Dual Targeting of TLR9 and cGAS-STING Pathways Attenuates Astrocyte Inflammatory Activation: Potential Implication in Multiple Sclerosis

Abstract

This study aims to investigate the role of Toll-like receptor 9 (TLR9), a deoxyribose nucleic acid (DNA) sensor, in astrocyte activation and its contribution to multiple sclerosis (MS) pathogenesis. Additionally, we examined whether TLR9 and the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathways act synergistically to promote astrocyte inflammatory activation and whether combined inhibition of both pathways offers superior protective effects. Human astrocytes were treated with unmethylated Cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides at varying concentrations. Intervention groups included treatment with a TLR9 blocker (anti-TLR9) and a STING inhibitor (RU.521). Cellular responses were assessed by cell counting kit (CCK)-8 viability assay and Annexin V-based apoptosis detection. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the secreted levels of lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interleukins (IL)-6, IL-1β, and IL-18. Protein expression levels of TLR9, nuclear factor kappa B (NF-κB), phosphorylated NF-κB (p-NF-κB), STING, and cGAS were analyzed by Western blot. Treatment with unmethylated CpG oligodeoxynucleotides significantly reduced astrocyte viability and promoted apoptosis, while upregulating the expression of TLR9, p-NF-κB, and STING proteins. Combined treatment with both the TLR9 blocker and the STING inhibitor significantly restored cell viability, reduced apoptosis, and decreased the production of inflammatory factors in astrocytes. These findings suggest that targeting TLR9 and the STING pathway can alleviate astrocyte overactivation, indicating a potential therapeutic role for targeting the TLR9-cGAS-STING pathway in MS pathogenesis.