SB202190 br Materials and methods br Results br

Materials and methods
Results
Discussion Japanese encephalitis caused by JEV is characterized by SB202190 neuron death. However, the underlying mechanism is not fully understood. We and other researchers have demonstrated that JEV induces apoptosis by ER stress-mediated IRE1/JNK and ROS-mediated ASK1-ERK/p38 MAPK signaling pathways (Huang et al., 2016; Yang et al., 2010). To identify novel apoptosis signaling pathways involved in Japanese encephalitis, we analyzed the global mRNA expression changes in response to in vitro and in vivo JEV infection by RNA-seq. mRNA profiling in response to JEV infection in vitro has been done using microarrays for human microglial cells and sequencing for swine testis cells (Kumari et al., 2016; Zhang et al., 2015). mRNA profiling in response to JEV infection in spleen and brain has been done using microarrays (Gupta and Rao, 2011; Yang et al., 2011). However, there is no report integrating mRNA profiles upon JEV infection in vitro and in vivo based on RNA-seq. In this study, we integrated mRNA profiles upon JEV infection in vitro and in vivo, and discovered that apoptosis-related Foxo signaling pathway was regulated by JEV infection in vitro and in vivo (Fig. 1). Foxo protein belongs to the class O of the Forkhead transcription factors. Foxo binds to the DBE binding site within the promoter of target genes, which include genes involved in apoptosis, cell-cycle, autophagy, metabolism, stress resistance, longevity, immune response and other cellular processes (Eijkelenboom and Burgering, 2013; Farhan et al., 2017; Link and Fernandez-Marcos, 2017). Foxo promoted neuron survival by preventing mitochondrial dysfunction in Drosophila PTEN-induced kinase 1 (PINK1) null mutant (Koh et al., 2012). T-cell survival was also increased by Foxo via limiting the production of IL-2 (Oh et al., 2011). Consistent with the role of Foxo in promoting cell survival, Foxo in Drosophila was found to extend life-span by regulating insulin signaling (Giannakou et al., 2004; Hwangbo et al., 2004). In response to oxidative stress, Foxo in mammals maintained the stem cell pool by reducing cell apoptosis (Tothova et al., 2007). In contrast, overwhelming evidence demonstrated that Foxo induced apoptosis of cells such as B cell, T cells, macrophage cells, neuronal cells and glioma cells (Cui et al., 2009; Lau et al., 2009; Su et al., 2011; Sullivan et al., 2012). Nitric Oxide-Foxo axis reduced neuron survival in the Drosophila model of Parkinson’s disease (Kanao et al., 2012). Foxo was also found to promote apoptosis by upregulating pro-apoptotic Daxx-like protein (DLP) in Drosophila (Hwang et al., 2013), Bim and TRAIL in Drosophila, granulosa cells, neuronal cells and tumor cells (Cui et al., 2009; Li et al., 2014; Wang et al., 2014). Here, we showed that Foxo plays an important role in reducing apoptosis and JEV induced cell apoptosis by inhibiting the expression of Foxo. In contrast, Foxo within the context of HIV infection induced lymphocyte apoptosis (Cui et al., 2009). Taken together, these findings suggest that the role of Foxo in apoptosis depends on the context. Foxo was activated in Cricket Paralysis Virus infected Drosophila S2 cells, resulting in the decrease of viral load by increasing RNA interference (RNAi) efficacy (Spellberg and Marr, 2015). However, Foxo had no effect on JEV replication (Fig. 2D), indicating that Foxo facilitates the survival of JEV-infected Neuro-2a cells in a virus replication-independent manner. Instead, we found that JEV induced cell apoptosis by repressing the expression of apoptosis-related Foxo target genes. Expression of apoptosis-related genes such as Bim, Bcl-6 and p21 can be upregulated by Foxo (Link and Fernandez-Marcos, 2017). Bim contains a Bcl-2 homology domain 3 (BH3) and act as an apoptotic activator (Banjara et al., 2018). Bcl-6 is a transcription repressor, which downregulates p53 expression by binding to its promoter region, thus inhibiting cell apoptosis (Wang et al., 2017). Cyclin-dependent kinase inhibitor p21 triggers G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis. Prevailing evidence indicates that p21 acts as anti-apoptotic agent (Georgakilas et al., 2017). Our data shows that Foxo in the context of JEV infection predominantly promotes cell survival via anti-apoptotic proteins Bcl-6 and p21 although it also induces the expression of pro-apoptotic Bim (Fig. 3).