Xu Yuhai, Zhu Ying, Shi Yue, Ye Bo, Bo Lulong, Tao Tianzhu
Department of Anesthesiology, Air Force Medical Center, Beijing, 100142, China.
Department of Pulmonary and Critical Care Medicine, 7Th Medical Center of Chinese PLA General Hospital, Beijing, 100700, China.
Mol Neurobiol. 2025 Apr;62(4):4452-4465. doi: 10.1007/s12035-024-04572-z. Epub 2024 Oct 25.
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.
T细胞活化的V结构域免疫球蛋白抑制因子（VISTA）已成为神经疾病发病机制中的关键因素。然而，VISTA调节小胶质细胞活化的具体机制仍不清楚。将脓毒症小鼠经脑室内注射激动性抗VISTA抗体或同型对照。为了研究差异基因表达谱，对这些小鼠的脑组织进行了RNA测序。在体外，使用短发夹RNA（shRNA）使BV2小胶质细胞中的VISTA沉默。进行免疫共沉淀试验以鉴定涉及己糖激酶2（HK2）的蛋白质-蛋白质相互作用，并使用泛素化试验检测HK2的泛素化状态。此外，用含三联基序的28过表达质粒（TRIM28-PcDNA3.1(+)）或TRIM28特异性小干扰RNA转染BV2细胞，以评估TRIM28对VISTA介导的小胶质细胞活化的影响。使用细胞外酸化率测定法测量细胞糖酵解活性，并对促炎细胞因子和趋化因子进行定量。用VISTA抗体治疗可显著减轻脓毒症小鼠的小胶质细胞活化并预防认知障碍。相反，BV2小胶质细胞中的VISTA沉默导致促炎细胞因子的过表达，并以HK2依赖的方式增强糖酵解。从机制上讲，HK2的表达受E3泛素连接酶TRIM28通过K63连接的泛素化调节，该泛素化将HK2靶向蛋白酶体降解。此外，敲低TRIM28可降低在VISTA沉默的小胶质细胞中观察到的糖酵解升高和促炎反应。VISTA通过TRIM28介导的K63连接的泛素化调节HK2表达，从而调节脓毒症相关性脑病中的小胶质细胞活化。这些发现突出了VISTA作为调节脓毒症中小胶质细胞活化的潜在治疗靶点。
