Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

Galectin-3 regulates inflammasome activation in cholestatic liver injury.

Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin-3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL-17 signaling. In liver tissues from patients with PBC and dnTGF-ßRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis-associated speck-like protein was induced, with the downstream activation of caspase-1 and IL-1ß. In wild-type hepatic macrophages, deoxycholic acid induced the association of Gal3 and NLRP3 with direct activation of the inflammasome, resulting in an increase in IL-1ß. Downstream retinoid-related orphan receptor C mRNA, IL-17A, and IL-17F were induced. In Gal3-/- macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF-ßRII/galectin-3-/- (dn/Gal3-/-) mice, which showed impaired inflammasome activation along with significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL-17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.-Tian, J., Yang, G., Chen, H.-Y., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F.-T., Gershwin, M. E., Török, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.