Immune checkpoint VISTA negatively regulates microglia glycolysis and activation via TRIM28 mediated ubiquitination of HK2 in sepsis associated encephalopathy

A mouse model of sepsis was established using cecal ligation and puncture (CLP), and an anti-VISTA antibody (MH5A) was administered intracerebroventricularly. RNA sequencing was used to explore differentially expressed genes following MH5A injection. V-domain Ig suppressor of T cell activation (VISTA) was silenced in BV-2 microglial cells using siRNA to elucidate its effects on microglial activation and metabolism. The production of cytokines (TNF-α and IL-6) and chemokines (CCL3 and CCL19) was quantified using RT-qPCR and ELISA. Co-immunoprecipitation and ubiquitination assays were performed to investigate the potential mechanisms underlying ubiquitination of hexokinase-2 (HK2). Furthermore, TRIM28 overexpression plasmids-TRIM28-PcDNA3.1(+) or siRNA was transfected into BV-2 cells, with or without VISTA silencing, to determine the role of TRIM28 in VISTA-mediated microglial activation.