Ramasamy Selvarani

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl^−/− mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl^+/− mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl^−/− and Mlkl^+/− mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase ( Sod1 ^−/− or Sod1KO mice) develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from fatty liver disease to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Because liver inflammation is the main mechanism that drives the disease progression in chronic liver disease (CLD) and because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and progression to fibrosis and HCC in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to WT mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1β, and Ccl2 that are associated with human NASH and HCC, were significantly increased. Markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, significantly reduced necroptosis, pro-inflammatory cytokines, fibrosis markers and STAT3 activation. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis and HCC progression in Sod1KO mice, a mouse model of accelerated aging and progressive HCC development. These findings suggest that necroptosis might be a target for treating NASH and HCC.