title: Interleukin-1 receptor antagonist mediates toll-like receptor 3-induced inhibition of trophoblast adhesion to endometrial cells in vitro.
abstract: Is interleukin-1 receptor antagonist (IL-1RA) involved in the toll-like receptor 3 (TLR 3)-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro?
  
  
  
  IL-1RA mediates the TLR 3-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro.
  
  
  
  It is well documented that endometrial TLR 3 activation leads to impairment of trophoblast binding to endometrial cells in vitro. IL-1RA is known as an anti-implantation factor, as its injection significantly reduced implantation rates in mice by an effect on endometrial receptivity.
  
  
  
  Poly I:C was used as a TLR3 specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. IL-1RA was applied to block IL-1 signal transduction. IL-1RA was knocked down by Accell Human IL1RN siRNA. Flagellin was used to stimulate TLR 5. SP600125 (JNK) was applied to inhibit the mitogen-activated protein kinases (MAPK) pathway. BAY11 -7082 was used to inhibit the nuclear factor-κB (NF-κB) pathway. The experiments were performed in three replicates on three separate days.
  
  
  
  An in vitro assay was developed using RL95-2 (an endometrial cell line) and JAr (a trophoblast cell line) cells. Initially, the production of IL-1RA in RL95-2 cells in response to TLR 3 activation was measured. To determine whether the TLR 3-induced inhibition of trophoblast binding was mediated through IL-1RA: (i) we evaluated the effect of IL-1RA on the attachment of trophoblast cells to endometrial cells; (ii) we knocked down TLR3-induced IL-1RA gene expression by IL-1RA Small interfering RNA (siRNA) and evaluated trophoblast attachment to endometrial cells. Finally, to clarify through which pathway TLR 3-induced inhibition of trophoblast binding occurs: (i) activation of NF-κB and MAPK was detected by transfecting the cells with secreted placental alkaline phosphatase reporter plasmids bearing promoter sequences for each transcription factor; (ii) the inhibitors for NF-κB and MAPK were used to block signaling; (iii) it was then investigated whether addition of these inhibitors could restore the TLR 3-induced impairment of trophoblast attachment to the endometrial cells.
  
  
  
  Our results showed that addition of polyinosinic:polycytidylic acid (Poly I:C) to RL95-2 cells significantly increased the production of IL-1RA (P < 0.05). Addition of human recombinant IL-1RA to RL95-2 cells remarkably decreased the adhesion rate of trophoblast cells to endometrial cells (P < 0.05). In addition, suppression of TLR3-induced IL-1RA gene expression in RL95-2 cells significantly restored trophoblast cells attachment to endometrial cells in the presence of Poly I:C (P < 0.05). Only TLR3 and not TLR5 induced MAPK activation (P < 0.05). TLR3 ligation did not affect NF-κB activation. Of NF-kB and MAPK inhibitors, only MAPK's inhibitor could achieve restoration of spheroid adhesion to endometrial cells (P < 0.05).
  
  
  
  This study has been only done in vitro. Future in vivo studies will confirm our data.
  
  
  
  The findings of this study have a potential clinical application in introducing IL-1RA as one of the diagnostic infertility markers in the endometrium, which can affect the process of embryo adhesion at the time of implantation. Moreover, based on the novel data obtained in the current study, blocking and regulating the MAPK pathway by its inhibitors can be used as a new strategy to prevent and treat virus-induced infertility cases in ART techniques.
  
  
  
  This study was partially funded by a Marie Curie IIF-253948 grant to I.C. and was partially funded by the author's institutions. The authors have no conflict of interest to declare.
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK.
authors.forename: M
authors.initials: M
authors.lastname: Montazeri
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Sanchez-Lopez
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK UMR1282 ISP, INRA, Nouzilly, France.
authors.forename: I
authors.initials: I
authors.lastname: Caballero
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK.
authors.forename: N
authors.initials: N
authors.lastname: Maslehat Lay
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK.
authors.forename: S
authors.initials: S
authors.lastname: Elliott
authors.affiliation: Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK a.fazeli@sheffield.ac.uk.
authors.forename: A
authors.initials: A
authors.lastname: Fazeli
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects*; D002460:Cell Line; D004717:Endometrium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011070:Poly I-C / Q000494:pharmacology; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D053590:Interleukin 1 Receptor Antagonist Protein; D034741:RNA, Small Interfering; D051196:Toll-Like Receptor 3; D011070:Poly I-C
keywords: IL-1RA; MAPK; NF-κB; Toll-like receptor 3; embryo implantation; implantation failure; infertility; trophoblast spheroids