Pyroptosis, A novel player for alcoholic hepatitis?
Authors: Shaogui Wang, Hua Wang, Wen‐Xing Ding
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. SEE ARTICLE ON PAGE 1737 Supported by the National Institutes of Health (U01 AA024733, R01 AA020518, R01 DK102142, and 8 P20 GM103549‐07). Alcoholic hepatitis (AH) is a clinical syndrome of jaundice and liver failure that develops over decades of excessive alcohol consumption, which represents 0.2% of all hospital admissions in the United States.1 Patients with a severe episode of AH have a potential for 30%‐40% 1‐month mortality. Recommended treatments for AH include corticosteroids, nutritional supplements, and pentoxifylline. However, these treatments provide only limited survival benefit.2 One of the main obstacles to developing effective therapeutic drugs to treat AH is the lack of understanding of the molecular mechanisms involved in the pathogenesis of AH. Difficulties in reproducing the histological and clinical features of AH in animal models represent a major limitation in advancing our knowledge of the pathogenesis of AH. Recently, Dr. Hidekazu Tsukamoto's group developed a hybrid feeding mouse model, which combined ad libitum Western diet feeding with an intragastric ethanol diet, two risk factors for AH. This hybrid feeding mouse model can lead to severe chronic alcoholic steatohepatitis in mice. When a weekly binge was added to this model without changing the total alcohol intake, key histological and clinical changes of AH were reproduced.3 Hepatocyte death after alcohol consumption is known to contribute to the pathogenesis of alcoholic liver disease. Several types of cell death, including apoptosis, necrosis, and necroptosis, have been observed in cultured hepatocytes and livers of rodents exposed to alcohol as well as in livers of alcoholic liver disease patients.4 In this issue of hepatology, Khanova et al. provide perspectives of a novel cell death named "pyroptosis" in the pathogenesis of AH.6 Pyroptosis is a type of programmed necrosis that was long thought to depend on the activation of caspase‐1, a proinflammatory caspase that is activated by inflammasome complexes.7 Pyroptosis has been considered an important innate immune response to certain bacterial insults. Pyroptosis is associated with cell swelling and disruption of the plasma membrane due to the formation of pores on the plasma membrane. In addition to caspase‐1, caspase‐11 (mouse) and caspase‐4/5 (human) can sense intracellular lipopolysaccharide to trigger pyroptosis.7 Mechanistically, activated caspase‐11 or caspase‐4/5 cleaves gasdermin D (GSDMD) within its linking loop to release its autoinhibition on its GSDMD‐N domain. The cleaved GSDMD‐N domain binds to phosphoinositides on the plasma membrane and lyses it to cause cell death.7 GSDMD is expressed in many other tissues and cell types, but it is predominantly expressed in the gastrointestinal tract and immune cells.8 GSDMD belongs to the gasdermin family of proteins, which has more than five different members in humans. All of them share a similar structure, with the most conserved gasdermin‐N domain and gasdermin‐C domain. All of the gasdermin proteins have similar pore‐forming activity, and thus, pyroptosis is now considered a gasdermin‐mediated program necrosis.7 In searching for the molecular mediators for the pathogenesis AH, Khanova et al. first performed a global unbiased RNA‐sequencing and proteomic analyses in their newly established hybrid feeding mouse AH model and AH patients.6 Through ontology and pathway analysis, a group of genes involved in the pathway activated by bacterial infection were consistently identified from the mouse AH model and human AH liver samples. Immunoblot and quantitative PCR analyses further confirmed the up‐regulation of caspase‐11 and GSDMD in the mouse AH model. Moreover, both the cleaved form and activities of caspase‐11 were significantly elevated in AH mouse livers but not in chronic alcoholic steatohepatitis mouse livers. Surprisingly, Khanova et al found that caspase‐1, the key player in the canonical inflammasome pathway, did not change in this AH mouse model. Consistent with the lack of caspase‐1 activation, serum interleukin 1β (IL‐1β) levels also tended to be lower in AH mice. Khanova et al. further detected the mature GSDMD forms from isolated hepatocytes and hepatic macrophages of the AH mouse model, suggesting activation of GSDMD and a possible involvement of pyroptosis in both macrophages and hepatocytes in the pathogenesis of AH in mice. More importantly, Khanova et al. found that levels of cleaved caspase‐4 and membrane translocation of mature GSDMD were also increased in human AH liver samples but not in healthy human livers.6 These data strongly support an association of the caspase‐11/4–GSDMD pathway and pyroptosis in mouse and human AH. To determine the causal role of caspase‐11/4‐GSDMD‐mediated pyroptosis in the pathogenesis of AH, Khanova et al. elegantly used two genetic approaches to modulate this pathway. In the first approach, Khanova et al. compared the liver pathogenesis of the caspase‐11 knockout (KO) mice with matched wild‐type mice in the AH model. They found that activation of GSDMD, rate of hepatocellular cell death, and liver bacterial load in experimental AH was blunted in capase‐11 KO mice compared with wild‐type mice. However, no significant changes were found in the histological grading for steatosis and inflammation as well as serum alanine aminotransferase and aspartate aminotransferase levels between capase‐11 KO mice and matched wild‐type mice. It should be noted that caspase‐11 KO mice are also defective for caspase‐1. Because Khanova et al. found that caspase‐1 was not active in AH mice, the phenotypes observed in caspase‐11 KO mice might be due primarily to the lack of caspase‐11. In a second approach, Khanova et al. overexpressed the N‐terminal active form of GSDMD into the mouse hepatocytes in vivo using an adeno‐associated virus (AAV) system followed by the AH model. GSDMD was driven by a transthyretin (Ttr) promoter, and thus, GSDMD was presumably only expressed in hepatocytes but not in other hepatic nonparenchymal cells. Compared to control mice that received the AAV‐Ttr‐enhanced green fluorescent protein, mice with overexpression of GSDMD in hepatocytes had significantly increased hepatocyte death and hepatic polymorphonuclear neutrophil infiltration. Similar to the caspase‐11 KO mice, liver histology such as parameters of macrovesicular or microvesicular steatosis were not different between mice that received AAV‐Ttr‐enhanced green fluorescent protein and AAV‐Ttr‐GSDMD after the AH model. Moreover, serum alanine aminotransferase and aspartate aminotransferase levels were only slightly elevated in mice that received AAV‐Ttr‐GSDMD compared to mice that received AAV‐Ttr‐enhanced green fluorescent protein.6 Together, these data from the genetic modulation of caspase‐11 and GSDMD support a role of caspase‐11‐GSDMD in contributing to the death of hepatocytes and the infiltration of polymorphonuclear neutrophils, which is dispensable for hepatic steatosis during the pathogenesis of AH. In addition to the key finding of activation of the caspase‐11/4‐GSDMD pathway in AH, Khanova et al. found that elevated levels of serum IL‐18 were associated with increased liver bacterial load in the mouse AH model.6 IL‐18 is a proinflammatory cytokine that belongs to the IL‐1 cytokine family. Although IL‐18 was initially identified as interferon‐γ‐inducing factor, it also acts as an antibacterial cytokine and plays a pivotal role in defense against bacterial pathogens.9 Indeed, Khanova et al. found that IL‐18 KO mice displayed increased liver bacterial load and GSDMD‐mediated pyroptosis of hepatocytes in the AH mouse model.6 These results are consistent with the hypothesis that endotoxin induces the noncanonical inflammasome caspase11/4–GSDMD–pyroptosis pathway in AH. In conclusion, this study highlights a new role of pyroptosis in AH. While this study has largely expanded our knowledge of the pathogenesis of alcoholic liver disease, many questions remain unanswered. As discussed above, GSDMD mainly expresses in immune cells and the gastrointestinal tract, and it is likely that its expression levels in hepatocytes are lower. Therefore, it is possible that the pyroptotic death of macrophages may contribute more to the pathogenesis of AH than the pyroptotic death of hepatocytes. Future work to use tissue‐specific GSDMD KO mice in immune cells versus hepatocytes will be helpful in defining the role of pyroptosis in immune cells versus hepatocytes in the pathogenesis of AH. Finally, development of small molecule GSDMD inhibitors is urgently needed, perhaps in combination with inhibitors for other modes of cell death, to further explore the translational potential of targeting cell death for treating AH. Author names in bold designate shared co‐first authorship.