STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

Yang Xiao; Chong Zhao; Yang Tai; Bei Li; Tian Lan; Enjiang Lai; Wenting Dai; Yangkun Guo; Can Gan; Enis Kostallari; Chengwei Tang; Jinhang Gao

doi:10.1016/j.redox.2023.102691

STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

Yang Xiao, Chong Zhao, Yang Tai, Bei Li, Tian Lan, Enjiang Lai, Wenting Dai, Yangkun Guo, Can Gan, Enis Kostallari, Chengwei Tang, Jinhang Gao

Gastroenterology and Hepatology

Research output: Contribution to journal › Article › peer-review

Abstract

The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, 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.

Original language	English (US)
Article number	102691
Journal	Redox Biology
Volume	62
DOIs	https://doi.org/10.1016/j.redox.2023.102691
State	Published - Jun 2023

Keywords

GSDMD
Histone methylation
IRF3
Liver cirrhosis
Metabolic reprogramming
Oxidative stress

ASJC Scopus subject areas

Organic Chemistry
Clinical Biochemistry

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10.1016/j.redox.2023.102691

Cite this

@article{9d680d7e994c4440840ac42a29a99d55,

title = "STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome",

abstract = "The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, 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.",

keywords = "GSDMD, Histone methylation, IRF3, Liver cirrhosis, Metabolic reprogramming, Oxidative stress",

author = "Yang Xiao and Chong Zhao and Yang Tai and Bei Li and Tian Lan and Enjiang Lai and Wenting Dai and Yangkun Guo and Can Gan and Enis Kostallari and Chengwei Tang and Jinhang Gao",

note = "Funding Information: The Supporting Materials include induction of liver fibrosis, human liver tissue collection, hematoxylin and eosin staining (H&E), immunofluorescence (IF), immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), serum biochemistry, Milliplex mouse multiplex assay, primary murine hepatocyte isolation, primary murine HSC (mHSC) isolation, cell culture and treatments, determination of hepatocyte pyroptosis, Primary mHSCs and hepatocyte co-culture, RNA-sequencing and data analysis, metabolomics, quantitative RT‒PCR (qPCR), Western blot (WB), co-immunoprecipitation (co-IP), histone-modifying enzyme compound screening system, and chromatin immunoprecipitation (ChIP), cytochromeP450 2E1 (CYP2E1) activity assay, measurement of hepatic O−2 contents and oxidative stress.To explore the genes and signaling pathways involved in liver fibrosis, bulk RNA sequencing of livers from a CCl4-induced murine model was performed. The ontology analysis of upregulated differentially expressed genes (DEGs) indicated that the cytosolic DNA-sensing pathway and NLRP3 inflammasome signaling pathway were significantly activated in fibrotic livers (Fig. 1A). The main molecule of the cytosolic DNA-sensing pathway is STING [17]. As shown by the heatmap of key DEGs, the mRNA levels of Sting (Tmem173), Nlrp3, Il1β, and Caspase 1 were significantly increased in fibrotic livers compared to control livers (Fig. 1B). Furthermore, compared to healthy livers, hepatocytes in fibrotic livers exhibited increased STING protein expression in murine and human livers (Fig. 1C, Supporting Figs. S1A–E). IRF3 and NFκB are crucial downstream transcription factors of the STING signaling pathway [17]. Hepatocyte nuclei were positive for both phosphorylated IRF3 (p-IRF3) and phosphorylated NFκB-p65 (p-p65) (Fig. 1C, Supporting Figs. S1A, C, F). However, only p-IRF3 was significantly increased in murine and human cirrhotic livers compared to the respective controls (Fig. 1C, Supporting Figs. S1A–G). These results indicate that the STING signaling pathway is associated with the development of liver fibrosis.The involvement of STING in liver fibrosis and hepatic inflammation was further confirmed using the STING pharmacological inhibition by C-176. CCl4-mediated liver fibrosis was significantly decreased in mice treated with C-176, as determined by Sirius red staining as well as collagen I and αSMA protein levels (Fig. 1I-L). The increased immune cell infiltration in the CCl4-induced fibrosis model was significantly attenuated in mice treated with C-176, as evidenced by H&E staining, IHC of MPO-positive neutrophils, and F4/80-positive macrophages (Supporting Fig. S3B). CYP2E1 is essential to the metabolic function of the liver [25]. CYP2E1 is mainly expressed in hepatocytes (Fig. 1M). The reduced CYP2E1 protein level in wild-type mice induced by CCl4 was restored after C-176 treatment (Fig. 1M). Comparable hepatic protein level and activity of CYP2E1 were observed in Sting+/+ and Sting−/− mice treated with olive oil (Fig. 1N, Supporting Fig. S3C). Additionally, a decreased CYP2E1 activity was induced by CCl4 treatment when compared to olive oil treatment in Sting+/+ mice (Fig. 1N). However, the decreased CYP2E1 activity induced by CCl4 was significantly attenuated by Sting knockout and C-176 treatment (Fig. 1N). In summary, STING promotes the development of liver fibrosis and hepatic inflammation in mice.There are two groups of inflammasomes in terms of receptors, with NLRP3 inflammasome being the most typical inflammasome [26]. Of all inflammasomes, the NLRP3 inflammasome signaling pathway was activated in fibrotic livers and downregulated after Sting knockout (Fig. 1A–B, H). The NLRP3 inflammasome, an NLRP3/ASC/Caspase 1 protein complex that cleaves GSDMD or IL1β/IL18, plays a pivotal role in pyroptosis and inflammation [14]. Whether the NLRP3 inflammasome and hepatocyte pyroptosis are activated in liver fibrosis remains controversial. In the TAA-induced murine fibrotic liver and human cirrhotic liver, the crucial pyroptotic molecules NLRP3, cleaved Caspase 1 (cl-Caspase 1), and cleaved GSDMD (cl-GSDMD) determined by WB and/or IHC were significantly increased, and NLRP3 and cl-Caspase 1 were mainly expressed in hepatocytes (Supporting Figs. S4A–D). Moreover, pyroptotic hepatocytes characterized by ruptured membranes and the formation of membrane vesicles were observed by H&E staining and TEM in murine fibrotic livers (Supporting Fig. S4E). In summary, NLRP3 inflammasome activation and hepatocyte pyroptosis are present in murine and human fibrotic livers.Next, we sought to understand whether STING is involved in NLRP3-mediated pyroptosis. In vivo, STING and NLRP3 were coexpressed in hepatocytes around fibrotic septa of the murine and human cirrhotic liver, as determined by colocalization of STING and NLRP3 (Fig. 2A and B). Additionally, hepatocyte pyroptosis was significantly reduced in Sting knockout mice, as determined by IHC of cl-Caspase 1 (Supporting Fig. S2B).In vitro, TNFα and lipopolysaccharide (LPS) are commonly used stimulators of pyroptosis [27]. However, TNFα (25, 50, 100 ng/mL) or LPS (1, 5, 10 μg/mL) alone could not induce primary murine hepatocyte pyroptosis as determined by cell morphology, Caspase 1/PI colocalization, and WB of NLRP3, cl-Caspase 1 and cl-GSDMD (Supporting Figs. S5A–D). To mimic the microenvironment of liver fibrosis, the TNFα plus STING agonist DMXAA (TNFα+DMXAA) was utilized to induce hepatocyte pyroptosis in murine primary hepatocytes. TNFα+DMXAA increased STING and NLRP3 expression and colocalization in primary murine hepatocytes (Fig. 2C). Meanwhile, TNFα+DMXAA treatment caused a typical pyroptotic cell morphology in primary murine hepatocytes, such as cell shrinkage, membrane rupture, and membrane blebbing, compared to the controls (Fig. 2D). Moreover, TNFα+DMXAA stimulation increased the double-positive cells of activated Caspase 1 and propidium iodide (PI, Fig. 2D). The protein levels of the NLRP3 inflammasome complex, such as NLRP3, cl-Caspase 1, and cl-GSDMD, were also significantly enhanced after TNFα+DMXAA stimulation (Fig. 2E and F). However, the pyroptotic cell morphology was significantly reversed by the STING inhibitor C-176 in primary murine hepatocytes (Fig. 2D). Additionally, the increase in the NLRP3 inflammasome and pyroptotic cell markers NLRP3, cl-Caspase 1, and cl-GSDMD induced by TNFα+DMXAA was also significantly decreased by the STING inhibitor C-176 in primary murine hepatocytes (Fig. 2E and F). Furthermore, as determined by the increased protein levels of STING and p-IRF3, TNFα+DMXAA treatment induced the activation of the STING-IRF3 signaling pathway in primary murine hepatocytes (Fig. 2E and F). However, the enhanced protein levels of STING and p-IRF3 caused by TNFα+DMXAA were markedly decreased by the STING inhibitor C-176 (Fig. 2E and F). Consistently, Sting knockout reduced the aforementioned pyroptotic changes, such as pyroptotic cell morphology and double-positive cells of activated Caspase 1/PI in primary murine hepatocytes (Fig. 2G). Moreover, TNFα stimulation in Sting-overexpressing AML12 cells also induced hepatocyte pyroptosis, as determined by morphological changes and increased mRNA and protein levels of NLRP3 and cl-Caspase 1 (Supporting Figs. S6A–D). In summary, STING induces hepatocyte pyroptosis via activating the NLRP3 inflammasome.The above results have uncovered the molecular mechanism of STING in NLRP3 regulation. However, the role of NLRP3 in hepatocyte pyroptosis and liver fibrosis remains controversial and is the focus of this section. In vitro, TNFα plus NLRP3 agonist nigericin treatment induced the pyroptotic phenotype, increased Caspase 1/PI colocalization, and enhanced protein levels of cl-Caspase 1 and cl-GSDMD in primary murine hepatocytes, and these pyroptotic changes were significantly attenuated by the NLRP3 inhibitor MCC950 (Supporting Figs. S8A–C). Activated HSCs are the major source of hepatic myofibroblasts and ECM in liver fibrosis [30]. To test the role of hepatocyte pyroptosis in the activation of HSCs, a transwell assay was setup by plating primary murine hepatocytes into the top well with corresponding treatments and primary murine HSCs into the bottom of the transwell apparatus. Pyroptoic hepatocytes induced by TNFα plus STING agonist DMXAA increased HSC activation, as determined by IF of αSMA in HSCs, which was reversed when STING inhibitor C-176 or NLRP3 inhibitor MCC950 was added in hepatocytes (Supporting Figs. S9A–B).In vivo, we next investigated the role of hepatocyte-specific Nlrp3 deletion and NLRP3 pharmacological inhibition by MCC950 in the development of liver fibrosis in TAA or CCl4 treatment or BDL surgery murine models. Hepatocyte-specific Nlrp3 deletion mice (Nlrp3△Hep) were generated by crossing Nlrp3flox mice with Alb-Cre mice and were confirmed by IHC of NLPR3 (Supporting Fig. S10A). As expected, pyroptotic hepatocytes induced by TAA were also significantly abrogated in Nlrp3△Hep mice, as indicated by IHC of cl-Caspase 1 (Supporting Fig. S10B). TAA or CCl4 treatment or BDL surgery led to increased liver fibrosis in control mice compared to vehicle or sham, as determined by Sirius red, IHC, and WB of collagen I and αSMA (Fig. 5A–F, Supporting Figs. S11A–D). However, liver fibrosis was significantly reduced in TAA- or BDL-treated Nlrp3△Hep mice compared to Nlrp3fl/fl mice (Fig. 5A–D, Supporting Figs. S11A–D). Similarly, liver fibrosis induced by CCl4 treatment was also significantly attenuated by NLRP3 inhibitor MCC950 (Fig. 5E and F).Unlike other types of cell death, pyroptosis can initiate local inflammation by releasing inflammatory cytokines and recruiting immune cells [8,12]. We next explored the impact of NLRP3 on hepatic inflammation. TAA or CCl4 treatment or BDL surgery on Nlrp3fl/fl or wild-type mice increased immune cell infiltration compared to that in the respective controls, as evidenced by H&E staining, IHC of MPO-positive neutrophils, and F4/80-positive macrophages (Fig. 5G, Supporting Figs. S11E–F, Supporting Fig. S12A). Consistently, the increase in hepatic inflammation in TAA treatment or BDL surgery was further confirmed by the enhanced hepatic cytokines and chemokines, including IL1β, TNFα, IFNγ, IL6, and C–C motif chemokine ligand 5 (CCL5, Fig. 5H, Supporting Fig. S11G). This increase in immune cell infiltration, hepatic cytokines and chemokines in the TAA- or BDL-induced fibrosis models were significantly attenuated in Nlrp3△Hep mice (Fig. 5G and H, Supporting Figs. S11E–G). The increase in immune cell infiltration induced by CCl4 treatment was also significantly ameliorated by NLRP3 pharmacological inhibition with MCC950 (Supporting Fig. S12A). Regarding different liver fibrosis models, the decreased activity and protein level of CYP2E1 induced by CCl4 were enhanced by MCC950 (Fig. 5I and J). Moreover, a similar hepatic protein level and activity of CYP2E1 were observed in Nlrp3fl/fl and Nlrp3△Hep mice treated with saline (Supporting Figs. S12B–D). In comparison, the decreased protein level of CYP2E1 induced by TAA treatment was also abrogated in Nlrp3△Hep mice (Supporting Figs. S12B–C). As defined by increased serum ALT and AST, liver injury was ameliorated in Nlrp3△Hep mice of TAA- or BDL-induced fibrosis models (Fig. 5K, Supporting Fig. S11H). Additionally, the increased total bilirubin in BDL surgery mice was also abrogated in Nlrp3△Hep mice (Supporting Fig. S11H). In summary, NLRP3 inhibition attenuates hepatocyte pyroptosis, and hepatocyte-specific Nlrp3 deletion reduces liver fibrosis, hepatic inflammation, and liver injury.NLPR3-dependent GSDMD cleavage is crucial to promoting cell pyroptosis [31]. To investigate the pro-fibrotic effect of pyroptosis, NLRP3 downstream Gsdmd knockout mice were subjected to TAA or BDL. Gsdmd knockout was confirmed by WB (Supporting Fig. S10C). By quantifying cl-Caspase 1, TAA-mediated hepatocyte pyroptosis was significantly attenuated in Gsdmd−/− mice (Supporting Fig. S10D). Compared to Gsdmd+/+ mice, TAA- or BDL-mediated liver fibrosis was significantly decreased in Gsdmd−/− mice, as determined by Sirius red staining as well as collagen I and αSMA protein levels (Supporting Figs. S13A–D, Supporting Figs. S14A–D). GSDMD cleaves the cell membrane of pyroptotic cells to release cell content and inflammatory factors [11]. We next confirmed that Gsdmd knockout inhibited hepatic inflammation and liver injury. The increased immune cell infiltration, hepatic cytokines and chemokines (IL1β, TNFα, IFNγ, IL6, and CCL5), as well as serum ALT, AST, and total bilirubin in the TAA- or BDL-induced fibrosis models were significantly attenuated in Gsdmd knockout mice (Supporting Figs. S13E–H, Supporting Figs. S14E–H). A comparable hepatic protein level of CYP2E1 was observed in Gsdmd+/+ and Gsdmd−/− mice treated with saline (Supporting Fig. S13I). However, the reduced protein level of CYP2E1 induced by TAA treatment was restored in Gsdmd−/− mice (Supporting Fig. S13I). In summary, inhibition of pyroptosis by Gsdmd knockout attenuates liver fibrosis, hepatic inflammation, and liver injury.The abovementioned results verified that blocking hepatocyte pyroptosis by NLRP3 and GSDMD deficiency can abrogate liver fibrosis. We next clarified how hepatocyte pyroptosis deficiency attenuates liver fibrosis using transcriptome and metabolomics. In vitro, primary murine hepatocytes treated with TNFα plus NLRP3 agonist nigericin and TNFα plus nigericin plus MCC950 were analyzed by RNA-sequencing (Supporting Fig. S8D). The DEG ontology analysis showed that 209 signaling pathways were significantly enriched, with the top being oxidative phosphorylation, ATP metabolism, ROS metabolism, metabolism of RNA, and the citric acid (TCA) cycle (Supporting Fig. S8D).This work was supported by the National Natural Science Fund of China (82170623, 82170625, U1702281, 81873584, 82000613, and 82000574), the National Key R&D Program of China (2017YFA0205404), Sichuan Science and Technology Program (2020YJ0084 and 2021YFS0147), and the 135 projects for disciplines of excellence of West China Hospital, Sichuan University (ZYGD18004). Funding Information: This work was supported by the National Natural Science Fund of China ( 82170623 , 82170625 , U1702281 , 81873584 , 82000613, and 82000574 ), the National Key R&D Program of China ( 2017YFA0205404 ), Sichuan Science and Technology Program ( 2020YJ0084 and 2021YFS0147 ), and the 135 projects for disciplines of excellence of West China Hospital, Sichuan University ( ZYGD18004 ). Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = jun,

doi = "10.1016/j.redox.2023.102691",

language = "English (US)",

volume = "62",

journal = "Redox Biology",

issn = "2213-2317",

publisher = "Elsevier BV",

}

TY - JOUR

T1 - STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

AU - Xiao, Yang

AU - Zhao, Chong

AU - Tai, Yang

AU - Li, Bei

AU - Lan, Tian

AU - Lai, Enjiang

AU - Dai, Wenting

AU - Guo, Yangkun

AU - Gan, Can

AU - Kostallari, Enis

AU - Tang, Chengwei

AU - Gao, Jinhang

N1 - Funding Information: The Supporting Materials include induction of liver fibrosis, human liver tissue collection, hematoxylin and eosin staining (H&E), immunofluorescence (IF), immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), serum biochemistry, Milliplex mouse multiplex assay, primary murine hepatocyte isolation, primary murine HSC (mHSC) isolation, cell culture and treatments, determination of hepatocyte pyroptosis, Primary mHSCs and hepatocyte co-culture, RNA-sequencing and data analysis, metabolomics, quantitative RT‒PCR (qPCR), Western blot (WB), co-immunoprecipitation (co-IP), histone-modifying enzyme compound screening system, and chromatin immunoprecipitation (ChIP), cytochromeP450 2E1 (CYP2E1) activity assay, measurement of hepatic O−2 contents and oxidative stress.To explore the genes and signaling pathways involved in liver fibrosis, bulk RNA sequencing of livers from a CCl4-induced murine model was performed. The ontology analysis of upregulated differentially expressed genes (DEGs) indicated that the cytosolic DNA-sensing pathway and NLRP3 inflammasome signaling pathway were significantly activated in fibrotic livers (Fig. 1A). The main molecule of the cytosolic DNA-sensing pathway is STING [17]. As shown by the heatmap of key DEGs, the mRNA levels of Sting (Tmem173), Nlrp3, Il1β, and Caspase 1 were significantly increased in fibrotic livers compared to control livers (Fig. 1B). Furthermore, compared to healthy livers, hepatocytes in fibrotic livers exhibited increased STING protein expression in murine and human livers (Fig. 1C, Supporting Figs. S1A–E). IRF3 and NFκB are crucial downstream transcription factors of the STING signaling pathway [17]. Hepatocyte nuclei were positive for both phosphorylated IRF3 (p-IRF3) and phosphorylated NFκB-p65 (p-p65) (Fig. 1C, Supporting Figs. S1A, C, F). However, only p-IRF3 was significantly increased in murine and human cirrhotic livers compared to the respective controls (Fig. 1C, Supporting Figs. S1A–G). These results indicate that the STING signaling pathway is associated with the development of liver fibrosis.The involvement of STING in liver fibrosis and hepatic inflammation was further confirmed using the STING pharmacological inhibition by C-176. CCl4-mediated liver fibrosis was significantly decreased in mice treated with C-176, as determined by Sirius red staining as well as collagen I and αSMA protein levels (Fig. 1I-L). The increased immune cell infiltration in the CCl4-induced fibrosis model was significantly attenuated in mice treated with C-176, as evidenced by H&E staining, IHC of MPO-positive neutrophils, and F4/80-positive macrophages (Supporting Fig. S3B). CYP2E1 is essential to the metabolic function of the liver [25]. CYP2E1 is mainly expressed in hepatocytes (Fig. 1M). The reduced CYP2E1 protein level in wild-type mice induced by CCl4 was restored after C-176 treatment (Fig. 1M). Comparable hepatic protein level and activity of CYP2E1 were observed in Sting+/+ and Sting−/− mice treated with olive oil (Fig. 1N, Supporting Fig. S3C). Additionally, a decreased CYP2E1 activity was induced by CCl4 treatment when compared to olive oil treatment in Sting+/+ mice (Fig. 1N). However, the decreased CYP2E1 activity induced by CCl4 was significantly attenuated by Sting knockout and C-176 treatment (Fig. 1N). In summary, STING promotes the development of liver fibrosis and hepatic inflammation in mice.There are two groups of inflammasomes in terms of receptors, with NLRP3 inflammasome being the most typical inflammasome [26]. Of all inflammasomes, the NLRP3 inflammasome signaling pathway was activated in fibrotic livers and downregulated after Sting knockout (Fig. 1A–B, H). The NLRP3 inflammasome, an NLRP3/ASC/Caspase 1 protein complex that cleaves GSDMD or IL1β/IL18, plays a pivotal role in pyroptosis and inflammation [14]. Whether the NLRP3 inflammasome and hepatocyte pyroptosis are activated in liver fibrosis remains controversial. In the TAA-induced murine fibrotic liver and human cirrhotic liver, the crucial pyroptotic molecules NLRP3, cleaved Caspase 1 (cl-Caspase 1), and cleaved GSDMD (cl-GSDMD) determined by WB and/or IHC were significantly increased, and NLRP3 and cl-Caspase 1 were mainly expressed in hepatocytes (Supporting Figs. S4A–D). Moreover, pyroptotic hepatocytes characterized by ruptured membranes and the formation of membrane vesicles were observed by H&E staining and TEM in murine fibrotic livers (Supporting Fig. S4E). In summary, NLRP3 inflammasome activation and hepatocyte pyroptosis are present in murine and human fibrotic livers.Next, we sought to understand whether STING is involved in NLRP3-mediated pyroptosis. In vivo, STING and NLRP3 were coexpressed in hepatocytes around fibrotic septa of the murine and human cirrhotic liver, as determined by colocalization of STING and NLRP3 (Fig. 2A and B). Additionally, hepatocyte pyroptosis was significantly reduced in Sting knockout mice, as determined by IHC of cl-Caspase 1 (Supporting Fig. S2B).In vitro, TNFα and lipopolysaccharide (LPS) are commonly used stimulators of pyroptosis [27]. However, TNFα (25, 50, 100 ng/mL) or LPS (1, 5, 10 μg/mL) alone could not induce primary murine hepatocyte pyroptosis as determined by cell morphology, Caspase 1/PI colocalization, and WB of NLRP3, cl-Caspase 1 and cl-GSDMD (Supporting Figs. S5A–D). To mimic the microenvironment of liver fibrosis, the TNFα plus STING agonist DMXAA (TNFα+DMXAA) was utilized to induce hepatocyte pyroptosis in murine primary hepatocytes. TNFα+DMXAA increased STING and NLRP3 expression and colocalization in primary murine hepatocytes (Fig. 2C). Meanwhile, TNFα+DMXAA treatment caused a typical pyroptotic cell morphology in primary murine hepatocytes, such as cell shrinkage, membrane rupture, and membrane blebbing, compared to the controls (Fig. 2D). Moreover, TNFα+DMXAA stimulation increased the double-positive cells of activated Caspase 1 and propidium iodide (PI, Fig. 2D). The protein levels of the NLRP3 inflammasome complex, such as NLRP3, cl-Caspase 1, and cl-GSDMD, were also significantly enhanced after TNFα+DMXAA stimulation (Fig. 2E and F). However, the pyroptotic cell morphology was significantly reversed by the STING inhibitor C-176 in primary murine hepatocytes (Fig. 2D). Additionally, the increase in the NLRP3 inflammasome and pyroptotic cell markers NLRP3, cl-Caspase 1, and cl-GSDMD induced by TNFα+DMXAA was also significantly decreased by the STING inhibitor C-176 in primary murine hepatocytes (Fig. 2E and F). Furthermore, as determined by the increased protein levels of STING and p-IRF3, TNFα+DMXAA treatment induced the activation of the STING-IRF3 signaling pathway in primary murine hepatocytes (Fig. 2E and F). However, the enhanced protein levels of STING and p-IRF3 caused by TNFα+DMXAA were markedly decreased by the STING inhibitor C-176 (Fig. 2E and F). Consistently, Sting knockout reduced the aforementioned pyroptotic changes, such as pyroptotic cell morphology and double-positive cells of activated Caspase 1/PI in primary murine hepatocytes (Fig. 2G). Moreover, TNFα stimulation in Sting-overexpressing AML12 cells also induced hepatocyte pyroptosis, as determined by morphological changes and increased mRNA and protein levels of NLRP3 and cl-Caspase 1 (Supporting Figs. S6A–D). In summary, STING induces hepatocyte pyroptosis via activating the NLRP3 inflammasome.The above results have uncovered the molecular mechanism of STING in NLRP3 regulation. However, the role of NLRP3 in hepatocyte pyroptosis and liver fibrosis remains controversial and is the focus of this section. In vitro, TNFα plus NLRP3 agonist nigericin treatment induced the pyroptotic phenotype, increased Caspase 1/PI colocalization, and enhanced protein levels of cl-Caspase 1 and cl-GSDMD in primary murine hepatocytes, and these pyroptotic changes were significantly attenuated by the NLRP3 inhibitor MCC950 (Supporting Figs. S8A–C). Activated HSCs are the major source of hepatic myofibroblasts and ECM in liver fibrosis [30]. To test the role of hepatocyte pyroptosis in the activation of HSCs, a transwell assay was setup by plating primary murine hepatocytes into the top well with corresponding treatments and primary murine HSCs into the bottom of the transwell apparatus. Pyroptoic hepatocytes induced by TNFα plus STING agonist DMXAA increased HSC activation, as determined by IF of αSMA in HSCs, which was reversed when STING inhibitor C-176 or NLRP3 inhibitor MCC950 was added in hepatocytes (Supporting Figs. S9A–B).In vivo, we next investigated the role of hepatocyte-specific Nlrp3 deletion and NLRP3 pharmacological inhibition by MCC950 in the development of liver fibrosis in TAA or CCl4 treatment or BDL surgery murine models. Hepatocyte-specific Nlrp3 deletion mice (Nlrp3△Hep) were generated by crossing Nlrp3flox mice with Alb-Cre mice and were confirmed by IHC of NLPR3 (Supporting Fig. S10A). As expected, pyroptotic hepatocytes induced by TAA were also significantly abrogated in Nlrp3△Hep mice, as indicated by IHC of cl-Caspase 1 (Supporting Fig. S10B). TAA or CCl4 treatment or BDL surgery led to increased liver fibrosis in control mice compared to vehicle or sham, as determined by Sirius red, IHC, and WB of collagen I and αSMA (Fig. 5A–F, Supporting Figs. S11A–D). However, liver fibrosis was significantly reduced in TAA- or BDL-treated Nlrp3△Hep mice compared to Nlrp3fl/fl mice (Fig. 5A–D, Supporting Figs. S11A–D). Similarly, liver fibrosis induced by CCl4 treatment was also significantly attenuated by NLRP3 inhibitor MCC950 (Fig. 5E and F).Unlike other types of cell death, pyroptosis can initiate local inflammation by releasing inflammatory cytokines and recruiting immune cells [8,12]. We next explored the impact of NLRP3 on hepatic inflammation. TAA or CCl4 treatment or BDL surgery on Nlrp3fl/fl or wild-type mice increased immune cell infiltration compared to that in the respective controls, as evidenced by H&E staining, IHC of MPO-positive neutrophils, and F4/80-positive macrophages (Fig. 5G, Supporting Figs. S11E–F, Supporting Fig. S12A). Consistently, the increase in hepatic inflammation in TAA treatment or BDL surgery was further confirmed by the enhanced hepatic cytokines and chemokines, including IL1β, TNFα, IFNγ, IL6, and C–C motif chemokine ligand 5 (CCL5, Fig. 5H, Supporting Fig. S11G). This increase in immune cell infiltration, hepatic cytokines and chemokines in the TAA- or BDL-induced fibrosis models were significantly attenuated in Nlrp3△Hep mice (Fig. 5G and H, Supporting Figs. S11E–G). The increase in immune cell infiltration induced by CCl4 treatment was also significantly ameliorated by NLRP3 pharmacological inhibition with MCC950 (Supporting Fig. S12A). Regarding different liver fibrosis models, the decreased activity and protein level of CYP2E1 induced by CCl4 were enhanced by MCC950 (Fig. 5I and J). Moreover, a similar hepatic protein level and activity of CYP2E1 were observed in Nlrp3fl/fl and Nlrp3△Hep mice treated with saline (Supporting Figs. S12B–D). In comparison, the decreased protein level of CYP2E1 induced by TAA treatment was also abrogated in Nlrp3△Hep mice (Supporting Figs. S12B–C). As defined by increased serum ALT and AST, liver injury was ameliorated in Nlrp3△Hep mice of TAA- or BDL-induced fibrosis models (Fig. 5K, Supporting Fig. S11H). Additionally, the increased total bilirubin in BDL surgery mice was also abrogated in Nlrp3△Hep mice (Supporting Fig. S11H). In summary, NLRP3 inhibition attenuates hepatocyte pyroptosis, and hepatocyte-specific Nlrp3 deletion reduces liver fibrosis, hepatic inflammation, and liver injury.NLPR3-dependent GSDMD cleavage is crucial to promoting cell pyroptosis [31]. To investigate the pro-fibrotic effect of pyroptosis, NLRP3 downstream Gsdmd knockout mice were subjected to TAA or BDL. Gsdmd knockout was confirmed by WB (Supporting Fig. S10C). By quantifying cl-Caspase 1, TAA-mediated hepatocyte pyroptosis was significantly attenuated in Gsdmd−/− mice (Supporting Fig. S10D). Compared to Gsdmd+/+ mice, TAA- or BDL-mediated liver fibrosis was significantly decreased in Gsdmd−/− mice, as determined by Sirius red staining as well as collagen I and αSMA protein levels (Supporting Figs. S13A–D, Supporting Figs. S14A–D). GSDMD cleaves the cell membrane of pyroptotic cells to release cell content and inflammatory factors [11]. We next confirmed that Gsdmd knockout inhibited hepatic inflammation and liver injury. The increased immune cell infiltration, hepatic cytokines and chemokines (IL1β, TNFα, IFNγ, IL6, and CCL5), as well as serum ALT, AST, and total bilirubin in the TAA- or BDL-induced fibrosis models were significantly attenuated in Gsdmd knockout mice (Supporting Figs. S13E–H, Supporting Figs. S14E–H). A comparable hepatic protein level of CYP2E1 was observed in Gsdmd+/+ and Gsdmd−/− mice treated with saline (Supporting Fig. S13I). However, the reduced protein level of CYP2E1 induced by TAA treatment was restored in Gsdmd−/− mice (Supporting Fig. S13I). In summary, inhibition of pyroptosis by Gsdmd knockout attenuates liver fibrosis, hepatic inflammation, and liver injury.The abovementioned results verified that blocking hepatocyte pyroptosis by NLRP3 and GSDMD deficiency can abrogate liver fibrosis. We next clarified how hepatocyte pyroptosis deficiency attenuates liver fibrosis using transcriptome and metabolomics. In vitro, primary murine hepatocytes treated with TNFα plus NLRP3 agonist nigericin and TNFα plus nigericin plus MCC950 were analyzed by RNA-sequencing (Supporting Fig. S8D). The DEG ontology analysis showed that 209 signaling pathways were significantly enriched, with the top being oxidative phosphorylation, ATP metabolism, ROS metabolism, metabolism of RNA, and the citric acid (TCA) cycle (Supporting Fig. S8D).This work was supported by the National Natural Science Fund of China (82170623, 82170625, U1702281, 81873584, 82000613, and 82000574), the National Key R&D Program of China (2017YFA0205404), Sichuan Science and Technology Program (2020YJ0084 and 2021YFS0147), and the 135 projects for disciplines of excellence of West China Hospital, Sichuan University (ZYGD18004). Funding Information: This work was supported by the National Natural Science Fund of China ( 82170623 , 82170625 , U1702281 , 81873584 , 82000613, and 82000574 ), the National Key R&D Program of China ( 2017YFA0205404 ), Sichuan Science and Technology Program ( 2020YJ0084 and 2021YFS0147 ), and the 135 projects for disciplines of excellence of West China Hospital, Sichuan University ( ZYGD18004 ). Publisher Copyright: © 2023 The Authors

PY - 2023/6

Y1 - 2023/6

N2 - The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, 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.

AB - The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, 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.

KW - GSDMD

KW - Histone methylation

KW - IRF3

KW - Liver cirrhosis

KW - Metabolic reprogramming

KW - Oxidative stress

UR - http://www.scopus.com/inward/record.url?scp=85151485451&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85151485451&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2023.102691

DO - 10.1016/j.redox.2023.102691

M3 - Article

AN - SCOPUS:85151485451

SN - 2213-2317

VL - 62

JO - Redox Biology

JF - Redox Biology

M1 - 102691

ER -

STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

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