Route of Administration Determines the Immunosuppressive Mechanism of Bifunctional Peptide Inhibitors in a Mouse EAE Model

Individuals diagnosed with multiple sclerosis (MS), an autoimmune disease, experience both physical and cognitive impairments due to the degeneration of the myelin sheath surrounding neurons triggered by autoreactive T cells and antibodies. Current MS therapies based on immunosuppressants often result in systemic immune suppression, known as immune tolerance, which leads to side effects including risk of infection and malignancy. Previously, we reported bifunctional peptide inhibitors (BPIs) that can induce antigen-specific immune tolerance and suppress disease progression in a mouse MS model of experimental autoimmune encephalomyelitis (EAE). While BPIs showed similar effectiveness via different routes of administration including intravenous (IV), subcutaneous (SC), and intraperitoneal (IP), whether the route of administration, specifically, mediates immunosuppressive mechanisms used to suppress EAE has not yet been studied. In this study, we prepared BPIs based on the model antigen ovalbumin (OVA₃₃₉-BPI) and myelin proteolipid protein (PLP-BPI) to evaluate immunomodulatory effects on splenocytes and the PLP-induced EAE model, respectively. The efficacy of BPIs inducing an immunomodulatory response was determined by measuring cytokine production as well as transcription factor and costimulatory molecule expression levels in microglia cells and splenocytes. In this study, PLP-BPI suppressed EAE in IV and SC treatment groups, with clinical scores reaching 0 (no clinical symptoms) by day 19. In contrast, the IP-treated group showed no suppression, with clinical scores similar to those of the EAE + no treatment group. Interestingly, IV and SC employed distinct immunomodulatory mechanisms: IV primarily reduces proinflammatory markers in microglia, while SC treatment increases the anti-inflammatory markers in microglia and transcription factor Foxp3⁺ in splenocytes. These results suggest that the route of BPI administration can determine the <i>in vivo</i> efficacy by differentially modulating the frequency and activity of immunosuppressive cell populations.