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RNAi induces innate immunity through multiple cellular signaling pathways.

ABSTRACT: BACKGROUND AIMS: Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi. METHODS: Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting. RESULTS: In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse ?-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-?, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2'-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction. CONCLUSIONS: RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.

SUBMITTER: Meng Z

PROVIDER: S-EPMC3659100 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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RNAi induces innate immunity through multiple cellular signaling pathways.

Meng Zhongji Z Zhang Xiaoyong X Wu Jun J Pei Rongjuan R Xu Yang Y Yang Dongliang D Roggendorf Michael M Lu Mengji M

PloS one 20130520 5

<h4>Background aims</h4>Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.<h4>Methods</h4>Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with d ...[more]

PMID: 23700487

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Project description:The transport of live fish is a necessary step for commercial production. The skin of teleost fish is the first non-specific immune barrier against exogenous stimuli, and it plays an important protective role under transport stress. Thus, the aim of this study was to explore the skin responses to transport stress in hybrid yellow catfish (Tachysurus fulvidraco♀ × Pseudobagrus vachellii♂) through transcriptome and biochemical analyses. Water samples were collected during a simulated transport treatment. Biochemical indexes and/or gene expression in blood, skin, and mucus in fish in control groups and transport-stress groups (0 h, 2 h, 4 h, 8 h, 16 h) were assayed. The levels of total ammonia–nitrogen and nitrite–nitrogen in the water increased with increasing transport time. Comparison of skin transcriptomes between the control group and the group subjected to 16 h of transport revealed 1547 differentially expressed genes (868 up-regulated and 679 down-regulated). The results of the transcriptome analysis were validated by analyses of the expression levels of selected genes by qRT-PCR. The results indicated that the toll-like receptors and nod-like receptors signaling pathways mediate the skin’s immune response to transport stress: tlr9, mfn2, and ikbke were significantly up-regulated and nfkbia and map3k7cl were significantly down-regulated under transport stress. With increasing transport time, lysozyme activity and the immunoglobulin M content in skin mucus first increased and then decreased. The number of mucous cells peaked at 8 h of transport stress, and then decreased. The mucus cells changed from types II and IV to types I, II, III, and IV. The amounts of red and white blood cells and the levels of hemoglobin and hematocrit first increased and then decreased during 16 h of transport stress. Together, the results showed that the skin responds to transport stress by activating the immune signaling pathway and regulating mucus secretion. These findings have important biological significance for selecting strains that tolerate transport, as well as economic significance for optimizing the transport conditions for scaleless fish.

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RNAi induces innate immunity through multiple cellular signaling pathways.

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RNAi induces innate immunity through multiple cellular signaling pathways.

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