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The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro–interleukin-1β (pro–IL-1β) into secreted IL-1β, is induced by endogenous and exogenous danger signals. Lipo-polysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline–deficient (MCD) diet–induced NASH and high-fat diet–induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains–containing protein 3 (NALP3; cryopyrin), apoptosis-associated specklike CARD-domain containing protein, pannexin-1, and pro–caspase-1] at the messenger RNA (mRNA) level increased caspase-1 activity, and mature IL-1β protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS-induced increases in NALP3, pannexin-1, IL-1β mRNA, and mature IL-1β protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS-induced IL-1β release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase-dependent manner. These hepatocyte-derived danger signals, in turn, activate inflammasome, IL-1β, and tumor necrosis factor α release in liver mononuclear cells. Conclusion Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up-regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL-β release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediatedincrease in TNF␣ production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcoholinduced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNF␣ induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNF␣ production in isolated KCs when compared with pairfed controls. The mechanistic role of miR-155 in TNF␣ regulation was indicated by decreased TNF␣ levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNF␣ production after miR-155 overexpression, respectively. We found that miR-155 affected TNF␣ mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNF␣ mRNA half-life. Using the NF-B inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-B activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-B and the increased miR-155 contributes to alcohol-induced elevation in TNF␣ production via increased mRNA stability.

Inflammation is a common element in the pathogenesis of most chronic liver diseases that lead to fibrosis and cirrhosis. Inflammation is characterized by activation of innate immune cells and production of pro-inflammatory cytokines IL-1α, IL-1β, and TNFα. Inflammasomes are intracellular multiprotein complexes expressed in both parenchymal and non-parenchymal cells of the liver that in response to cellular danger signals activate caspase-1, and release IL-1β and IL-18. The importance of inflammasome activation in various forms of liver diseases in relation to liver damage, steatosis, inflammation and fibrosis is discussed in this review.

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3), a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis. A lcoholic liver disease (ALD) affects over 140 million people worldwide, and currently there is no effective treatment. Acute alcohol consumption induces fatty liver and excessive alcohol use causes progression to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Dysregulation of innate immunity and liver inflammation, triggered by the translocation of gut-derived endotoxin [lipopolysaccharide (LPS)] to the liver, represent major contributors to ALD (1). The recognition of gut-derived LPS by Kupffer cells (KC) requires the Toll-like receptor 4 (TLR4), which triggers two downstream pathways. The TLR4/ myeloid differentiation primary response gene 88 (MyD88) pathway activates transcription of inflammatory cytokines, whereas TLR4/TRAM/TRIF [TIR domain-containing adaptor inducing interferon-beta (TRIF)-related adaptor molecule (TRAM)] triggers Type-I interferons (IFN) regulatory factor 3 (IRF3) to induce IFN (2). The essential role of the TLR4 signaling in ALD was demonstrated in mice lacking functional TLR4 that showed attenuation of alcoholic steatohepatitis (3, 4). In previous studies, we reported that the MyD88-dependent pathway was dispensable for ALD (4) and observed complete protection from alcohol-induced inflammation, steatosis, and injury in mice deficient in IRF3 (5), suggesting that the pathogenic effects of TLR4 in ALD were mediated via the TRAM/TRIF-dependent pathway. However, the mechanisms by which IRF3 causes ALD remain obscure.IRF3 is a constitutively expressed transcription factor that resides in the cytoplasm and dimerizes and translocates to the nucleus upon phosphorylation (6). Phosphorylated IRF3 induces IFN-β during viral infection, but may also contribute to inflammatory cytokine response to LPS (7). IRF3 also promotes apoptosis in virus-infected cells through association with proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] (8). To elucidate the mechanism by which IRF3 determines ALD, we asked three fundamental questions. F...