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‐βRII 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 downs tream activation of caspase‐1 and IL‐1β. Inwild‐typehepaticmacrophages, deoxycholic acid induced the association of Gal3 and NLRP3with direct activation of the inflammasome, resulting in an increase in IL‐1β. Downstreamretinoid‐related orphan receptor CmRNA, IL‐17A, and IL‐17Fwere 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‐βRII/galectin‐3‐/ (dn/Gal3‐/) mice, which showed impaired inflammasome activation alongwith 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. FASEB J. 30, 4202–4213 (2016). http://www.fasebj.org

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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 p47 phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47 phox , 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/p47 phox 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 p47 phox subunit that results in redox stress and accelerated fibrosis in the aged. (Hepatology 2020;72:1204-1218). N ASH and associated cirrhosis are the leading causes of liver-related mortality in the United States and worldwide. (1) The incidence of NASH rises strikingly with age, contributing significantly to elder mortality. (2) Older patients are often excluded from liver transplantation because of comorbidities and the age limit for transplantation; therefore, a significant increase in NASH-related mortality is expected because NASH has no approved medical treatment. Most of the preclinical data in NASH/fibrosis research is derived from experiments employing young mice. In clinical trials, however, the target population is usually middle-aged or elderly. To

Recruitment of inflammatory cells is a major feature of alcoholic liver injury however; the signals and cellular sources regulating this are not well defined. C-C chemokine receptor type 2 (CCR2) is expressed by active hepatic stellate cells (HSC) and is a key monocyte recruitment signal. Activated HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been addressed, we studied NOX4-mediated regulation of CCR2/CCL2 mRNA stability. NOX4 mRNA was significantly induced in patients with alcoholic liver injury, and was co-localized with αSMA-expressing activated HSC. We generated HSC-specific NOX4 KO mice and these were pair-fed on alcohol diet. Lipid peroxidation have not changed significantly however, the expression of CCR2, CCL2, Ly6C, TNFα, and IL-6 was significantly reduced in NOX4HSCKO compared to fl/fl mice. NOX4 promoter was induced in HSC by acetaldehyde treatment, and NOX4 has significantly increased mRNA half-life of CCR2 and CCL2 in conjunction with Ser221 phosphorylation and cytoplasmic shuttling of HuR. In conclusion, NOX4 is induced in early alcoholic liver injury and regulates CCR2/CCL2 mRNA stability thereby promoting recruitment of inflammatory cells and production of proinflammatory cytokines.

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