雙股RNA病毒經由腫瘤壞死因子α (TNFα) 調控宿主細胞表現細胞素與細胞凋亡之途徑

Abstract

The infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family of viruses and causes acute contagious diseases in a number of economically important freshwater and marine fish. Previous studies have shown that IPNV induces both atypical apoptosis and secondary necrosis in fish cells. The expression of the survival factor Mcl-1 has been shown to be down-regulated and that of the pro-apoptotic bcl-2 family gene Bad has been shown to be up-regulated by IPNV. IPNV infection can trigger the tyrosine kinase-mediated death pathway and cause the activation of caspase-8 and -3 in virus-infected cells. IPNV can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. Using DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage, we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then during the early-middle replication stages, the TNFα level was eight-fold higher, and the pro-apoptotic Bcl-2 family members Bad and Bid were also up-regulated. Specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the activity of the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection. We also infected zebrafish embryonic (ZF4) cells with IPNV and analyzed the gene expression patterns of normal and infected cells using quantitative real-time PCR. We identified a number of immune response genes, including ifna, ifng, mx, irf1, irf2, irf4, tnfa, tnfb, il-1b, il-15, il-26, ccl4 and mmp family genes, that are induced after viral infection. Transcriptional regulators, including cebpb, junb, nfkb and stat1, stat4 and stat5, were also up-regulated in IPNV-infected cells. In addition, we used Pathway Studio software to identify TNFα as the factor with the greatest downstream influence among these altered genes. Treating virus-infected cells with an siRNA targeting TNFα inhibited NF-κB expression. To further interrupt the TNFα/NF-κB-mediated pathway, the expression levels of cytokines and metalloproteinases were inhibited in IPNV-infected cells. Using microRNA array and real-time quantitative PCR assays, the expression patterns of microRNAs in IPNV-infected fish cells were characterized during different replication stages of IPNV. We found that the gene transcription levels of miR-132, miR-146a and miR-155 were up-regulated, and miR-125b was down-regulated. Previous studies have shown that the 3’-untranslated regions of TNFα transcripts can be targeted by miR-125b. A miR-125b mimic or a TNFα-specific siRNA was able to down-regulate the expression of TNFα in IPNV-infected cells. Following miR-125b mimic treatment, the viability of IPNV-infected cells was increased. The percentages of apoptotic and necrotic IPNV-infected ZF4 cells that were pretreated with a miR-125b mimic or a TNFα-specific siRNA were decreased, as shown by fluorescence images of Annexin V-fluorescein and PI staining. The activation of caspase-3, -8, and -9 and the formation of ROS were inhibited following miR-125b mimic or TNFα-specific siRNA treatment of ZF4 cells infected with IPNV. Therefore, this work indicates that miR-125b is suppressed in response to IPNV and acts as a negative regulator of TNFα-mediated apoptosis and secondary necrosis induced by IPNV. Taken together, our results indicate that IPNV triggers two death pathways via upstream induction of the pro-inflammatory cytokine TNFα and that the expression of cytokines and metalloproteinases might be initiated through the TNFα/NF-κB-mediated pathway. TNFα plays important roles in cell death and immune responses during IPNV infection. These results may provide new insights into the pathogenesis of RNA viruses.