STING mediates hepatocyte pyroptosis in liver fibrosis via epigenetic activating NLRP3 inflammasome

Background and Aims: The activation of stimulator of interferon genes (STING) and NOD-like receptors protein 3 (NLRP3) inflammasomes-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnection between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown and is the focus of this study. Approach and Results: Liver fibrosis was induced in Sting knockout, Gasdermin D (Gsdmd) knockout mice, and in mice with hepatocyte-specific Nlrp3 deletion. RNA-sequencing, metabolomics, epigenetic compound screening system, and chromatin immunoprecipitation were utilized. STING and NLRP3 inflammasome signaling pathways were activated in cirrhotic livers but were suppressed by Sting knockout. Sting knockout also ameliorated hepatic pyroptosis, inflammation, and fibrosis in the murine cirrhotic model. In vitro, STING induced pyroptosis in primary murine hepatocytes via activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) were identified to regulate NLRP3 expression in STING-overexpressed AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhanced interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promoted STING-induced Nlrp3 transcription in hepatocytes. The RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes showed that metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gsdmd knockout attenuated hepatic pyroptosis, inflammation, and fibrosis in murine cirrhotic models. Conclusions: This study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis. Overall design: 1.The primary murine hepatocytes were stimulated with LPS (1 µg/mL) for 6 hours and nigericin (10 µM) for 30 minutes. The NLRP3 inhibitor MCC950 (10 µM) were added 2 hours before stimulation 2. One liver fibrosis model was induced by intraperitoneal injection of thioacetamide (TAA) in mice with hepatocyte-specific Nlrp3 deletion and wild type for 8 weeks. Control mice received the same volume of normal saline (NS). Another model was induced by intraperitoneal injection of carbon tetrachloride (CCl4) in mice with Sting knock out and wild for 8weeks. Control mice received the same volume of olive oil.

背景与目的：干扰素基因刺激因子（STING）与NOD样受体蛋白3（NLRP3）炎症小体介导的细胞焦亡信号通路，是肝脏疾病中两种截然不同的核心致病机制。然而，这两条通路间的相互关联，以及肝纤维化进程中肝细胞焦亡里STING-NLRP3轴的表观遗传调控机制，目前仍未阐明，这也是本研究的核心聚焦点。

方法与结果：本研究通过构建Sting基因敲除、焦亡素D（Gasdermin D，Gsdmd）基因敲除小鼠，以及肝细胞特异性Nlrp3敲除小鼠，诱导肝纤维化模型。实验采用RNA测序、代谢组学、表观遗传化合物筛选系统以及染色质免疫沉淀技术。研究发现，肝硬化肝脏中STING与NLRP3炎症小体信号通路均被激活，而Sting基因敲除可抑制这两条通路的活化。此外，Sting基因敲除能够改善小鼠肝硬化模型中的肝细胞焦亡、炎症反应与肝纤维化程度。体外实验表明，STING可通过激活NLRP3炎症小体，诱导原代小鼠肝细胞发生细胞焦亡。在过表达STING的AML12肝细胞中，研究者鉴定出H3K4特异性组蛋白甲基转移酶含WD重复结构域蛋白5（WDR5）与DOT1样组蛋白H3K79甲基转移酶（DOT1L）可调控NLRP3的表达。WDR5/DOT1L介导的组蛋白甲基化，能够增强干扰素调节转录因子3（IRF3）与Nlrp3启动子的结合，并促进STING诱导的肝细胞中Nlrp3的转录。对小鼠肝脏与原代肝细胞的RNA测序及代谢组学分析显示，代谢重编程可能参与NLRP3介导的肝细胞焦亡与肝纤维化过程。进一步实验证实，肝细胞特异性Nlrp3敲除与下游Gsdmd敲除，均可减轻小鼠肝硬化模型中的肝细胞焦亡、炎症反应与肝纤维化程度。

结论：本研究阐明了一种全新的表观遗传机制，即STING-WDR5/DOT1L/IRF3-NLRP3信号通路可增强肝纤维化中的肝细胞焦亡与肝脏炎症反应。

实验整体设计：
1. 将原代小鼠肝细胞用脂多糖（LPS，1 μg/mL）刺激6小时，随后用尼日利亚菌素（10 μM）刺激30分钟；NLRP3抑制剂MCC950（10 μM）需在刺激前2小时加入。
2. 第一种肝纤维化模型：对肝细胞特异性Nlrp3敲除小鼠与野生型小鼠腹腔注射硫代乙酰胺（TAA），连续给药8周以诱导肝纤维化，对照组小鼠给予等体积生理盐水（NS）。第二种肝纤维化模型：对Sting敲除小鼠与野生型小鼠腹腔注射四氯化碳（CCl4），连续给药8周，对照组小鼠给予等体积橄榄油。