IntroductionToll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis.MethodsArthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-α) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR.ResultsBy screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-β) and TNF-α by pristane stimulation was blocked by anti-TLR3 antibody in NR8383.ConclusionsTLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.

IntroductionToll-like receptors (TLRs) are likely to play crucial roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the key TLRs in synovium and explore their roles in the activation of fibroblast-like synoviocytes (FLSs) mediated by T cells in arthritis.MethodsPristane-induced arthritis (PIA) was established by subcutaneous injection with pristane at the base of the rat's tail. TLR expression in synovium from PIA rats was detected at different time points by performing real-time PCR. Polyinosinic:polycytidylic acid (poly(I:C)) was intra-articularly administrated to PIA rats, and arthritis was monitored macroscopically and microscopically. Synovial TLR3 was detected by immunohistochemical staining. Rat FLSs were stimulated with pristane-primed T cells or pristane-primed, T-cell conditioned medium. The intervention of TLR3 in FLSs was achieved by specific short-hairpin RNA (shRNA) or an antibody. The migration ability of FLSs was measured by using the scratch test, and gene expression was detected by using real-time PCR. FLSs from RA patients were stimulated with various cytokines and TLR ligands, and TLR3 expression was detected by performing real-time PCR. In addition, with different concentrations of poly(I:C) stimulation, TLR3 expression of FLSs from RA patients and patients with osteoarthritis (OA) was compared.ResultsSynovium TLR3 displayed early and persistent overexpression in PIA rats. TLR3 was expressed in FLSs, and local treatment with poly(I:C) synergistically aggravated the arthritis. Rat FLSs co-cultured with pristane-primed T cells showed strengthened migration ability and significant upregulation of TLR3, IFN-β, IL-6 and matrix metalloproteinase 3 (MMP3) expression, which could also be induced by pristane-primed, T-cell conditioned medium. The upregulation of cytokines and MMPs was blocked by shRNA or TLR3 antibodies. In RA FLSs with cytokine or TLR ligand stimulation, TLR3 expression exhibited remarkable upregulation. Furthermore, RA FLSs showed higher reactivity than OA FLSs to poly(I:C).ConclusionsTLR3 in the synovium of PIA rats was overexpressed, and activation of the TLR3 signaling pathway could aggravate this arthritis. The induction of TLR3 in FLSs resulted from T cell-derived inflammatory stimulation and could further mediate FLS activation in arthritis. We conclude that TLR3 upregulation of FLSs activated by T cells results in articular inflammation.

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