The end of RIPK1‐RIPK3‐MLKL–mediated necroptosis in acetaminophen‐induced hepatotoxicity?

Potential conflict of interest: Nothing to report. To the Editor: We read with great interest the recently published article by Dara et al.1 in which they provided novel, mechanistic insights regarding the emerging role of receptor‐interacting protein kinase (RIPK)‐mediated necroptosis in acetaminophen (APAP)‐induced liver injury. Necroptosis is regulated by the RIPK1‐RIPK3‐MLKL (mixed lineage kinase domain‐like protein) signaling cascade and has been implicated in APAP‐induced liver injury.2 Dara et al. found that APAP‐induced necrosis was dependent on RIPK1, but not RIPK3, or MLKL. This finding suggests that necroptosis is not an important mediator of APAP‐induced liver injury. One major argument by Dara et al. for the lack of RIPK3 involvement in APAP‐induced cell death was that hepatocytes do not express RIPK3; however, their experimental design may have led to the failure to detect RIPK3 in primary mouse hepatocytes (PMHs). PMHs dedifferentiate rapidly in culture and thus quickly lose many hepatic proteins, such as macromolecule transporters, and drug‐metabolizing enzymes. It was unclear how long the PMHs were cultured by Dara et al., before the cells were harvested, but we found levels of RIPK3 protein in PMHs markedly decreased after 6 hours in culture. This may be the reason that Dara et al. failed to detect RIPK3 in PMHs. We used an RIPK3 antibody from ProSci, which was validated using RIPK3‐deficient liver tissues, and mouse embryonic fibroblasts.2 Although Dara et al. used the same antibody as we did, it is unclear why they failed to detect RIPK3 in the liver. Another finding from the study by Dara et al. was that RIPK1 acted upstream of c‐Jun N‐terminal kinase (JNK). This brings up the interesting question of whether RIPK1 directly interacts with and phosphorylates JNK, or is JNK activation a secondary effect of RIPK1 knockdown. Studies performed by our group and others, including Dara et al. themselves, indicate that the key necroptosis components (RIPK1, RIPK3, and MLKL) are primarily expressed in spleen and thymus, and not in the liver.4 This could mean that RIPK‐MLKL–mediated necroptosis may play a major role in regulating innate immunity. The innate immune system has been implicated in regulating the progression of APAP‐induced liver injury. The authors may like to comment on the possibility that RIPK1 knockdown in nonparenchymal cells, such as Kupffer cells and infiltrating neutrophils, also plays a protective role against APAP‐induced liver injury.