Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.

introductionThis study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).

methodsInvasion, 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.

resultsPam3CSK4 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.

conclusionsTLR2 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.