Loss Of MLKL Ameliorates Liver Fibrosis By Inhibiting Hepatocyte Necroptosis And Hepatic Stellate Cell Activation

Background:Liver fibrosis affects millions of people worldwide without an effective treatment.Multiple forms of cell death,including necrosis,apoptosis,and necroptosis,have been reported to co-exist,in liver diseases.Mixed lineage kinase domain-like protein（MLKL）is the terminal effector in necroptosis pathway.Although necroptosis has been reported to play an important role in many diseases,the function of MLKL in liver fibrosis has yet to be unraveled.Here we sought to elucidate the function of MLKL in liver fibrosis and underlying mechanism.Methods:Human fibrotic liver tissues were examined to estimate correlations between MLKL and liver fibrosis.To further investigate the role of MLKL in liver fibrosis,we used different mouse fibrosis models:CCl₄ treatment and bile duct ligation（BDL）in wild type（WT）and Mlkl knockout(Mlkl^-/-)mice.Hepatocytes,macrophages,and hepatic stellate cells（HSCs）were isolated from WT and Mlkl^-/-mice to identify potential mechanisms of action.We explored the restoration of liver injury by AAV8-mediated specific knockdown of Mlkl in hepatocytes in CCl₄ induced mouse model.Results:Here we reported that MLKL level is positively correlated with a number of fibrotic markers in liver samples from both patients with liver fibrosis and animal models.We then generated Mlkl^-/-mice with the CRISPR-Cas9 system.We first studied immune-related events in WT and Mlkl^-/-mice treated with CCl₄.Fluorescence-activated cell sorting（FACS）analysis showed that after acute CCl₄ treatment,the numbers of total infiltrating leucocytes,neutrophils,monocyte-derived macrophages（Mo MFs）,pro-inflammatory macrophages,and anti-inflammatory macrophages were significantly lower in Mlkl^-/-mice versus WT mice.After acute CCl₄ treatment,the Mlkl^-/-mice showed less hepatocellular injury.Chronic damage of hepatocytes leads to liver fibrosis.Mlkl deletion in mice significantly reduces clinical symptoms of CCl₄-and BDL-induced liver injury and fibrosis.Liver fibrosis is a multicellular response to liver injury.We found that MLKL is ubiquitously expressed in isolated primary liver parenchyma cells（hepatocytes）,CD11b⁺cells,and hepatic stellate cells（HSCs）.Macrophage is one of the most abundant cell types in CD11b⁺cells.Previous studies have shown that liver injury triggers monocytes migration from bone marrow to the site of injury,then monocytes polarize into either pro-inflammatory or anti-inflammatory macrophages.To examine the role of MLKL in macrophage polarization,WT and Mlkl^-/-bone marrow-derived macrophages（BMDMs,M0）were stimulated to induce classical（pro-inflammatory,M1）or alternative（anti-inflammatory,pro-healing,M2）polarization.Our data indicate that knock out of Mlkl does not seem to affect macrophage polarization and function.MLKL was widely acknowledged to regulate cell necroptosis through phosphorylation and oligomerization at the plasma membrane.Hepatotoxicity is the initial cause of liver injury and hepatic fibrosis.We hypothesized that Mlkl^-/--hepatocytes may exhibit different responses to chemical-induced injury.Mlkl^-/--and WT-hepatocytes were exposed to CCl₄ for 24 hours to induce cell damage.WT-hepatocytes displayed obvious cell death,while Mlkl^-/--hepatocytes showed significantly less damage.HSC_S activation is a key step leading to liver fibrosis.Previous reports have revealed that HSCs cultured in vitro will become activated with morphology changes.After being cultured in vitro for 5 days,HSCs underwent a characteristic fibrotic phenotype change to differentiate into myofibroblasts and became activated to express fibrotic markersα-SMA and Vimentin.In vitro culture for5 days led to increased protein levels of MLKL andα-SMA,more interestingly,although the total Smad2/3 level did not change,the p-Smad2/3 was significantly increased.Deletion of Mlkl reduced p-Smad2/3 andα-SMA levels,indicating MLKL may promote the activation of TGF-β/Smad pathway.Considering hepatocyte damage is the first step in liver fibrosis,we generated adeno-associated virus（AAV）type 8 carrying Mlkl sh RNA（AAV8-sh Mlkl）or scramble sh RNA（AAV8-Scramble）under the control of the liver-specific thyroid hormone-binding globulin（TBG）promoter.The AAV also contained a GFP sequence controlled by a TBG promoter.Eight weeks after tail vein injection of AAV8-sh Mlkl or AAV8-scramble,GFP signal could be detected throughout the liver but not other tissues.AAV8-sh Mlkl was given both in prophylactic and therapeutic ways,and 8weeks after CCl₄ treatment,mice were sacrificed.As expected,knockdown of Mlkl with AAV-sh RNA in hepatocytes markedly ameliorates CCl₄-induced hepatocyte damage,liver inflammation,and hepatic fibrosis in both preventative and therapeutic ways.Conclusion:MLKL may participate in liver fibrosis via two mechanisms:1.MLKL-mediated hepatocyte necroptosis initiates the immune response,which in turn activates HSCs and leads to fibrosis;2.MLKL may directly participate in HSCs activation by regulating the TGFβ/Smad pathway.Deletion or knockdown of Mlkl protects hepatocytes from environmental insults and reduces HSCs activation,thus reducing liver fibrosis in animal models.Our results show that MLKL-mediated signalings play an important role in liver damage and fibrosis,and targeting MLKL might be an effective way to treat liver fibrosis.Novelties:The role of MLKL in hepatic fibrosis has not been reported,and we found that MLKL knockout can reduce liver inflammation and liver fibrosis in mice induced by carbon tetrachloride and bile duct ligation.In-depth mechanistic studies have found that knockout of MLKL can alleviate hepatocyte injury and activation of hepatic stellate cells.Based on the results above,we used gene therapy to target MLKL to treat liver fibrosis,and the treatment achieved a good effect.At present,there are no anti-fibrosis drugs approved for marketing,and our research results provide new targets and methods for the clinical treatment of liver fibrosis.