Synoviocyte innate immune responses: TANK-binding kinase-1 as a potential therapeutic target in rheumatoid arthritis

Objectives. Innate immune responses in the rheumatoid synovium contribute to inflammation and joint destruction in RA. Two IkB kinase (IKK)-related kinases, TNF receptor associated factor (TRAF) family member-associated nuclear factor k-light-chain enhancer of activated B cells (NF-kB) activator (TANK)binding kinase 1 (TBK1) and IKKe, potentially regulate synovitis by activating IFN response genes. These kinases induce the expression of inflammatory mediators such as C-X-C motif ligand 10 (CXCL10)/ IFN-ginduced protein 10 kDa (IP-10) in fibroblast-like synoviocytes (FLS). Since IP-10 is a promising therapeutic target in RA, we evaluated whether blocking TBK1 might be an effective way to modulate IP-10 expression. Methods. Wild-type (WT) and IKK epsilon(-/-) FLS were transfected with TBK1 or control small interfering RNA (siRNA) and stimulated with polyinosinic acid : polycytidylic acid [poly(I:C)]. Gene expression was assayed using quantitative PCR. Cytokine production in culture supernatants was measured by Luminex multiplex analysis. IFN-regulatory factor (IRF3) dimerization was determined by native PAGE. IFN-beta and IP-10 promoter activity was measured using luciferase reporter constructs. Results. Initial studies showed that siRNA markedly decreased TBK1 expression in cultured FLS. Poly(I:C)-induced IRF7 gene expression was inhibited in the absence of TBK1, but not IKK epsilon. IRF3 gene expression was similar to WT cells in TBK1 or IKK epsilon-deficient FLS. IRF3 dimerization required both TBK1 and IKK epsilon. Surprisingly, IRF3-mediated gene and protein expression of IFN-beta and IP-10 was dependent on TBK1, not IKK epsilon. Promoter constructs showed that TBK1 decreased IP-10 gene transcription and IP-10 mRNA stability was unaffected by TBK1 deficiency. Conclusion. Based on the selective regulation of IP-10 in FLS, TBK1 appears to be the optimal IKK-related kinase to target in RA.