title: RNAi induces innate immunity through multiple cellular signaling pathways.
abstract: OBJECTIVE
  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.
authors.affiliation: Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany.
authors.forename: Zhongji
authors.initials: Z
authors.lastname: Meng
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Rongjuan
authors.initials: R
authors.lastname: Pei
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Dongliang
authors.initials: D
authors.lastname: Yang
authors.forename: Michael
authors.initials: M
authors.lastname: Roggendorf
authors.forename: Mengji
authors.initials: M
authors.lastname: Lu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D020043:Genes, Essential; D005814:Genes, Viral; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics / Q000276:immunology; D022781:Hepatocytes / Q000276:immunology / Q000378:metabolism / Q000821:virology; D007113:Immunity, Innate / Q000187:drug effects / Q000235:genetics*; D007369:Interferon Inducers / Q000494:pharmacology; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008392:Marmota; D051379:Mice; D008822:Mice, Transgenic; D064547:Myxovirus Resistance Proteins / Q000235:genetics / Q000378:metabolism; D011070:Poly I-C / Q000494:pharmacology; D061251:Primary Cell Culture; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D015533:Transcriptional Activation; D015854:Up-Regulation; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D054357:Chemokine CXCL10; C485433:Cxcl10 protein, mouse; D007369:Interferon Inducers; D064547:Myxovirus Resistance Proteins; D034741:RNA, Small Interfering; D016899:Interferon-beta; D019892:eIF-2 Kinase; D011070:Poly I-C