title: Activation of Toll-like receptor 3 reduces actin polymerization and adhesion molecule expression in endometrial cells, a potential mechanism for viral-induced implantation failure.
abstract: OBJECTIVE
  Does activation of endometrial Toll-like receptor 3 (TLR 3) affect cell receptivity to trophoblast adhesion?
  
  
  CONCLUSIONS
  TLR 3 activation in vitro reduces the attachment of trophoblast cells to endometrial cells by altering the cell cytoskeleton and reducing the expression of adhesion molecules in human endometrial cells.
  
  
  BACKGROUND
  It is well documented that the presence of an infection at the time of implantation can lead to implantation failure. The female reproductive tract recognizes invading micro-organisms through the innate pathogen recognition receptors such as the TLRs.
  
  
  METHODS
  Poly I:C was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. The experiments were performed in three replicates on three separate days.
  
  
  METHODS
  An in vitro assay was developed using RL95-2 (a human endometrial cell line) and JAr (a human trophoblast cell line) cells. Initially, the percentage of attached JAr spheroids to RL95-2 was measured in response to TLR 3 activation. Next, actin polymerization in RL95-2 cells was assessed in response to TLR 2/6, 3 and 5 activation. Phalloidin was used to assess the mean fluorescence intensity of F-actin by flow cytometry or confocal microscopy. Secondly, the influence of TLR 2/6, 3 and 5 activation on the expression of cluster of differentiation 98 (CD98) and β3 integrin was determined. To further understand through which pathways the TLR 3-induced alterations occur, inhibitors were applied for Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), myeloid differentiation primary response 88 (MYD88), mitogen-activated protein kinases (MAPK) and nuclear factor pathways.
  
  
  RESULTS
  We observed that stimulation of TLR 3 in endometrial cells with different concentrations of Poly I:C led to a reduction in the percentage of trophoblasts attached to the endometrial cells in a dose-dependent manner (P < 0.05). This decrease was consistent in the Poly I:C treated group regardless of the co-incubation time (P < 0.05). In addition, our results demonstrated that actin polymerization and CD98 expression significantly decreased only in response to TLR 3 activation (P < 0.05). Activation of endometrial cells with TLR 2/6, 3 and 5 significantly reduced β3 integrin expression (P < 0.05). These alterations were shown to work via MYD88-MAPK pathways (P < 0.05).
  
  
  CONCLUSIONS
  This study has been performed in vitro. Future in vivo studies will be required in order to confirm our data.
  
  
  CONCLUSIONS
  This is a novel discovery which extends our current knowledge concerning diagnosis and treatment of viral-induced infertility cases.
  
  
  BACKGROUND
  This research was supported by the COST Action FA1201 (GEMINI) by granting a Short Term Scientific Mission and the Instituto de Salud Carlos III by granting Grant PI11/01645. 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: Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria Princesa, C/Maestro Amadeo Vives 2, 28009 Madrid, Spain.
authors.forename: S
authors.initials: S
authors.lastname: López-Martín
authors.affiliation: Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria Princesa, C/Maestro Amadeo Vives 2, 28009 Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Yáñez-Mó
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: D000199:Actins / Q000737:chemistry*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism; D010064:Embryo Implantation / Q000502:physiology*; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D027261:Fusion Regulatory Protein-1 / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D014327:Trophoblasts / Q000166:cytology*; D014777:Virus Diseases / Q000150:complications*
chemical_list: D000199:Actins; D015815:Cell Adhesion Molecules; D027261:Fusion Regulatory Protein-1; C061248:ITGB3 protein, human; D039661:Integrin beta3; D008024:Ligands; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D020928:Mitogen-Activated Protein Kinases; D011070:Poly I-C
keywords: Toll-like receptors; actin polymerization; cluster of differentiation 98; implantation failure; β3 integrin