Hepatocyte Autophagy: Maintaining a Toxic‐Free Environment
Authors: Heather Francis, Nan Wu, Gianfranco Alpini, Fanyin Meng
Journal: Hepatology (2020)

Potential conflict of interest: Nothing to report. See Article on Page 596 Autophagy is a profoundly preserved homeostatic process for the sequestration and degradation of cytosolic macromolecules, damaged organelles, and a few pathogens. It is a catabolic lysosomal process that degrades cytoplasmic materials including misfolded proteins and damaged organelles as a cellular response and survival mechanism in reaction to an assortment of stress conditions.(1) Controlled by the products of autophagy‐related genes (Atg), autophagy occurs under normal physiological conditions but is regulated by several factors, including cytokines such as interferon‐gamma, tumor necrosis factor‐alpha (TNF‐α; up‐regulation), as well as interleukin‐4 (IL‐4) and IL‐13 (down‐regulation). Inhibition of autophagy in macrophages by knockdown of Atg7 or Atg16L1 promotes IL‐1β secretion in response to lipopolysaccharide (LPS).(2) Three types of autophagy have been described (Fig. 1): macroautophagy, microautophagy, and chaperone‐mediated autophagy. In this editorial we will discuss macroautophagy and its role within liver homeostasis. To streamline the editorial, the term "autophagy" will be used throughout to allude to "macroautophagy."Fig. 1: Interactions between hepatocyte autophagy and inflammatory cytokines. Interactions between hepatocyte autophagy and inflammatory cytokines such as TNF and IL‐1β are mainly through free fatty acid and reactive oxygen species through ATP‐dependent mechanisms. Three types of autophagy have been illustrated at the bottom. Abbreviations: LC3, microtubule‐associated protein 1 light chain; LAMP2A, lysosomal‐associated membrane protein 2A; FFA, free fatty acid; HSC90, heat shock cognate 90‐kDa protein; ROS, reactive oxygen species.Within the liver, hepatocyte basal autophagy is necessary to preserve liver homeostasis for proteins, lipids, and organelles; and dysregulation of basal hepatocyte autophagy leads to liver damage, inflammation, fibrosis, and tumorigenesis.(3) Furthermore, hepatocyte autophagy protects against both acute and chronic liver injuries. Silencing of autophagy genes in hepatocytes and hepatic nonparenchymal cells, such as stellate cells and Kupffer cells, induces different phenotypic changes. Autophagy‐deficient mice made by crossing mice with a floxed autophagy gene to albumin‐cyclization recombination (Alb‐Cre) transgenic mice have depletion of the floxed autophagy gene not only in hepatocytes but also in cholangiocytes. However, by crossing the same floxed mice to transgenic mice expressing a modified tamoxifen‐inducible Alb‐Cre promoter (Alb‐CreERT2), one can induce deletion of the floxed autophagy gene in adult mice so that the impact is limited to hepatocytes. Over time these mice develop a complex pathology including hepatomegaly, injury, inflammation, ductular reaction, fibrosis, and tumorigenesis.(4) The phenotype has been largely replicated in Atg7ΔHep‐ERT2 mice, suggesting that autophagy deficiency in hepatocytes is the main driving force for dysregulation. In this manner, the liver phenotypes caused by autophagy deficiency in parenchymal cells mimic those seen in clinically common unremitting liver illnesses caused by other etiologies, such as hepatitis virus, alcohol consumption, or a high‐fat/high‐cholesterol diet. In this way, it is conceivable that these pathologies share comparable or common molecular mechanisms despite the fact that interesting signaling components may be required to trigger these inflammatory and apoptotic changes in reaction to particular etiologies. Understanding the mechanisms of how autophagy deficiency alters liver homeostasis may give rise to important information regarding the treatment of other chronic liver diseases. In the study by Shen et al., the authors evaluated the effect of decreased autophagy on hepatocyte death from TNF and IL‐1ß.(5) The in vitro studies show that decreased autophagy sensitized alpha mouse liver 12 (AML12) hepatocytes to TNF‐dependent IL‐1ß toxicity. Additionally, cellular death occurred through cathepsin‐dependent necrosis triggered by impaired energy homeostasis. Cathepsin B (Cat B) is released from lysosomes during TNF cytotoxic signaling in hepatocytes and contributes to cell death. It has been suggested that targeting sphingomyelinases and/or ceramidases responsible for sphingosine generation, inhibiting Cat B activity, and using lysosomal stabilizing agents are all potential therapeutic maneuvers to inhibit TNF‐mediated hepatocyte apoptosis.(6) However, little is known about the function of hepatocyte autophagy in the liver's response to inflammatory cytokines. The studies of TNF‐dependent liver injury from the hepatotoxin galactosamine have demonstrated that autophagy protects against hepatocyte injury and cell death from TNF, which suggested that autophagy may modulate hepatocyte injury and death from the TNF cytotoxicity.(7) Communication between the autophagic and apoptotic pathways has been shown at multiple levels. Although autophagy can mediate cell death under certain conditions, this pathway is mainly considered to be an antideath, prosurvival mechanism.(8) Production of TNF is regulated by a number of signaling pathways, including nuclear factor kappa B, Akt, and the mitogen‐activated protein kinase c‐Jun N‐terminal kinase. Previous studies from the same research team have demonstrated that mice with a hepatocyte‐specific knockout of Atg7‐dependent autophagy are sensitized to LPS‐induced liver injury and hepatocyte death. Liver injury was associated with altered Akt signaling pathway, and primary hepatocytes had impaired Akt activation in response to a number of stimuli, including TNF. Inhibition of Akt activation in response to LPS stimulation induced TNF‐dependent liver injury as it was abrogated by the treatment with anti‐TNF antibodies.(7) The paper by Shen et al. also sheds light on the pathogenesis of IL‐1ß‐induced liver injury by finding that, with decreased autophagy, IL‐1ß becomes cytotoxic and proinflammatory to hepatocytes. During chronic liver injury, the release of two damage‐associated molecular patterns (DAMPs), adenosine triphosphate (ATP) and uric acid by damaged hepatocytes,(9) increases IL‐1β activity, which leads to inflammation, steatosis, and additional damage. Although IL‐1ß can induce death in normal, cultured hepatocytes, the mechanism of IL‐1ß cytotoxicity is still under investigation. The current in vivo studies demonstrate that mice with decreased hepatocyte autophagy were similarly sensitized to IL‐1ß/TNF‐induced liver injury and inflammation. Furthermore, decreased autophagy led to AML12 cell proinflammatory exosomal DAMP secretion in response to IL‐1ß/TNF, suggesting that autophagy also regulates hepatocyte inflammation from these cytokines. The authors also showed a lack of IL‐1ß‐induced death in normal mice but sensitization to IL‐1ß toxicity in the presence of TNF and decreased autophagy. Overall these findings identify a critical role for autophagy in blocking IL‐1ß‐induced hepatocyte death and inflammation, as well as a novel mechanism of synergistic IL‐1ß/TNF hepatotoxicity. The study highlights that the IL‐1ß hepatotoxicity resulted from necrosis rather than apoptosis. Previous studies have shown that hepatic parenchymal cells, especially hepatocytes, are particularly susceptible to death receptor–mediated apoptosis but not necrosis, given the ubiquitous expression of the death receptors in the organ.(10) Hepatocyte apoptosis and necrosis occur in models of acetaminophen (APAP) toxicity. Toll‐like receptor 9 was activated by APAP‐induced DAMPs, leading to increased expression of the precursor forms of the proinflammatory cytokine IL‐1β. The NACHT, leucine‐rich repeat, and PYD domains containing protein 3 inflammasome was also activated by APAP, leading to enhanced proteolytic cleavage of the precursors to mature IL‐1β.(11) The unique findings of the current studies are necessary to demonstrate that necrosis is dependent on the copresence of TNF, which, like IL‐1ß, is itself nontoxic to normal hepatocytes. Death from IL‐1ß/TNF results from compromised energy homeostasis, which only occurs in autophagy‐defective hepatocytes. Another important result of the study by Shen et al. is establishment of a distinct, IL‐1ß‐induced, caspase‐independent necrotic death pathway. The work is novel because the authors are showing a TNF necrotic pathway, albeit an IL‐1β‐dependent one. Regulation of apoptosis by caspase‐8 specifically resulted in the processing and release of IL‐1β. Treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as Cats B and L impair autophagy, enhancing the caspase‐dependent apoptosis. The current study has demonstrated that hepatocyte death was Cats B and L–dependent as silencing of either cathepsin effectively blocked cell death. Furthermore, the authors have discovered that lysosomal permeabilization and release into the cytosol of Cats B and L occur in the livers of IL‐1ß/TNF‐treated autophagy knockout mice due to the impairment in energy homeostasis from decreased autophagy (Fig. 1). Overall, the article by Shen et al. opens new perspectives into therapies targeted to increase autophagy and the management of liver diseases, such as steatohepatitis. Further studies are warranted to fully understand and appreciate the multifactorial roles of hepatocyte autophagy and inflammation both in normal conditions and during liver disease progression.