title: mRNA induces RANTES production in trophoblast cells via TLR3 only when delivered intracellularly using lipid membrane encapsulation.
abstract: BACKGROUND
  Trophoblasts express Toll-like receptor 3 (TLR3). The artificial TLR3 ligand, PolyI:C, induces an inflammatory response in trophoblasts but an endogenous ligand has not been identified. Notably, inflammatory disorders of pregnancy are associated with increased circulating placenta-derived mRNA. Endogenous degraded, uncapped mRNA is recognized by TLR3 in other cell lines.
  
  
  OBJECTIVE
  We tested the hypothesis that plasma-derived mRNA induces an inflammatory response in a trophoblast cell line via TLR3.
  
  
  METHODS
  Experiments were performed in the human first trimester extravillous trophoblast cell line HTR-8/SV neo. Plasma-derived mRNA was amplified using modified template switching and final in vitro transcription. We compared free mRNA (which favors cell surface interaction) to liposomally encapsulated mRNA (which favors intracellular mRNA delivery). We tested for the specific requirement of TLR3 signaling using siRNA. We tested for involvement of the canonical signaling pathway downstream of TLR3 by measuring NF-κB and IFN regulatory factor transcriptional activity using firefly-luciferase constructs.
  
  
  RESULTS
  Free mRNA did not induce RANTES production. In contrast, liposomal mRNA resulted in marked induction of RANTES production (non-stimulated control 3.4 ± 0.6 pg/mL, liposomal mRNA 169.7 ± 26.2 pg/mL, p < 0.001), and this RANTES production was abolished by siRNA for TLR3. Downstream of TLR3, liposomal mRNA-induced dose-response NF-κB and IFN regulatory factor transcriptional activity, and IFN beta production.
  
  
  CONCLUSIONS
  Plasma-derived 5' uncapped mRNA delivered intracellularly signals to induce NF-κB activation and increase RANTES production via TLR3.
authors.affiliation: University of British Columbia, Critical Care Research Laboratories, Heart + Lung Institute, St. Paul's Hospital, Vancouver, BC, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Nakada
authors.forename: T A
authors.initials: TA
authors.lastname: Nakada
authors.forename: K R
authors.initials: KR
authors.lastname: Walley
authors.forename: M
authors.initials: M
authors.lastname: McConechy
authors.forename: L
authors.initials: L
authors.lastname: McLaughlin
authors.forename: P
authors.initials: P
authors.lastname: von Dadelszen
authors.forename: M
authors.initials: M
authors.lastname: Shozu
authors.forename: J H
authors.initials: JH
authors.lastname: Boyd
mesh_terms: D002460:Cell Line; D018946:Chemokine CCL5 / Q000096:biosynthesis*; D006801:Humans; D050835:Interferon Regulatory Factors / Q000502:physiology; D016899:Interferon-beta / Q000096:biosynthesis; D008081:Liposomes; D008297:Male; D016328:NF-kappa B; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000502:physiology*; D014327:Trophoblasts / Q000378:metabolism*
chemical_list: D018946:Chemokine CCL5; D050835:Interferon Regulatory Factors; D008081:Liposomes; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D016899:Interferon-beta