STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Abstract

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...

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes

“…In hepatocytes, we demonstrated earlier that STING and IRF3 mediate the pathogenesis in alcoholic liver disease, a role independent of microbial components (8). In the present study, our data demonstrate a hepatocyte-specific role of STING and IRF3 in mediating hepatic apoptosis as an early event in the development of liver fibrosis.…”

“…Protein Quantification-Liver whole cell lysates and nuclear preparations were extracted as described previously (8). Mitochondrial and total ER fractions were prepared using specific extraction kits from Imgenex (San Diego, CA) as per the manufacturer's protocol.…”

“…We previously showed that IRF3-deficient mice were protected from alcohol-induced liver disease and that IRF3 deficiency in hepatocytes prevented fibrogenesis in the liver in alcoholic liver disease (8). We evaluated this hypothesis using a mouse model of liver injury and fibrosis using CCl 4 or vehicle, in a 6-week regimen of biweekly intraperitoneal injections.…”

Acute organ failure following the loss of anti-apoptotic cellular FLICE-inhibitory protein involves activation of innate immune receptors

“…31 The importance of TLR9 for the sensing of DNA from apoptotic hepatocytes and subsequent liver damage was already shown by Watanabe et al 32 In line with this, STING-IRF3 signaling has recently been implied in alcoholic liver disease and STING deficiency prevented interferon regulatory transcription factor (IRF) 3-mediated mitochondrial hepatocellular apoptosis. 33 In this context, cytosolic dsDNA, which activates STING via cGMP-AMP synthase (cGAS), has been suggested to be crucial for cell death in vitro. 34 dsDNA is normally non-immunogenic due to its rapid extracellular degradation and intracellular presence of DNA recognizing receptors.…”

Hitting the right button: MAVS-mediated defense against HAV infection

“…Infection by DNA viruses, on the other hand, is detected by the cGAS-STING pathway [7], which also elicits IRF3/7 phosphorylation and type-I-Interferon production (see Figure 1). While there is no evidence for DNA viruses causing STING-mediated damage in infected hepatocytes, alcohol-induced and STING-mediated IRF3 activation can cause hepatocyte apoptosis in the context of ER stress [8]. The mechanisms that determine the outcome of RIG-I/MDA5/MAVS or cGAS/STING activation for induction of cell death remain unclear but may be related to the distinct subcellular localization of MAVS to mitochondria and STING to the ER in combination with hepatocyte stress conditions.…”