title: Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.
abstract: BACKGROUND
  This study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).
  
  
  METHODS
  Invasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.
  
  
  RESULTS
  Pam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.
  
  
  CONCLUSIONS
  TLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. trudy.mc-garry@ucdconnect.ie.
authors.forename: Trudy
authors.initials: T
authors.lastname: McGarry
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. douglas.veale@ucd.ie.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Veale
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. wei.gao2@mail.dcu.ie.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. carl.orr@ucd.ie.
authors.forename: Carl
authors.initials: C
authors.lastname: Orr
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. ursula.fearon@ucd.ie.
authors.forename: Ursula
authors.initials: U
authors.lastname: Fearon
authors.affiliation: Department of Rheumatology, St. Vincent's University Hospital, Elm Park, Dublin Academic Health Care and The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. mary.connolly@ucd.ie.
authors.forename: Mary
authors.initials: M
authors.lastname: Connolly
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology*; D015153:Blotting, Western; D002465:Cell Movement; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051195:Toll-Like Receptor 2 / Q000378:metabolism*
chemical_list: C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2