Objective: The rheumatoid synovium displays characteristics of Toll-like receptor (TLR) activation and antiviral gene expression, including production of RANTES and interferon-beta (IFNbeta). The mechanism of this activation in rheumatoid synovial tissue is unknown. This study was designed to investigate the role of the IKK-related kinase IKKepsilon and IFN regulatory factor 3 (IRF-3) in the activation of antiviral genes in rheumatoid arthritis (RA).
Methods: Kinase assay and immunostaining were performed on synovial tissue. Dominant-negative (DN) IKKepsilon adenoviral infection of human fibroblast-like synoviocytes (FLS) was followed by poly(I-C) stimulation and Western blotting. Quantitative polymerase chain reaction was performed on DN IKKepsilon-infected FLS and IKKepsilon(-/-) and IKKepsilon(+/+) mouse FLS.
Results: Western blotting showed that IKKepsilon phosphorylation was significantly greater in RA synovium compared with osteoarthritis synovium. Kinase assay confirmed that IKKepsilon was activated in RA synovium, and immunostaining showed localization of pIKKepsilon to the intimal lining. Western blot analysis demonstrated that activation of IRF-3 was also increased in RA synovium. Poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha (TNFalpha) activated phosphorylation of IKKepsilon and IRF-3 in FLS. DN IKKepsilon inhibited IRF-3 phosphorylation as well as RANTES and IFNbeta protein production in synoviocytes. Antiviral gene expression was also reduced in FLS from IKKepsilon(-/-) mice compared with IKKepsilon(+/+) mice.
Conclusion: Antiviral gene expression in RA, especially due to TLR ligands and TNFalpha, is dependent on IKKepsilon and IRF-3, and this pathway plays a key role in the production of type I IFNs and chemokines such as RANTES. These findings indicate that the IKKepsilon pathway may have potential as a therapeutic target in RA.