Differential role of MLKL in alcohol-associated and non–alcohol-associated fatty liver diseases in mice and humans

Abstract

Hepatocellular death contributes to progression of alcohol–associated (ALD-associated) and non–alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain–like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl –/– mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.

The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

“…These findings offer a tantalising clue that necroptosis may contribute to this disease, depending on the trigger. In contrast to NAFLD, MLKL does not appear to play a statistically significant role in acute or chronic alcoholic liver disease [58].…”

The role of MLKL in Hepatic Ischemia-Reperfusion Injury of Alcoholic Steatotic Livers

“…The difference could be caused by different steatotic models utilized in the studies, as well as the lipid composition and the extent of inflammation in NAFLD vs ALD as discussed above. Notably, a recent study by Miyata et al found that MLKL played a differential role in ALD and NAFLD [14]. Although MLKL protein level and plasma membrane translocation were both significantly increased in HFF-fed mouse livers, there was no change in MLKL protein level or plasma membrane translocation in chronic plus binge ethanol mouse livers.…”

“…However, how fatty liver increases IR injury remains unclear, which has halted the use of fatty livers in liver transplantation. Accumulating evidence suggests that necroptosis is critical in the pathogenesis of inflammatory liver diseases including NAFLD, ALD, and hepatic IR injury [10][11][12][13][14][15]. Necroptosis is a form of regulated cell death, which is mediated by three key molecules: receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL) [16,17].…”

Roles of necroptosis in alcoholic liver disease and hepatic pathogenesis

“…RIPK3, the key molecule of necroptosis signalling, was induced in murine models after ethanol exposure 3,50–52 . The biopsy samples from ALD and alcoholic liver cirrhosis patients showed increased RIPK3 and p‐MLKL expression, but not RIPK1, implying the important role of necroptosis in the occurrence and development of ALD 3,51,53–55 …”

“…Compared with WT mice, the expression of inflammatory cytokines after Gao‐binge ethanol feeding was decreased in MLKL −/− mice, whereas the expression of chemokines was independent of MLKL genotype, as detected by chemokine (C‐X‐C motif) ligand 1 (Cxcl1) and Cxcl2 expression. Similarly, Gao‐binge ethanol–induced neutrophil accumulation was not ameliorated by MLKL knockout 54 . In addition, Gao‐binge feeding–stimulated cytochrome P450 2E1 (CYP2E1) induction and endoplasmic reticulum (ER) stress were not affected by MLKL deletion, indicating that MLKL is not critical for ethanol‐induced liver injury.…”