| The intricate balance between cell death and pro-survival pathways is critical for regulating liver injury and inflammation during progression of ALD or NAFL/NASH. Previous work identified a critical role for rip3, a kinase regulating necroptotic cell death, in mediating chronic ethanol-induced, but not high fat diet-induced, liver injury in mice. In humans, RIP3 expression is low in healthy livers, but is increased in livers of patients with ALD and NAFL/NASH. However, very little data are available on direct comparisons of expression of the RIP1-RIP3-MLKL molecular machinery between patients with different etiologies of liver disease. Based on these data, here we investigated two questions: 1) is there a differential role for mlkl, the down-stream effector of RIP3 in the necroptotic pathway, in murine models of AH and NASH and 2) can RIP1-RIP3-MLKL be used as biomarkers to distinguish AH from NASH in patients. Mlkl-/- mice and their wild-type littermates were exposed to Gao-binge (acute on chronic) ethanol or high fat fructose and cholesterol diets (FFC). Liver tissue and plasma from patients with AH and NASH, as well as healthy controls (HC) were used for Western blot and ELISA.Despite the protective effect of rip3-deficiency in murine models of chronic ethanol exposure, mlkl-deficient mice were not protected from Gao binge-induced liver injury. In contrast, mlkl-/-, but not rip3-/-, mice were protected from FFC-induced liver injury. Western blot studies of liver samples from patients with NASH, AH, and healthy controls revealed distinct differences in expression patterns of phospho-RIP1, RIP1, RIP3, phospho-MLKL, and MLKL between HC, AH and NASH patients. pMLKL was higher in NASH vs AH and HC while phospho RIP1 was higher in AH vs NASH and HC. RIP1 and RIP3 concentration, assessed by ELISA in plasma, was also different between AH and NASH, with RIP3 increased in AH vs NASH and HC and RIP1 increased in NASH vs AH and HC. No differences in circulating MLKL were identified between AH, NASH and healthy controls.Overall, the data from murine models indicate important non-canonical functions of RIP3 and MLKL differentially contribute to ethanol- and FFC diet-induced injury. Differential expression of proteins in the RIP1-RIP3-MLKL signaling pathway in liver and plasma from patients suggest that these differences might be leveraged to develop predictive models to distinguish AH and NASH. |
