Decreased Hepatocyte Autophagy Leads to Synergistic IL‐1β and TNF Mouse Liver Injury and Inflammation

Abstract

Background and Aims The proinflammatory cytokine IL‐1β has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL‐1β promotes liver injury in these diseases is unclear, as no IL‐1β receptor‐linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy’s function in hepatotoxicity from proinflammatory cytokines. Approach and Results AML12 cells with decreased autophagy from a lentiviral autophagy‐related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL‐1β, which was markedly amplified by TNF co‐treatment. IL‐1β/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin‐1s to prevent death. IL‐1β/TNF depleted autophagy‐deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL‐1β/TNF‐induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy‐deficient cells. Mice with a tamoxifen‐inducible, hepatocyte‐specific Atg5 knockout were similarly sensitized to cathepsin‐dependent hepatocellular injury and death from IL‐1β/TNF in combination, but neither IL‐1β nor TNF alone. Knockout mice had increased hepatic inflammation, and IL‐1β/TNF‐treated, autophagy‐deficient AML12 cells secreted exosomes with proinflammatory damage–associated molecular patterns. Conclusions The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL‐1β/TNF‐induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage–associated molecular patterns.

Hepatocyte Autophagy: Maintaining a Toxic‐Free Environment

“…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.…”

Exosomes in Sepsis

“…In addition, injection of GW4869, exosome inhibitor, or knockdown of Rab27, a GTPase required for exosome release, into LPS-challenged mice resulted in significant lower levels of HMGB1 in the plasma, suggesting the importance of exosomes as couriers of HMGB1 during sepsis (31). Several mechanisms of the release of exosomal HMGB1 from cells have been revealed so far, such as the activation of TLR4 and caspase-11/gasdermin D (GSDMD) signaling, decreased autophagy and endoplasmic reticulum (ER) stress, all of which are important cellular mechanisms of sepsis pathophysiology (31)(32)(33). HMGB1-enriched exosomes released from autophagy-deficient cells treated with IL-1β and TNF-α induced pro-inflammatory genes, including IL-1α, IL-1β, IL-6, Ccl2, and Cox2, when it was co-cultured with cells (32).…”

“…HSP90 also works as a DAMP by inducing inflammatory signaling or prolonging neutrophil survival, and its inhibition is shown to attenuate organ dysfunction and improve survival in experimental model of sepsis (38,39). Exosomal HSP90 was shown to be elevated under autophagy deficiency with IL-1β and TNF-α treatment, and was associated with the induction of pro-inflammatory genes, including IL-1α, IL-1β, IL-6, Ccl2, and Cox2, of its affecting cells (32).…”

“…Several mechanisms of the release of exosomal HMGB1 from cells have been revealed so far, such as the activation of TLR4 and caspase-11/gasdermin D (GSDMD) signaling, decreased autophagy and endoplasmic reticulum (ER) stress, all of which are important cellular mechanisms of sepsis pathophysiology ( 31 – 33 ). HMGB1-enriched exosomes released from autophagy-deficient cells treated with IL-1β and TNF-α induced pro-inflammatory genes, including IL-1α, IL-1β, IL-6, Ccl2, and Cox2, when it was co-cultured with cells ( 32 ). Depletion of HMGB1 resulted in attenuated NF-κB activation induced by the exosomes ( 24 ).…”

“…Exosomes of LPS-treated macrophages were taken up by the hepatocytes and, subsequently, induced the activation of NLRP3 inflammasome and caspase-1, the infiltration of macrophages and neutrophils, and the elevation of AST, ALT, and LDH in the serum ( 28 ). Exosomes released from autophagy-deficient cells contained DAMPs, including HMGB1 and HSP90, and increased pro-inflammatory genes, including IL-1α, IL-1β, IL-6, Ccl2, and Cox2, of Kupffer cells ( 32 ). Exosomal miR-155, a micro-RNA with pro-inflammatory activity, was elevated in the liver of mice subjected to LPS and/or the TLR9 ligand cytidine-phosphate-guanosine (CpG), suggesting its contribution to liver dysfunction ( 71 ).…”