The Lipopolysaccharide-Sensing Caspase(s)-4/11 Are Activated in Cirrhosis and Are Causally Associated With Progression to Multi-Organ Injury

Background and Aims The development of multi-organ injury in cirrhosis is associated with increased intestinal permeability, translocation of gut-derived bacterial products [e.g., lipopolysaccharide (LPS)] into the circulation, and increased non-apoptotic hepatocyte cell death. Pyroptosis is a non-apoptotic, lytic form of cell death mediated by the LPS-sensing caspase(s)-4/11 (caspase-4 in humans, caspase-11 in mice), which leads to activation of the effector protein Gasdermin D (GSDMD) and subsequent formation of pores in the plasma membrane. Endoplasmic reticulum (ER) stress, a feature of cirrhosis, has been identified as a factor promoting the activation of caspase-11, thus increasing sensitivity of the cell to LPS-mediated pyroptosis. The aim of this study was to determine the role of bacterial LPS in the activation of hepatic caspase(s)-4/11 and progression of hepatic and extra-hepatic organ injury in cirrhosis. Materials and Methods Human liver samples from patients with stable cirrhosis (SC) or acutely decompensated cirrhosis (AD) were analyzed for caspase-4 activation by immunohistochemistry. Wild-type and Casp11–/– mice underwent CCl4 treatment by gavage to induce advanced liver fibrosis, and subsequently low-dose injection of LPS to mimic bacterial translocation and induce multi-organ injury. Liver, kidney, and brain function were assessed by plasma ALT/creatinine and brain water respectively. The activity of inflammatory caspases was assessed by fluorometric assay and the occurrence of pyroptosis and overall cell death in liver tissue by GSDMD cleavage and TUNEL assay, respectively. Primary human hepatocytes were cultured according to standard techniques. Results Human liver samples demonstrated increased caspase-4 activation in AD cirrhosis. Caspase-4 activation was associated with MELD score and circulating levels of LDH. Wild-type mice treated with CCl4 developed significant multi-organ injury (increased ALT, creatinine, and brain water) upon LPS injection, and showed increased hepatic GSDMD cleavage compared to mice treated with CCl4 alone. Primary human hepatocytes could be sensitized to pyroptosis by pre-treatment with the ER-stress inducer tunicamycin and LPS. Casp11–/– mice treated with CCl4 + LPS were significantly protected from multi-organ injury compared to wild-type CCl4 + LPS. Conclusion These data demonstrate for the first time a causal relationship between LPS-mediated activation of caspase(s)-4/11 and development of hepatic and extra-hepatic injury in cirrhosis. Graphical Abstract Model of caspase-4/11 activation in cirrhosis. Gut-derived bacterial LPS (1) enters the portal circulation after translocation across the gut epithelium and is internalized by hepatocytes. Cytoplasmic LPS is recognized by caspase-4/11 (2), which undergoes self-activation upon ligand binding. The activity of caspase-4/11 is enhanced by endoplasmic reticulum stress (3), which occurs in fibrosis/cirrhosis, leading to “sensitization” of this pathway. Active caspase-4/11 cleaves the dimeric protein Gasdermin D (GSDMD) (4), and freeing the N-terminal domain to migrate to the plasma membrane and form GSDMD N-terminal octameric pores (5). GSDMD pores insert themselves into the plasma membrane, allowing the deregulated passage of molecules and causing cell swelling and membrane rupture, eventually resulting in pyroptotic cell death (6).