Background Rheumatoid arthritis (RA) is characterised by inflammation and proliferation of synovial tissue, leading to progressive degradation of articular cartilage and bone.
Objectives This study examines the effect of Toll-like Receptor (TLR) 2 activation on the inflammasome, cell migration/invasion and Notch signalling pathways.
Methods RASFC and RA ex vivo explant cytokine release and NLRP3 expression in response to Pam3CSK4 (TLR2-ligand) (1μg/ml) were measured by ELISA, MSD multiplex assay and Western Blotting. TLR2-induced RASFC invasion, migration/cytoskeletal rearrangement, MMP-3 and TIMP-1 expression were assessed by wound repair assays, F-actin immunofluorescence and ELISA. Notch-1 expression in RASFC and whole RA ex vivo explant in response to Pam3CSK4 (1μg/ml) was determined by Western Blotting and RT-PCR. Expression of Notch signaling components (Notch-1, Dll-4, Jagged-1 and HRT-1) were assessed in RA, osteoarthritis (OA) and healthy control synovial tissue by immunohistochemistry. Finally, TLR2-induced cytokine production, cell migration and cytoskeletal rearrangement in the presence or absence of Notch-1 siRNA or a γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) was assessed.
Results Pam3CSK4 specifically induced IL-6, IL-8, TNFα and IL-1β in RA ex vivo explants and RASFC (p<0.05), with no increase in IFNγ. Pam3CSK4 significantly induced NLRP3 expression and downstream inflammasome-related cytokines IL-1β and IL-18 (p<0.05). Pam3CSK4 significantly induced cell migration, ECM degradation and increased the MMP-3/TIMP-1 ratio (all p<0.05). Pam3CSK4 induced RA synovial explant outgrowth consistent with TLR2-induced RASFC migration and ECM degradation. Notch signalling components were significantly increased in RA synovium compared to OA and control synovium (p<0.05). Pam3CSK4 induced Notch-1 and Jagged-1 mRNA expression with differential effects observed for active Notch-1 intracellular (IC) protein expression. Pam3CSK4-induced cytokine production was in part inhibited in the presence of either Notch-1 siRNA or DAPT.
Conclusions TLR2 activation in RA activates inflammasome, cytokine production and migrational/invasive mechanisms, effects that may in part be mediated by the Notch-1 signalling pathway.
Disclosure of Interest T. Mcgarry: None declared, W. Gao: None declared, M. Connolly: None declared, G. Walsh: None declared, J. McCormick: None declared, D. Veale Grant/research support: MSD, Abbvie, MSD, Pfizer, Roche, Consultant for: Pfizer, Roche, Speakers bureau: Abbott, MSD, Pfizer, Roche, U. Fearon: None declared