摘要
Plasmid DNA transfection is one of the fundamental tools of biomedical research.Here,we found that plasmid DNA transfection mediated by liposomes activates multiple innate immune responses in several widely used cell lines.Their activations were visible by the detection of stress granules(SG)and cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)-DNA condensates(cGC)in the transfected cells in a plasmid DNA dose-dependent manner.The elevated levels of phosphorylated eukaryotic translation initiation factor 2 subunit alpha(eIF2a),interferon regulatory factor 3(IRF3),and signal transducer and activator of transcription 1(STAT1)were induced in plasmid DNAtransfected cells.The formation of SG but not cGC required active transcription and the formation of double-stranded RNA in transfected cells.Plasmid DNA-induced SG or cGC were mutually exclusive because they triggered two distinct pathways.Knockdown(KD)of protein kinase R(PKR)before plasmid DNA transfection led to abolishing SG without affecting cGC formation.Conversely,cGAS KD could prevent cGC without affecting SG formation.In addition,plasmid DNA-induced SG and cGC formation could be prevented,respectively,by co-expression of Kaposi’s sarcoma-associated herpesvirus proteins ORF57(PKR inhibitor)and ORF52(cGAS inhibitor).Inhibition of SG formation mediated by PKR KD,but not cGC KD,also led to increased expression of transgenes,indicating that PKR activation represents a major roadblock to gene expression.Together,these data indicate that plasmid DNA triggers innate immune responses in the transfected cells and causes a significant cellular perturbation that should be considered during experiment design and data interpretation.
出处
《hLife》
2025年第1期21-37,共17页
微生物与宿主健康(英文)
基金
supported by the Intramural Research Program of the National Institutes of Health,the National Cancer Institute,and the Center for Cancer Research(ZIASC010357 to Z.M.Z.).
