and perform hepatic functions, and specific cell-cell junctions are required for the maintenance of the polarized cell layers (Gissen and Arias, 2015). In some liver diseases, these junctions and the polarity of hepatocytes are affected. However, it is difficult to fully elucidate the pathophysiological features of hepatocytes using hepatoma cell lines because hepatocellular epithelial to mesenchymal transition and degeneration are common events in hepatocellular carcinoma progression, which result in the loss of epithelial features and some hepatic functions (van Zijl et al., 2009). In the case of DENV infection, it is has been reported that DENV can damage hepatocytes and disrupt liver functions, leading to elevations of serum bilirubin and transaminases, prolonged prothrombin time, and reduced serum albumin level (Huy et al., 2013; Roy et al., 2013; Samanta and Sharma, 2015). In our study, we utilized stem cell-derived HLCs to investigate the interactions between DENV and hepatocytes and observed downregulation of albumin, several coagulation factors, and a component of the complement pathway by DENV infection. These virus-induced changes in hepatic features may contribute to the effect of DENV on liver in vivo. The precise mechanism of the direct downregulation of these genes remains unclear at this time. DENV infection activates an antiviral response in vitro and in vivo (Green et al., 2014). Our results showed that many reported anti-DENV ISGs, including IFITM1, IFTM3, IRF1, IRF7, and IFI6 (Schoggins et al., 2012; Brass et al., 2009), were highly increased in the DENV-treated HLCs, which probably is the main reason why DENV infection of hepatic cells is self-limiting. In vivo, the IFN response generated by the hepatic cells is likely complemented by similar responses from resident immune cells in the liver; in fact, IFN secreted from infected immune cells can also trigger IFN signaling in the hepatocytes and protect liver from massive infection. Together, these data may help explain the rareness of acute liver injuries in DENV infection.