Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases and affects more than one-third of the population of the Western world.1,2 The histopathological spectrum of NAFLD includes steatosis alone, steatosis with inflammation, and steatohepatitis with necroinflammation (with or without fibrosis).1 The last form, which is progressive, can lead to cirrhosis and even hepatocellular carcinoma.1 In 1998, the two-hit hypothesis of nonalcoholic steatohepatitis (NASH) pathogenesis was proposed. The initial step involves fat accumulation in the liver as a result of the excessive delivery of free fatty acids (FFAs) from the adipose tissue and an imbalance between lipid synthesis and export in hepatocytes.3 However, the role of fat accumulation as a component of the first hit and the implications for liver sensitization to further insults are not fully understood. Increased levels of saturated and monounsaturated FFAs have been reported in NASH patients4,5 and have been implicated in triggering inflammation and thus potentially serving as endogenous danger signals.6 Endotoxin or lipopolysaccharide (LPS), a bacterial wall component sensed by toll-like receptor 4 (TLR4), can act as a second hit and result in progressive liver injury. Increased plasma endotoxin levels have been detected in mice with methionine choline–deficient (MCD) steatohepatitis7 and in humans with NAFLD.8 Importantly, the fatty liver is highly sensitive to LPS, and a TLR4 deficiency has attenuated hepatic steatosis and inflammation in an animal model of NASH.9 Inflammasomes are major contributors to inflammation. They are large caspase-1–activating multiprotein complexes that sense both exogenous and endogenous danger signals through intracellular NOD-like receptors (NLRs).10 Among the three prototypes of inflammasomes, NACHT, LRR, and PYD domains–containing protein 3 (NALP3) is involved in sensing endogenous danger signals, including uric acid crystals and amyloid-β protein.11 In response to danger signals, NALP3 interacts with pro–caspase-1 through its adaptor molecule, apoptosis-associated speck-like CARD-domain containing protein (ASC), which leads to the activation of cas-pase-1. Active caspase-1 promotes the cleavage and, therefore, maturation of proinflammatory cytokines [pro–interleukin-1β (pro–IL-1β), pro–IL-18, and IL-33] to promote/sustain inflammation.10,11 Multiple studies have demonstrated that inflammasome activation is the result of two distinct signals: one that activates the transcription of pro–IL-1β, usually provided by TLR ligands, and another that mediates the assembly of the inflammasome.10,11 In some cells, such as macrophages, caspase-1 can be activated via the release of endogenous adenosine triphosphate (ATP), so a single dose of the TLR4 ligand LPS is sufficient to induce the prompt release of IL-1β.12 The expression and role of the inflammasome in Kupffer cells or hepatocytes in the liver have yet to be evaluated in NASH. In this study, we postulated that fatty acids (FAs) may act as danger-associated molecular patterns (DAMPs), activate the inflammasome, and thus act as a first hit in steatohepatitis. We also tested the possibility that FAs may act differentially in liver parenchymal cells and immune cells and facilitate the release of other proinflammatory factors to induce inflammasome activation in a paracrine fashion; in the latter case, FAs may induce sensitization to LPS-induced inflammasome activation (a second hit) in cells initially insensitive to LPS. We thus employed mice with MCD-induced or high-fat diet (HFD)–induced steatohepatitis and leptin-deficient mice with steatosis to test the hypothesis that inflammasome activation occurs in NASH. In addition to the mouse models of NASH, we also evaluated human liver samples from patients with NASH. Our novel data demonstrate that saturated FAs induce up-regulation of the inflammasome in hepatocytes and lead to sensitization to LPS-induced inflammasome activation and inflammatory injury. These results indicate novel mechanisms of persistent inflammation and identify possible new therapeutic targets for NASH.