Inhibition of HSPA8 alleviates experimental autoimmune encephalomyelitis via dual modulation of NLRP3 inflammasome activation: suppressing both NF-κB-mediated priming and ASC-dependent assembly.

Abstract

The pathological processes of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are closely associated with excessive activation of inflammasomes. HSPA8 is a constitutively expressed molecular chaperone involved in cellular signaling and immune-inflammatory regulation, but its role in EAE-associated neuroinflammation remains unclear. In this study, we found that HSPA8 was significantly upregulated in the spinal cord tissues of EAE mice and positively correlated with disease severity. Immunofluorescence co-staining showed that HSPA8 upregulation was more prominently associated with Iba1-positive microglia/macrophage-enriched regions than with GFAP- or NeuN-positive regions. Intrathecal knockdown of HSPA8 attenuated EAE progression, reduced inflammatory responses, and alleviated demyelination and axonal injury. In vitro, HSPA8 knockdown reduced NLRP3 inflammasome-mediated IL-1β/IL-18 release, caspase-1 activation, GSDMD-N formation, and pyroptosis in THP-1 cells, bone marrow-derived macrophages, and BV2 microglia, and these effects were partially restored by siRNA-resistant HSPA8 rescue. Mechanistically, HSPA8 knockdown was associated with impaired NF-κB-dependent priming, as reflected by reduced p65 phosphorylation, nuclear translocation, NF-κB transcriptional activity, and pro-IL-1β expression. HSPA8 is also associated with ASC, and HSPA8 knockdown impaired NLRP3-ASC interaction, ASC oligomerization, and ASC speck formation during inflammasome assembly. These findings suggest that HSPA8 is functionally associated with NLRP3 inflammasome activation through both NF-κB‑dependent priming and ASC‑associated assembly, thereby contributing to neuroinflammation in EAE. HSPA8 may represent a potential therapeutic target for MS-related and other inflammasome-driven neuroinflammatory disorders.