title: Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells.
abstract: BACKGROUND
  The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs.
  
  
  METHODS
  In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta).
  
  
  RESULTS
  mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation.
  
  
  CONCLUSIONS
  Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.
authors.affiliation: Institute of Virology and Immunoprophylaxis (IVI), Mittelhäusern, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Ceppi
authors.forename: N
authors.initials: N
authors.lastname: Ruggli
authors.forename: V
authors.initials: V
authors.lastname: Tache
authors.forename: H
authors.initials: H
authors.lastname: Gerber
authors.forename: K C
authors.initials: KC
authors.lastname: McCullough
authors.forename: A
authors.initials: A
authors.lastname: Summerfield
mesh_terms: D000818:Animals; D003713:Dendritic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D016898:Interferon-alpha / Q000096:biosynthesis*; D016899:Interferon-beta / Q000096:biosynthesis*; D018613:Microscopy, Confocal; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D013552:Swine; D014162:Transfection
chemical_list: D016898:Interferon-alpha; D012333:RNA, Messenger; D016899:Interferon-beta