(M1030-07-41) Elucidating Immunomodulatory Mechanism of Bifunctional Peptide Inhibitors Using Mouse Model of EAE

Purpose: According to the National Institutes of Health, up to 23.5 million people in the United States suffer from autoimmune diseases and their prevalence is rising.¹ Current treatments rely on immunosuppressants which halt immune cells from recognizing and attacking autoantigens. However, this broad suppression of the patients’ immune system can increase their risk of infection and make them susceptible to other diseases. Hence, there is an urgent need to identify strategies that can regulate autoimmunity without systemic tolerance. This study aims to evaluate and understand the effects of different routes of administration for bifunctional peptide inhibitors (BPI), previously known to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. The rationale for using BPI molecules is that it consists of an antigenic peptide conjugated to LABL peptide and hypothesized to bind to the MHC-II to block the immunological synapse from forming and suppresses the autoreactive response.

Methods: Mouse bone-marrow derived dendritic cells (BMDCs) were harvested from C57BL/6 mice. In in vitro BMDC activation assays, cells were plated and treated with myelin oligodendrocyte glycoprotein (MOG38–50) and its bifunctional peptide inhibitor (MOG-BPI) at a concentration of 10 µM. After 48 h incubation, supernatants were collected for cytokine analysis (ELISA), and cells were stained for flow cytometry analysis. To investigate in vivo efficacy of BPIs in different routes of administration, we established mouse multiple sclerosis model, which auto-reactive T cells attack the myelin sheath. On days 4 and 7 mice were injected with MOG-BPI either through intravenous injection (i.v.), subcutaneous (s.c.), or intraperitoneal (i.p.) (n=5). The control groups with and without the induction of EAE were administered PBS via i.v. injection.

Results: When evaluating the cytotoxicity of the BPIs we used various concentrations (10 µM, 1 µM, and 100 nM) on RAW 264.7 cells. When evaluating these peptides in vivo we observed an increase in Foxp3+ for all routes of administration but no difference amongst the groups. There was a difference among IL-10 an anti-inflammatory cytokine expression within the cells with the i.v. treated group having higher expression than the control or i.p. treated group. Additionally observed there was a significant decrease in expression for pro-inflammatory cytokine IL-17a among all groups but no significant difference among the different routes of administration. When observing the microglia extracted from brain tissue, we saw a decrease in expression for costimulatory molecule CD86 known to be a pro-inflammatory marker through each route of administration. Additionally, we saw a decrease in pro-inflammatory cytokine TNF-α expression when administered through IV, however, all routes of expression did decrease the expression.

Conclusion: In this study the route of administration affecting the function of the BPI was evaluated. Future studies can be done to improve the delivery of the peptides through nanoparticle formulation for local or systemic delivery. Pharmacokinetic studies to evaluate the clearance of the drug based on the route of administration. These improvements can help sustain the peptides for longer periods, therefore leading to long term therapeutic and reducing any relapses in EAE while the immune system keeps the specificity to fight other infections.

References: 1. NIH Autoimmune Diseases Coordinating Committee: Autoimmune Diseases Research Plan, March 2005, https://www.niaid.nih.gov/sites/default/files/adccfinal.pdf
2. Mahadik, R. Kiptoo, P. Tolbert, T. Siahaan, T. (2022) Immune modulation by antigenic peptides and Antigenic peptide conjugates for treatment of Multiple Sclerosis. Med Res Arch. doi:10.18103/mra.v10i5.2804

Acknowledgements: This work was supported in part by a grant from the National Institute of Health (P20GM113117) (H.K). T.J.S received Funding from National Institutes of Health (R01-AG08227).


Intracellular staining was performed to identify Foxp3+ within splenocyte CD4+T cells from in vivo study.


Intracellular staining was performed to identify IL-17a within splenocyte CD4+T cells from in vivo study.


Intracellular staining was performed to identify TNF-a within microglia cells from in vivo study.