Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis

Abstract

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression

“…Th17 cells gen, whereas the downregulated cluster was enriched for genes involved in lipid metabolism ( Figure 2E). Consistently, mRNA expression of genes involved in liver fibrosis, including Col1a1, Acta2, Tgfb1, and Mmp13, and hepatic inflammation, including Previous studies have demonstrated that treatments of primary hepatocytes with a combination of palmitic acid (PA) and TNF-α (PA/TNF-α) robustly triggered cell death that mimics NASHinduced liver injury (21,37). To investigate whether Nrg4 plays a direct role in the regulation of hepatocyte death, we treated primary hepatocytes transduced with GFP or ErbB4 adenovirus with PA/TNF-α.…”

“…Necroptosis releases intracellular danger-associated molecular patterns, such as high-mobility group B1 (HMGB1), that have potential to trigger robust inflammatory response (13,19). RIPK3 deficiency attenuates liver injury, steatosis, inflammation, and fibrosis in an MCD diet-induced NASH mouse model, implicating necroptosis in the liver as a contributor to NASH (20,21). As such, targeting hepatocyte cell death may provide an effective approach to alleviating or reversing NASH progression (22).…”

Molecular Imaging with Kupffer Cell-Targeting Nanobodies for Diagnosis and Prognosis in Mouse Models of Liver Pathogenesis

“…Correspondingly, SPECT/μCT imaging using both Tc-99m-labelled Nbs clearly showed an increase in the signal in mice fed a MCD diet. Since this disease associated with massive shrinkage of the liver, the increase in both the KC numbers/g of liver and the SPECT/μCT signal could be largely due to a documented clustering of CD11b + immune cells [25] combined with hepatocyte necroptosis [32]. Supplementing the mice for 1 week with standard chow reverted the MCD phenotype such that there were no longer differences in any of the above mentioned investigated parameters, suggesting that these mice are tending towards recovery from disease.…”

Necroptosis: Mechanisms and Relevance to Disease

“…Thus, the Ripk3 −/− genotype, the intraperitoneal delivery of Ripk3 -targeting antisense oligonucleotides, or the administration of dabrafenib (an FDA-approved inhibitor of oncogenic BRAF that also suppresses RIPK3 kinase activity) provides considerable hepatoprotection in mouse models of acetaminophen toxicity (134, 170, 171), concanavalin A–driven hepatitis (134), ethanol and dietary intoxication (172, 173), and nonalcoholic steatohepatitis (NASH) (174, 175). Along similar lines, mice treated with Nec-1 exhibit an increased resistance to the hepatotoxic effects of acetaminophen as compared with control animals (134).…”