Background and Aims The proinflammatory cytokine IL‐1β has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL‐1β promotes liver injury in these diseases is unclear, as no IL‐1β receptor‐linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy’s function in hepatotoxicity from proinflammatory cytokines. Approach and Results AML12 cells with decreased autophagy from a lentiviral autophagy‐related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL‐1β, which was markedly amplified by TNF co‐treatment. IL‐1β/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin‐1s to prevent death. IL‐1β/TNF depleted autophagy‐deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL‐1β/TNF‐induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy‐deficient cells. Mice with a tamoxifen‐inducible, hepatocyte‐specific Atg5 knockout were similarly sensitized to cathepsin‐dependent hepatocellular injury and death from IL‐1β/TNF in combination, but neither IL‐1β nor TNF alone. Knockout mice had increased hepatic inflammation, and IL‐1β/TNF‐treated, autophagy‐deficient AML12 cells secreted exosomes with proinflammatory damage–associated molecular patterns. Conclusions The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL‐1β/TNF‐induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage–associated molecular patterns.

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Abbreviations: CEB, colonic epithelial barrier; MetS, Metabolic Syndrome; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis. AbstractThere is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)-induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r −/− . WD-fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD-fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD-fed mice, increased paracellular permeability, while the BA-binding resin sevelamer hydrochloride protected against CDCA-induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD-fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD-induced impairment of the intestinal epithelial barrier in NASH. K E Y W O R D S bile acids, intestinal permeability, microbiome, NAFLD, NASH, tight junction 7090 | GUPTA eT Al. | MATERIALS AND METHODS | MiceJunctional adhesion molecule A (JAM-A) knock out mice (F11r −/− ) were generated as previously described. 9 Mice were bred and maintained at Emory University and the University of Pittsburgh Divisions of Animal Resources. All animal studies were approved by the Institutional Animal Care and Use Committees. | BA feeding experimentC57BL/6 mice were fed CDCA (3mg/gm body weight, Millipore Sigma, St. Louis, MO) mixed with FITC-conjugated dextran (4 kDa) (0.6 mg/gm body weight, Millipore Sigma, St. Louis, MO) solution by oral gavage following a 6 hours fast. After 3 hours, blood was collected and fluorescence intensity was measured using Fluorescence Spectrophotometer (Synergy 2, BioTek, Winooski, VT) as described previously. 9

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1−/− and µMT mice, suggesting B cells as a major ATX‐producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.—Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El‐Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon. FASEB J. 33, 3623–3635 (2019). http://www.fasebj.org

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Adiponectin inhibits hepatic stellate cell (HSC) activation and subsequent development of liver fibrosis via multiple mechanisms. Phosphatase and tensin homolog deletion 10 (PTEN) plays a crucial role in suppression of HSC activation, but its regulation by adiponectin is not fully understood. Here, we investigated the effect of adiponectin on PTEN in LX-2 cells, a human cell line and examined the underlying molecular mechanisms involved in adiponectin-mediated upregulation of PTEN activity during fibrosis. PTEN expression was found to be significantly reduced in the livers of mice treated with CCl4, whereas its expression was rescued by adiponectin treatment. The DNA methylation proteins DNMT1, DNMT3A, and DNMT3B are all highly expressed in activated primary HSCs compared to quiescent HSCs, and thus represent additional regulatory targets during liver fibrogenesis. Expression of DNMT proteins was significantly induced in the presence of fibrotic stimuli; however, only DNMT3B expression was reduced in the presence of adiponectin. Adiponectin-induced suppression of DNMT3B was found to be mediated by enhanced miR-29b expression. Furthermore, PTEN expression was significantly increased by overexpression of miR-29b, whereas its expression was markedly reduced by a miR-29b inhibitor in LX-2 cells. These findings suggest that adiponectin-induced upregulation of miR-29b can suppress DNMT3B transcription in LX-2 cells, thus resulting in reduced methylation of PTEN CpG islands and ultimately suppressing the PI3K/AKT pathway. Together, these data suggest a possible new explanation for the inhibitory effect of adiponectin on HSC activation and liver fibrogenesis.

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