Immune Checkpoint VISTA Negatively Regulates Microglia Glycolysis and Activation via TRIM28-Mediated Ubiquitination of HK2 in Sepsis-Associated Encephalopathy.

V-domain immunoglobulin suppressor of T cell activation (VISTA) has emerged as a crucial player in the pathogenesis of neurological disorders. However, the specific mechanism by which VISTA regulates microglial activation remains unclear. Septic mice were intracerebroventricularly injected with an agonistic anti-VISTA antibody or isotype control. To investigate the differential gene expression profiles, RNA sequencing was conducted on brain tissues from these mice. In vitro, VISTA was silenced in BV2 microglial cells using shRNA. Co-immunoprecipitation assays were performed to identify protein-protein interactions involving hexokinase 2 (HK2), and ubiquitination assays were used to examine the ubiquitination status of HK2. Additionally, BV2 cells were transfected with either tripartite motif-containing 28 overexpression plasmids (TRIM28-PcDNA3.1( +)) or TRIM28-specific siRNA to assess the impact of TRIM28 on VISTA-mediated microglial activation. The cellular glycolytic activity was measured using extracellular acidification rate assays, and proinflammatory cytokine and chemokines were quantified. Treatment with VISTA antibodies significantly alleviated microglial activation and prevented cognitive impairment in septic mice. In contrast, VISTA silencing in BV2 microglia led to the overexpression of proinflammatory cytokines and enhanced glycolysis in an HK2-dependent manner. Mechanistically, HK2 expression was regulated by the E3 ubiquitin ligase TRIM28 through K63-linked ubiquitination, which targeted HK2 for proteasomal degradation. Furthermore, knockdown of TRIM28 reduced the elevated glycolysis and proinflammatory response observed in VISTA-silenced microglia. VISTA modulates microglial activation in sepsis-associated encephalopathy by regulating HK2 expression through TRIM28-mediated K63-linked ubiquitination. These findings highlight VISTA as a potential therapeutic target for modulating microglial activation in sepsis.