Objective Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH.
Design Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4).
Results HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis.
Conclusion SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.
- immunology in hepatology
- nonalcoholic steatohepatitis
- lipid mediators
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EHK, JEY and MSK contributed equally.
Correction notice This article has been corrected since it published Online First. The author Laura Conde de la Rosa's name has been corrected.
Contributors EHK conceptualised the study, designed the study and wrote the manuscript. JEY designed the study, performed in vivo and in vitro experiments and wrote the manuscript. MSK designed the study, performed in vitro and molecular experiments and wrote the manuscript. J-YY and CHH performed in vivo experiments. YKC, JEJ, SEL, JYB and JSL analysed the data and participated in writing of the manuscript. COS provided critical review of liver histology. HJY and SJK measured lipid metabolite levels. ST, ESV, LCR and CG-R performed and analysed in vivo studies in mice and examined the human samples. W-IJ, SHB, AF and JCF-C critically reviewed the manuscript, provided suggestions and contributed to the discussion. I-JB generated the Nlrp3 K/O mice. K-UL and JCF-C conceptualised the study, wrote the manuscript and are responsible for the integrity of this work. All authors discussed the results and commented on the manuscript.
Funding This study was supported by grants from the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology, Korea (2017R1E1A1A01073206: KUL, 2017R1E1A1A01074207: EHK) and Asan Institute for Life Sciences, Korea (2018IP0557-1). We acknowledge the support from NIH grants R01 DK113592, R01 AA024206 to AEF; grants SAF2017-85877R and PID2019-1116691RB from Plan Nacional de I+D, Spain and by the CIBEREHD, Instituto de Salud Carlos III, Spain; the center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH; support from AGAUR of the Generalitat de Catalunya SGR-2017–1112 and the 'ER stress-mitochondrial cholesterol axis in obesity-associated insulin resistance and comorbidities'-Ayudas Fundación BBVA (35_2018) a Equipos de Investigación Científica 2017, the Red Nacional 2018–1 02 799 T 'Enfermedades metabólicas y cáncer' and the Fundació Marató TV3 201916-30-31.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
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