Ex Vivo Dual-Hit Method for Inflammasome Activation in Liver

Methods Mol Biol. 2022:2455:255-265. doi: 10.1007/978-1-0716-2128-8_20.


Activation of the inflammasome in hepatocytes and the liver-resident macrophages is associated with drug-induced hepatotoxicity and a plethora of metabolic diseases including nonalcoholic steatohepatitis (NASH). Initiation of this innate immune response requires two concomitant signals resulting in the formation of a molecular assembly that post-transcriptionally maturates a specific set of cytokines. While signal 1 results from the engagement and activation of pattern recognition receptors, signal 2 can be induced by diverse stimuli including adenosine triphosphate (ATP). Among various modules, NOD-like receptor 3 (NLRP3) inflammasome activation followed by caspase-1-dependent proIL-1β maturation has been observed in both preclinical models and NASH patients suggesting the crucial importance of inflammasome activation in NAFLD progression. The protocol reported here depicts an ex vivo method for investigating the role of inflammasome activation in macrophages and its impact on hepatocytes. We first described a rapid protocol for the isolation of primary Kupffer cells (KC) and hepatocytes from the murine liver. Next, to investigate the crosstalks between KCs and hepatocytes in the context of inflammasome activation, isolated KCs were activated with lipopolysaccharide (LPS), alone or in tandem with ATP, which resulted in inflammasome activation in KCs evident by abundant IL-1β secretion. Isolated primary hepatocytes were treated with conditioned medium (CM) from activated KCs to investigate the effect of inflammasome activation by various readouts. Moreover, this model also enabled us to investigate the role of specific cytokines by neutralizing them in the CM of inflammasome-activated KC. This precise ex vivo method provides a comprehensive protocol for investigating hepatocellular inflammasome activation.

Keywords: Conditioned medium; Hepatocyte; IL-1β; Inflammasome; Kupffer cells; NASH; TNF-α.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Humans
  • Inflammasomes* / metabolism
  • Interleukin-1beta / metabolism
  • Kupffer Cells / metabolism
  • Lipopolysaccharides / metabolism
  • Lipopolysaccharides / pharmacology
  • Liver / metabolism
  • Mice
  • Mice, Inbred C57BL
  • NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
  • Non-alcoholic Fatty Liver Disease* / metabolism


  • Inflammasomes
  • Interleukin-1beta
  • Lipopolysaccharides
  • NLR Family, Pyrin Domain-Containing 3 Protein