Background: Epstein–Barr virus (EBV) infection alternates between latency and reactivation, accompanied by EBV DNA shedding that can trigger proinflammatory immune responses implicated in autoimmune diseases such as rheumatoid arthritis (RA). Our group previously showed that EBV DNA increases the production of IL-17A, a proinflammatory cytokine, in mice through endosomal toll-like receptor (TLR) signaling. We also demonstrated that EBV DNA enhances the incidence and severity of arthritis in a collagen-induced arthritis (CIA) model using C57BL/6J mice. This study aimed to determine the effect of endosomal TLR inhibition on EBV DNA-exacerbated arthritis in this model.

Methods: Female C57BL/6J mice were injected with type II chicken collagen and treated with EBV DNA alone or combined with inhibitors of TLR3, TLR7, or TLR9. Control groups received each inhibitor alone. Arthritis severity was evaluated by paw thickness, clinical scoring, and grip strength. Histological analyses of footpads, ankle joints, and colons were performed, and the frequency of IL-17A, IFN-γ, and FOXP3 co-expressing immune cells in ankle joints was assessed using immunofluorescence and confocal microscopy.

Results: Mice treated with EBV DNA, collagen, and an endosomal TLR inhibitor exhibited significant reductions in paw thickness, arthritis scores, and histological damage, alongside improved grip strength, compared to those treated with EBV DNA and collagen alone. Among the inhibitors, TLR9 blockade produced the most pronounced reduction in clinical and histological severity. Inhibitor treatment also decreased the number of IL-17A⁺IFN-γ⁺FOXP3⁺ cells in ankle tissues.

Conclusions: Inhibition of TLR3, TLR7, or TLR9 attenuates EBV DNA-exacerbated arthritis in a CIA mouse model, with TLR9 inhibition showing the strongest therapeutic benefit. These findings highlight endosomal TLRs, particularly TLR9, as promising therapeutic targets for RA management in EBV-infected individuals.