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

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.


Mouse handling and tissue collection
Animals were housed in 2 per cage for ethanol feeding or 3 per cage for FFC feeding in standard microisolator cages and maintained in a temperature regulated facility with a 12h:12h light/dark cycle. In ethanol models, mice were weighed twice a week and food intake per cage measured daily, with Nylabones provided for environmental enrichment. In high-fat models, mice were weighed and food intake per cage measured weekly, and water was changed twice a week.
At the end of the feeding protocols, mice were anesthetized, livers perfused with saline and then portions of liver were flash frozen in liquid nitrogen and stored at -80°C, fixed in 10% formalin or frozen in optimal cutting temperature washed with RPMI 1640/10% FBS and centrifuged at 300 g for 7 min. To enrich the NPC fraction, cells were centrifuged at 50 g for 10 min without brake in a 30% Percoll gradient. The cell layer was removed carefully and cells collected by centrifugation at 300 g for 10 min. Cells were washed with PBS and centrifuged at 300 g for 10 min and prepared for western blots.

AML-12 hepatocyte Cell Culture
The murine hepatocyte cell line, alpha mouse liver 12 (AML-12), was purchased through the American Tissue Culture Collection (ATCC) and grown in

Preparation of bone marrow-derived macrophages (BMDMs)
Primary cultures of BMDMs were generated from tibia and fibula of WT and Mlkl -/mice. BM cells were cultured with DMEM supplemented with 10% FBS and 20 ng/mL macrophage colony stimulating factor (M-CSF) at 37°C/5% CO2. Fresh culture medium was replaced on day 3, 6 and 9. On day 10, adherent cells were seeded and then treated with LPS (10 ng/mL). 24 h later, RNA was isolated to measure cytokine/chemokine expression.

Biochemical assays
Plasma samples were assayed for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using commercially available enzymatic assay kits (Sekisui Diagnostics, Seacacus, NJ, USA) following the manufacturer's instructions. Total hepatic triglycerides were assayed using the Triglyceride Reagent Kit from Pointe Scientific Inc. (Lincoln Park, MI, USA).

Histopathology and immunohistochemistry
Formalin-fixed liver tissues were paraffin-embedded, sectioned and stained with hematoxylin and eosin for histological analysis. Formalin-fixed paraffin-  Table 4.

Western blot analysis
Frozen liver tissues (50-80mg) from mice were homogenized in lysis buffer (10 ml/ g tissue) and protein concentration measured using the DC Lowry assay 7 .
Samples were prepared in Laemmli buffer and denatured at 37° for 15 min for RIP1, RIP3 and MLKL; samples for all other proteins were denatured by boiling for 5 min. Proteins were separated on 8 to 10% gels by SDS-PAGE and liver lysates were used for Western blot analysis against antibodies described in Supplemental Table 5. Western blot analysis was performed using enhanced chemiluminescence for signal detection. Signal intensities were quantified by densitometry using Image J software (NIH, Bethesda, MD, USA).

Measurement of RIP1, RIP3 and MLKL in human plasma
Human plasma samples were collected and stored at -80℃. Plasma RIP1, RIP3 and MLKL concentrations were quantified by ELISA using the following kits:  Values represent means ±SEM *Data are from two independent feeding trial, n = 8-12 animals per group. † Data are from one feeding trial, n = 4-6 animals per group.