title: Lentiviral-mediated shRNA against RelB induces the generation of tolerogenic dendritic cells.
abstract: OBJECTIVE
  Lentiviral-mediated shRNA against RelB was used to produce tolerogenic dendritic cells from murine bone marrow derived dendritic cells (BMDCs).
  
  
  METHODS
  RelB expression in the BMDCs was silenced by lentivirus carrying RelB shRNA. The apoptosis rate and surface markers of DCs were assessed by flow cytometry. IL-12,IL-10,TGF-β1 secreted by DCs and DNA binding capacity of NF-κB subunits in the nucleus were measured by ELISA, independently. MLR was used to analyze the capacity of DCs to inhibit immune response.
  
  
  RESULTS
  RelB expression was significantly inhibited in DCs following lentiviral mediated delivery of RelB specific shRNA. The RelB shRNA-DC produced lower IL-12 and higher IL-10 than mature dendritic cells (mDCs) and silencing control DCs. There was no difference in the apoptosis rate between shRNA RelB-DCs and mDCs. The expression levels of co-stimulatory molecules (CD80, CD86 and CD83) and MHC-II class molecule were lower in the RelB shRNA-DCs than in the mDCs and silencing control DCs. In addition, RelB shRNA also inhibited the RelB DNA binding capacity but had no effect on other NF-κB subunits. The shRNA RelB-DCs can significantly inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines.
  
  
  CONCLUSIONS
  Our results indicate RelB shRNA transfection of DCs can induce the immature status, and produce tolerogenic DCs.
authors.affiliation: Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430060, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Qiu
authors.forename: Heng-cheng
authors.initials: HC
authors.lastname: Zhu
authors.forename: Xiu-heng
authors.initials: XH
authors.lastname: Liu
authors.forename: Wang-chao
authors.initials: WC
authors.lastname: Dong
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Weng
authors.forename: Chun-hai
authors.initials: CH
authors.lastname: Hu
authors.forename: You-lin
authors.initials: YL
authors.lastname: Kuang
authors.forename: Rui-hui
authors.initials: RH
authors.lastname: Gao
authors.forename: Chao
authors.initials: C
authors.lastname: Dan
authors.forename: Tao
authors.initials: T
authors.lastname: Tao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects*; D005434:Flow Cytometry; D020868:Gene Silencing; D005822:Genetic Vectors; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D007959:Lymphocyte Culture Test, Mixed; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D018417:Th1 Cells / Q000187:drug effects / Q000378:metabolism; D018418:Th2 Cells / Q000187:drug effects / Q000378:metabolism; D051841:Transcription Factor RelB / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D016753:Interleukin-10; D051841:Transcription Factor RelB; D018664:Interleukin-12