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Letter to the Editor| Volume 73, ISSUE 3, P699-701, September 2020

Intrahepatocytic necroptosis is dispensable for hepatocyte death in murine immune-mediated hepatitis

      Letter to the Editor:
      Hundreds of millions of people globally are affected by acute or chronic hepatitis, for which there are no effective treatments. Hepatocyte death is a key element in both initiation and progression of these liver diseases. Necroptosis emerged recently as one of the possible cell death programs involved in liver damage. This pathway is a regulated modality of necrosis that occurs in the presence of caspase inhibition. It involves RIP1/3 (receptor interacting protein) kinases and a final effector, the pseudokinase MLKL (mixed lineage kinase domain-like protein).
      • Petrie E.J.
      • Czabotar P.E.
      • Murphy J.M.
      The structural basis of necroptotic cell death signaling.
      Many studies have investigated the role of these necroptosis-associated proteins in different liver injury models, sometimes with conflicting results.
      • Dara L.
      • Liu Z.-X.
      • Kaplowitz N.
      Questions and controversies: the role of necroptosis in liver disease.
      Thus, the involvement of necroptosis in different liver diseases remains controversial and a hot topic in hepatology research.
      In 2016, Günther et al. reported that necroptosis mediates hepatocyte death in a murine model of immune-mediated hepatitis.
      • Günther C.
      • He G.-W.
      • Kremer A.E.
      • Murphy J.M.
      • Petrie E.J.
      • Amann K.
      • et al.
      The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis.
      Indeed, using mice with a total ablation of Mlkl (Mlkl−/− mice) challenged by Concanavalin A (ConA), a lectin triggering severe inflammation and T-cell mediated liver injury, they found that Mlkl−/− mice are completely protected from ConA-induced damage compare to wild-type mice.
      • Heymann F.
      • Hamesch K.
      • Weiskirchen R.
      • Tacke F.
      The concanavalin A model of acute hepatitis in mice.
      The authors concluded that the pseudokinase MLKL is an essential mediator of hepatocellular necrosis in ConA-induced hepatitis in mice.
      To deepen the understanding of the role of MLKL in ConA-induced hepatitis and particularly in the hepatocyte itself, we have generated a new mouse line with conditional ablation of Mlkl specifically in liver parenchymal cells (MlklLPC-KO mice). As expected, these MlklLPC-KO mice have reduced levels of MLKL in the whole liver and a total absence of MLKL in isolated hepatocytes, as shown by western blotting analyses (Fig. 1A). Importantly, intravenous treatment with ConA at a dose of 12 mg/kg for 6 h, 11 h and 16 h did not reveal any differences in levels of serum transaminases and degree of necrotic damage of the liver between MlklLPC-KO mice and Mlklfl/fl littermate controls (Fig. 1B and 1C). These results show that MlklLPC-KO mice are not protected from ConA-induced hepatitis demonstrating that hepatocyte-intrinsic MLKL is dispensable for ConA-induced hepatolysis in contrast to what has been advanced by Günther et al.
      • Günther C.
      • He G.-W.
      • Kremer A.E.
      • Murphy J.M.
      • Petrie E.J.
      • Amann K.
      • et al.
      The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis.
      Figure thumbnail gr1
      Fig.1MlklLPC-KO mice are not protected from ConA-induced hepatitis.
      Mice with conditional ablation of Mlkl in liver parenchymal cells (MlklLPC-KO mice) were generated by crossing Mlkl-floxed mice with Alfp-cre transgenic mice. (A) MLKL depletion in parenchymal liver cells is efficient. Western blot analyses were performed on whole liver or isolated hepatocyte protein extracts using anti-Mlkl antibody and anti-Hsc70 antibody as loading control. (B and C) Lack of MLKL in parenchymal cells does not confer protection against ConA-induced hepatitis. MlklLPC-KO mice and their littermate controls (Mlklfl/fl) were injected intravenously with vehicle (PBS, 1 mM CaCl2, 0.5 mM MnCl2) (NT: non-treated; 7 mice per genotype) or with 12 mg/kg of ConA and euthanized at 6 h, 11 h or 16 h post-injection (14, 7 or 8 mice per genotype, respectively). (B) Levels of serum alanine aminotransferase. Each dot and square represent an individual; error bars are expressed as mean ± SEM; each time point of ConA-treatment was compared to the NT related mouse strain and comparisons were made between the 2 mouse strains at each time point. Mean differences between experimental groups were assessed using the non-parametric Mann–Whitney U test. Significance is shown as follows: ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001; ns: non-significant. (C) Representative pictures of liver tissue sections analyzed by hematoxylin and eosin staining. Black bars represent 250 μm. ConA, Concanavalin A.
      The contradictory results obtained with the Mlkl−/− and the MlklLPC-KO mice, explained by the use of a constitute knockout model on the one hand and specific knockout model on the other hand, suggest that necroptosis occurring in other cell population(s) is involved in hepatolysis in response to ConA injection, in which immunity is essential to hepatocyte damage. Günther et al. excluded the involvement of immune cells by performing bone marrow graft experiments.
      • Günther C.
      • He G.-W.
      • Kremer A.E.
      • Murphy J.M.
      • Petrie E.J.
      • Amann K.
      • et al.
      The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis.
      However, further investigations studying the effect of ConA in mice lacking MLKL in other intrahepatic cell populations are needed to better understand the liver damage process. Liver sinusoidal endothelial cells are potentially interesting candidates to investigate, since necroptosis could be induced in endothelial cells.
      • Strilic B.
      • Yang L.
      • Albarrán-Juárez J.
      • Wachsmuth L.
      • Han K.
      • Müller U.
      • et al.
      Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis.
      Moreover, our new data show that other types of cell death than necroptosis can lead to hepatocyte death during ConA-induced hepatitis. Like Günther et al., we did not observe apoptotic cells following ConA injection either in Mlklfl/fl or in MlklLPC-KO mice (data not shown), in contrast to what we already observed in immune-mediated hepatitis in Ripk1LPC-KO mice.
      • Filliol A.
      • Piquet-Pellorce C.
      • Le Seyec J.
      • Farooq M.
      • Genet V.
      • Lucas-Clerc C.
      • et al.
      RIPK1 protects from TNF-α-mediated liver damage during hepatitis.
      ,
      • Filliol A.
      • Piquet-Pellorce C.
      • Raguénès-Nicol C.
      • Dion S.
      • Farooq M.
      • Lucas-Clerc C.
      • et al.
      RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis.
      Other cell death pathways must now be considered. Luan et al., in 2018, suggested that pyroptosis could be one of the cell death programs involved in ConA-induced hepatitis.
      • Luan J.
      • Zhang X.
      • Wang S.
      • Li Y.
      • Fan J.
      • Chen W.
      • et al.
      NOD-like receptor protein 3 inflammasome-dependent IL-1β accelerated ConA-induced hepatitis.
      We think that we have to reconsider the research on hepatocyte death, taking into account the fact that different pathways could act in synergy and compensate each other as addressed in the new concept of PAN-optosis.
      • Malireddi R.K.S.
      • Kesavardhana S.
      • Kanneganti T.-D.
      ZBP1 and TAK1: master regulators of NLRP3 inflammasome/pyroptosis, apoptosis, and necroptosis (PAN-optosis).
      ,
      • Schwarzer R.
      • Laurien L.
      • Pasparakis M.
      New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8.
      To conclude, our data demonstrate that intrahepatocytic MLKL and then necroptosis are dispensable for hepatocyte death in this model of immune-mediated hepatitis. Moreover, our results open new insights on the involvement of other intrahepatic cell populations in hepatocellular necrosis in this context. Thus, the design of therapeutic approaches integrating prevention of liver tissue cell death will have to consider the disparity of induced death types and their interconnections.

      Financial support

      This work was supported by the “Contrat de Plan Etat-Région” (CPER) grant named “Infectio”; the “Ligue Contre le Cancer, Comités du Grand Ouest”; the Biology and Health Federative Research Structure of Rennes (Biosit, UMS CNRS 3480 / US INSERM 018); the “Agence Nationale de la Recherche” (ANR Labcom Oncotrial) and the “Fondation pour la Recherche Médicale” (FRM # DEQ20180339216). A.H. was supported by a postdoctoral fellowship from the “Fondation pour la Recherche Médicale”.

      Authors' contributions

      Conceptualization, A.H., C.P.P., M.T.D.B., M.S. and J.L.S.; Methodology, A.H., C.P.P., M.T.D.B., M.S. and J.L.S.; Validation, A.H., C.P.P., M.T.D.B., M.S. and J.L.S.; Formal Analysis, A.H.; Investigation, A.H., C.P.P. and J.L.S.; Data Curation, A.H.; Writing – Original Draft Preparation, A.H.; Writing – Review & Editing, M.S. and J.L.S.; Visualization, A.H.; Supervision, M.S. and J.L.S.; Project Administration, A.H., C.P.P., M.T.D.B., M.S. and J.L.S.; Funding Acquisition, M.T.D.B., M.S. and J.L.S.

      Conflict of interest

      The authors have no conflicts of interest to declare.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgment

      We would like to thank Prof. Warren S. Alexander (The Walter and Eliza Hall Institute of Medical Research, Australia) for the gift of the 592MLKL/Flp mice used to establish the MlklLPC-KO mouse line. We also extend our thanks to Mélanie Simoes-Eugénio, Christelle Devisme, Ghania H. Kara-Ali, Huma Hameed and Abderrahman Chafik for their technical assistance and to Dr. Muhammad Farooq for his fruitful scientific discussions. We acknowledge the “Laboratoire de Biochimie-Toxicologie” from the “CHU de Rennes” for transaminase measurements. For histological analysis and animal house facilities, we would like to thank the dedicated platforms (i.e. H2P2 and animal house platforms) of SFR Biosit – UMS 3480, US_S 018, France.

      Supplementary data

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