Susanne Gaul1, Aleksandra Leszczynska2, Fernando Alegre3, Benedikt Kaufmann2, Casey D Johnson4, Leon A Adams5, Alexander Wree6, Georg Damm7, Daniel Seehofer7, Carolina J Calvente2, Davide Povero8, Tatiana Kisseleva9, Akiko Eguchi10, Matthew D McGeough2, Hal M Hoffman2, Pablo Pelegrin11, Ulrich Laufs12, Ariel E Feldstein13. 1. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA; Universität Leipzig, Klinik und Poliklinik für Kardiologie, Leipzig, Germany. 2. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA. 3. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA; Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain. 4. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA; University of California Irvine, Irvine, CA, USA. 5. Medical School, University of Western Australia, Perth, Australia. 6. Department of Hepatology and Gastroenterology, Charité, Campus Virchow Klinikum and Charité, Universitätsmedizin Berlin, Berlin, Germany. 7. Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany. 8. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. 9. Department of Surgery, University of California San Diego, La Jolla, CA, USA. 10. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA; Department of Gastroenterology and Hepatology, Mie University, Tsu, Mie, Japan. 11. Biomedical Research Institute of Murcia, Clinical University Hospital Virgen de la Arrixaca (IMIB-Arrixaca), Murcia, Spain. 12. Universität Leipzig, Klinik und Poliklinik für Kardiologie, Leipzig, Germany. 13. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA. Electronic address: afeldstein@ucsd.edu.
Abstract
BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.
BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.
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