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AB0051 Toll-Like Receptor 2 Activation Induces Pro-Inflammatory, Inflammasome and Notch Signalling Pathways in Rheumatoid Arthritis
  1. T. Mcgarry,
  2. W. Gao,
  3. M. Connolly,
  4. G. Walsh,
  5. J. McCormick,
  6. D.J. Veale,
  7. U. Fearon
  1. Rheumatology Department, St. Vincent's University Hospital, Dublin Academic Medical Centre and The Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland, Dublin, Ireland

Abstract

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

DOI 10.1136/annrheumdis-2014-eular.5131

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