We initially set out to identify the cellular immune responses to DV infection. Using global gene expression profiling, we found that IFN-α and IFN-γ signaling pathway-inducible genes were commonly regulated in several DV-susceptible cell types. TRAIL, one of the common response genes, was identified as a novel antiviral molecule against DV. Furthermore, our data suggest that the rTRAIL-mediated decrease in DV infection is mediated by a novel apoptosis-independent mechanism.
DV infection is an acute infection cleared within approximately 1 week (22
). We hypothesized that there is a set of cellular genes which is necessary to clear the virus in susceptible cells. We identified a common gene expression profile of 23 differentially regulated genes from GeneChip analysis of DV-infected HUVECs and peripheral blood mononuclear cell subsets (B cells and monocytes). These findings extend previous studies of gene expression in DV-infected HUVECs (46
) and macrophages (30
) that were limited to global gene expression analysis in one cell type. All of the 23 common response genes were among the list of differentially expressed genes detected in HUVECs using differential display and GeneChip microarrays (46
). These genes were not reported as up-regulated by DV in the cytokine gene array study of Moreno-Altamirano et al. (30
). However, their study did not investigate these specific genes.
Of the 23 genes that comprised the common DV response profile, functions and/or inducers of 19 genes have been described in the literature (Table ). These include classical antiviral response genes (OAS3 and IRF7), more recently identified antiviral genes (ISG15, HERC5, RSAD2, TRIM5, TRAIL, OASL, and ISG20), genes regulating ubiquitination (USP18), cell adhesion and cyclic ADP-ribose metabolism (CD38), apoptosis (XAF1), immune suppression (IFITM1), immune activation (LGALs3BP), and nine other genes (FLJ20035, FLJ38348, HERC6, IFI44, IFI44L, IFIT1, IFIT3, LY6E, and SAMD9) with unknown function. Functions of the genes identified in this study may be pivotal in understanding the role of the cellular responses to DV infection. Of the common response genes, G1p2, Mx1, and OAS3 were also associated with dengue shock in patients with severe DV infection (41
One of the common response genes, TRAIL, was identified as a potential link between IFN-α and IFN-γ signaling pathways. We selected TRAIL for further study based on its previously documented antiviral and antitumor functions (2
). This member of the TNF family of ligands is capable of initiating apoptosis through the engagement of its death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) (4
). In vitro and in vivo studies have demonstrated tumoricidal activity of TRAIL without significant toxicity toward normal cells or tissues (35
). TRAIL-mediated killing by activated CD4+
T cells, NK cells, and B cells has been shown to occur with influenza virus and others (17
). IFNs enhance expression of TRAIL while, on the other hand, TRAIL treatment can enhance expression of IFN-inducible genes like IFITM1, IFIT1, STAT1, LGal3BP, and PRKR as well as IFN-β itself (20
). The molecular cross-talk and functional synergy observed between the TRAIL and IFN signaling pathways is not limited to the genes involved in apoptosis and may have implications for the physiologic role and mechanism of action of TRAIL protein. Findings in this study (Fig. and ) support the idea that type I IFN, which is known to inhibit DV, might induce TRAIL as a part of the type I IFN response against DV.
Primary monocytes, DCs, B cells, and HUVECs infected with DV induced TRAIL mRNA expression. TRAIL protein levels were also found to be highly induced in DV-infected monocyte cell lysates, but using fluorescence-activated cell sorting, we were unable to detect TRAIL on monocyte and DC surfaces after DV infection or secreted TRAIL protein in the supernatant from DV-infected primary monocytes. Recently, Matsuda et al. reported that TRAIL protein secreted by HepG2 cells after DV infection was partly responsible for apoptosis of uninfected HepG2 cells (28
). Global gene expression analysis in DV-infected HepG2 cells and primary cells like monocytes, B cells, and HUVECs performed in our laboratory has shown a distinct set of differentially regulated genes in HepG2 versus primary human cells (data not shown). Unlike primary cells, TRAIL mRNA levels were not found to be up-regulated in DV-infected HepG2 cells by GeneChip analysis (data not shown). DV infection of primary cells may better reflect a physiological response. In the previous study of Matsuda et al., 80% of DV-infected HepG2 cells stained positive for cell death (28
), raising the possibility that the TRAIL protein detected in the supernatant of DV-infected cells was not secreted but resulted from cell death. DCs infected with DV stained minimally for apoptosis detection dye (Live/Dead Aqua) and markers (PARP-1 and caspase-3) (Fig. ). Our results indicate that under the conditions tested, DV infection does not induce apoptosis in monocytes and DCs.
The concentration of rTRAIL (5 to 20 μg/ml) in this study that was able to inhibit DV replication was similar to the one used in studies demonstrating TRAIL-induced apoptosis of tumor cells (1
). Hence, we hypothesized that TRAIL might be inhibiting DV copy number by acting as an antiviral agent and not by inducing apoptosis. An apoptosis-independent TRAIL antiviral function is a novel finding which should be further studied. One possible mechanism of TRAIL antiviral function could be TRAIL-mediated increased expression of known or novel antiviral cellular protein(s) which are secreted by cells.
TRAIL has been shown to mediate antiviral functions in vivo in mouse models of influenza and encephalomyocarditis virus infection (15
). Influenza viral clearance was prolonged in mice injected with anti-TRAIL antibody. TRAIL expressed by NK cells was crucial to limit encephalomyocarditis virus replication in vivo (38
). In this study, we observed TRAIL-dependent inhibition of DV replication. Previous studies have shown that both type I and type II IFNs are critical in controlling different stages of DV infection in mice (40
), although the precise mechanism(s) by which IFNs mediate an antiviral response is unknown. We postulated that TRAIL protein was induced by and contributed to the type I IFN-mediated antiviral function. Using wild-type (2fTGH) and type I IFN mutant (U1A, U3A, U4A, and U5A) fibroblast cells (24
), we found that induction of TRAIL gene expression by DV was type I IFN dependent. Furthermore, TRAIL inhibited DV in a novel apoptosis-independent manner. This is the first study to show that TRAIL might regulate a pathogen in an apoptosis-independent manner. Activation of the TRAIL signaling pathway might therefore be used as an antiviral therapy in the future.