Effect of Endosomal Toll-Like Receptor Inhibition on the Severity of Epstein Barr Virus DNA-Exacerbated Arthritis in a Mouse Model

Abstract

Background: Epstein-Barr Virus (EBV) infection is known to have periods of latency and reactivation accompanied by the shedding of EBV DNA. This may induce proinflammatory immune responses that are implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). RA is a chronic inflammatory disorder that affects the joints and may progress to cause disability and premature death. Moreover, previous studies conducted by our group showed that EBV DNA results in an increase in the production of IL-17A, a proinflammatory cytokine thought to play a key role in the pathogenesis of RA, in mice through endosomal toll-like receptor (TLR) signaling. Additionally, our group recently showed that EBV DNA increases the incidence and severity of arthritis in a collagen-induced arthritis (CIA) murine model using C57BL/6J mice administered type II chicken collagen. Based on these observations, the aim of this study was to determine the effects of the inhibition of endosomal TLRs on the severity of EBV DNA-exacerbated arthritis in a type II collagen-induced arthritis C57BL/6J mouse model. Methods: Collagen-injected female C57BL/6J mice, 12 weeks of age, were treated with either EBV DNA, EBV DNA in combination with a TLR3 inhibitor, EBV DNA with a TLR7 inhibitor, EBV DNA with a TLR9 inhibitor, a TLR3 inhibitor, a TLR7 inhibitor, or a TLR9 inhibitor. Arthritis severity was assessed through paw thickness measurements, clinical scoring, and grip strength testing. Furthermore, histological analysis was performed on tissue sections from affected footpads, ankle joints, and colons. Additionally, the number of pathogenic immune cells co-expressing interleukin 17A (IL-17A), interferon gamma (IFN-γ), and Forkhead box P3 (FOXP3) in ankle joint tissues was determined using immunofluorescence (IF) and confocal microscopy. Results: A significant reduction in paw thickness, clinical arthritis scores, and histological scores, alongside a marked improvement in grip strength, was observed in the groups that received EBV DNA, collagen, and an endosomal TLR inhibitor, as compared to mice treated with EBV DNA and collagen alone. Among the groups treated with EBV DNA, collagen, and an endosomal TLR inhibitor, the TLR9-inhibited group exhibited significantly lower phenotypic manifestations, compared to the groups that received a TLR3 or TLR7 inhibitor alongside EBV DNA and collagen. Similarly, the administration of an endosomal TLR inhibitor, EBV DNA, and collagen resulted in a decrease in the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in ankle joints. Conclusions: This study demonstrates that the inhibition of TLR3, TLR7, or TLR9 can attenuate the exacerbation of arthritis triggered by EBV DNA in a CIA mouse model. Notably, TLR9 inhibition resulted in the most pronounced therapeutic effects, as evidenced by significantly improved clinical outcomes. These findings suggest that endosomal TLRs, particularly TLR9, represent promising therapeutic targets for managing RA in EBV-infected individuals. The therapeutic benefits observed with endosomal TLR inhibition, highlight the need for further investigation into optimized dosing regimens, timing of intervention, and delivery strategies to support the clinical translation of endosomal TLR-targeted therapies.