Necroptosis in Nonalcoholic Steatohepatitis

Dear Editor:

One fundamental step along the sequence from benign liver steatosis toward progressive steatohepatitis is the occurrence of hepatocyte cell death, currently classified as apoptosis. However, necroptosis, governed by the kinase receptor-interacting protein 3 (RIP3), has emerged as an alternative programmed cell-death pathway.¹ Petra Hirsova and Gregory J. Gores, who have made many important contributions to the field of cell death in the liver, published an interesting article in Cellular and Molecular Gastroenterology and Hepatology² in which they provided a comprehensive overview on the role of programmed cell death pathways in NASH. We would like to take the chance to discuss their statement that “it is unlikely that necroptosis contributes to liver injury and inflammation in NASH” because we believe that it might be too early to exclude this important new cell death pathway from further exploration as a potential target in NASH patients.

The ideal model to study NASH in mice is a matter of intensive debate. We studied the role of necroptosis in NASH by using the model of methionine-choline-deficient (MCD) diet³ because this model was used as the experimental basis for the investigation of the role of apoptosis in NASH, leading even to clinical studies in NASH patients.⁴ We could demonstrate that mice deficient for Rip3 show a clear reduction in MCD-diet-induced liver injury, inflammation, and liver fibrosis compared with wild-type mice.³ We do not know of any other published study challenging this result in the MCD model. In addition, this finding is consistent with a previous study showing similar protective effects of Rip3-deletion in a mouse model of alcoholic steatohepatitis.⁵ Together, these findings suggest that necroptosis might play an important role in the processes linking NASH with fibrosis.

The authors mentioned inconsistent findings in our study and another study on the role of caspase-8 in the MCD model.⁶ Caspase-8 represents an important upstream caspase, initiating the apoptosis process downstream of death receptors such as FAS or tumor necrosis factor (TNF).⁷ A newly discovered function of caspase-8 is the suppression of RIP3-dependent necroptosis through cleavage of RIP3 and CYLD.1, 4, 8 Consequently, efficient deletion of caspase-8 sensitizes cells to necroptosis through up-regulation of RIP3 expression. This was shown in many different experimental settings and was the case in our studies in which we used a well-established caspase-8 floxed line⁹ and the alfp-cre line mediating robust and efficient deletion of floxed genes in parenchymal livers.³ Efficient caspase-8-deletion triggered spontaneous RIP3 overexpression (as seen previously in many other organs such as gut or skin¹⁰), mild liver injury, and inflammation without any stimulation.¹¹ Upon MCD diet feeding, caspase-8-deficient mice developed massive RIP3 elevation in hepatocytes and consequently strong liver injury and fibrosis,³ providing evidence that necroptosis is the main cell death pathway in MCD-diet-induced NASH.

The other important study on caspase-8 in the MCD model cited by the authors used the albumin-cre line for deletion of Caspase-8,⁶ which did not lead to overexpression of RIP3.³ Therefore, and for the fact that RIP3 knockout mice were not used, the study by Hatting et al⁶ is not suitable to speculate on the role of necroptosis in the MCD model. The authors of the present review article mention that they have preliminary results on a protective role of caspase-8 in a nutritional model of hepatic steatosis (not MCD diet).² However, in our work, caspase-8 deletion did not have any effect in a purely nutritional NAFLD model in mice (data not shown). A careful comparison of methodological differences might bring further light into the controversial function of caspase-8 in different NASH models.

Finally, in our study, we had first shown stainings for RIP3 in liver samples of NASH patients, exhibiting a typical granular staining pattern in hepatocytes surrounding areas of high lipid storage.³ Of note, Western blot analysis and stainings for RIP3 showed a much clearer result than experiments assessing the levels of the cleaved form of the executioner caspase-3 in human NASH livers,³ suggesting that necroptosis might be at least equally abundant in human NASH as apoptosis. It should be noted that RIP3 overexpression cannot be taken as equal with execution of necroptosis. However, we can now demonstrate that the phosphorylated form of the protein mixed-lineage-like kinase (p-MLKL), which is considered to be the executional kinase of necroptosis,¹² can be detected in human NASH livers (Figure 1) and that it strikingly resembles the granular staining patterns of RIP3 in NASH patients.³

Figure 1.

Necroptosis is activated in human nonalcoholic steatohepatitis (NASH) samples. Representative immunohistochemical staining for phospho-MLKL on livers from different controls (Ctl) and NASH patients. Note the specific granular cytoplasmatic staining. See Gautheron et al³ for the specifics of the NASH cohort as well as the technical and ethical aspects.

Independently of experimental questions in mouse models, we believe that the activation of a known cell-death pathway in hepatocytes in human NASH patients justifies further exploration as a pharmacologic target, potentially as a complementary target to mediators of apoptosis.