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RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

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 re...

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Autores principales: Meng, Zhongji, Zhang, Xiaoyong, Wu, Jun, Pei, Rongjuan, Xu, Yang, Yang, Dongliang, Roggendorf, Michael, Lu, Mengji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659100/
https://www.ncbi.nlm.nih.gov/pubmed/23700487
http://dx.doi.org/10.1371/journal.pone.0064708
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author Meng, Zhongji
Zhang, Xiaoyong
Wu, Jun
Pei, Rongjuan
Xu, Yang
Yang, Dongliang
Roggendorf, Michael
Lu, Mengji
author_facet Meng, Zhongji
Zhang, Xiaoyong
Wu, Jun
Pei, Rongjuan
Xu, Yang
Yang, Dongliang
Roggendorf, Michael
Lu, Mengji
author_sort Meng, Zhongji
collection PubMed
description 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 β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, 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.
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spelling pubmed-36591002013-05-22 RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways Meng, Zhongji Zhang, Xiaoyong Wu, Jun Pei, Rongjuan Xu, Yang Yang, Dongliang Roggendorf, Michael Lu, Mengji PLoS One Research Article 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 β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, 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. Public Library of Science 2013-05-20 /pmc/articles/PMC3659100/ /pubmed/23700487 http://dx.doi.org/10.1371/journal.pone.0064708 Text en © 2013 Meng et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Meng, Zhongji
Zhang, Xiaoyong
Wu, Jun
Pei, Rongjuan
Xu, Yang
Yang, Dongliang
Roggendorf, Michael
Lu, Mengji
RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title_full RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title_fullStr RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title_full_unstemmed RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title_short RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways
title_sort rnai induces innate immunity through multiple cellular signaling pathways
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659100/
https://www.ncbi.nlm.nih.gov/pubmed/23700487
http://dx.doi.org/10.1371/journal.pone.0064708
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