Shi Yin

In vivo consequences of liver-specific interleukin-22 expression in mice: Implications for human liver disease progression

Interleukin‐22 (IL‐22), which acts as either a proinflammatory or anti‐inflammatory cytokine in various disease models, is markedly up‐regulated in chronic liver diseases, including hepatitis B and C. In this report, we demonstrate a strong correlation between IL‐22 expression in the liver with active, inflammatory human liver disease. To clarify the role of IL‐22 up‐regulation in the pathogenesis of liver diseases, liver‐specific IL‐22 transgenic (IL‐22TG) mice, under the control of albumin promoter, were developed. Despite elevated IL‐22 serum levels ranging from 4,000 to 7,000 pg/mL, IL‐22TG mice developed normally without obvious adverse phenotypes or evidence of chronic inflammation (except for slightly thicker epidermis and minor inflammation of the skin) compared with wild‐type mice. Interestingly, IL‐22TG mice were completely resistant to concanavalin A–induced T cell hepatitis with minimal effect on liver inflammation and had accelerated liver regeneration after partial hepatectomy. Although they did not spontaneously develop liver tumors, IL‐22TG mice were more susceptible to diethylnitrosamine‐induced liver cancer. Microarray analyses revealed that a variety of antioxidant, mitogenic, acute phase genes were up‐regulated in the livers of IL‐22TG mice compared with those from wild‐type mice. Conclusion: These findings indicate that localized production of IL‐22 in the liver promotes hepatocyte survival and proliferation but primes the liver to be more susceptible to tumor development without significantly affecting liver inflammation. (HEPATOLOGY 2011;)

Hepatic cannabinoid receptor-1 mediates diet-induced insulin resistance via inhibition of insulin signaling and clearance in mice.

BACKGROUND & AIMS Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB(1)) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB(1) in insulin resistance and inhibition of insulin signaling pathways. METHODS Wild-type mice and mice with disruption of CB(1) (CB(1)(-/-) mice) or with hepatocyte-specific deletion or transgenic overexpression of CB(1) were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB(1) in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS The HFD induced hepatic insulin resistance in wild-type mice, but not in CB(1)(-/-) mice or mice with hepatocyte-specific deletion of CB(1). CB(1)(-/-) mice that overexpressed CB(1) specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB(1), the HFD or CB(1) activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB(1) activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB(1) was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB(1)-mediated inhibition of insulin signaling and clearance.

Myeloid STAT3 Inhibits T Cell-Mediated Hepatitis by Regulating T Helper 1 Cytokine and Interleukin-17 Production

BACKGROUND & AIMS T cell-mediated hepatitis is a leading cause of acute liver failure; there is no effective treatment, and the mechanisms underlying its pathogenesis are obscure. The aim of this study was to investigate the immune cell-signaling pathways involved-specifically the role of signal transducer and activator of transcription 3 (STAT3)-in T cell-mediated hepatitis in mice. METHODS T cell-mediated hepatitis was induced in mice by injection of concanavalin A (Con A). Mice with myeloid cell-specific and T-cell-specific deletion of STAT3 were generated. RESULTS STAT3 was activated in myeloid and T cells following Con A injection. Deletion of STAT3 specifically from myeloid cells exacerbated T-cell hepatitis and induced STAT1-dependent production of a T helper cell (Th)1 cytokine (interferon [IFN]-gamma) and to a lesser extent of Th17 cytokines (interleukin [IL]-17 and IL-22) in a STAT1-independent manner. In contrast, deletion of STAT3 in T cells reduced T cell-mediated hepatitis and IL-17 production. Furthermore, deletion of IFN-gamma completely abolished Con A-induced T-cell hepatitis, whereas deletion of IL-17 slightly but significantly reduced such injury. In vitro experiments indicated that IL-17 promoted liver inflammation but inhibited hepatocyte apoptosis. CONCLUSIONS Myeloid STAT3 activation inhibits T cell-mediated hepatitis via suppression of a Th1 cytokine (IFN-gamma) in a STAT1-dependent manner, whereas STAT3 activation in T cells promotes T-cell hepatitis to a lesser extent, via induction of IL-17. Therefore, activation of STAT3 in myeloid cells could be a novel therapeutic strategy for patients with T-cell hepatitis.

Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10–deficient mice†‡

Alcoholic and nonalcoholic steatohepatitis are characterized by fatty liver plus inflammation. It is generally believed that steatosis promotes inflammation, whereas inflammation in turn aggregates steatosis. Thus, we hypothesized the deletion of interleukin (IL)‐10, a key anti‐inflammatory cytokine, exacerbates liver inflammation, steatosis, and hepatocellular damage in alcoholic and nonalcoholic fatty liver disease models that were achieved via feeding mice with a liquid diet containing 5% ethanol for 4 weeks or a high‐fat diet (HFD) for 12 weeks, respectively. IL‐10 knockout (IL‐10−/−) mice and several other strains of genetically modified mice were generated and used. Compared with wild‐type mice, IL‐10−/− mice had greater liver inflammatory response with higher levels of IL‐6 and hepatic signal transducer and activator of transcription 3 (STAT3) activation, but less steatosis and hepatocellular damage after alcohol or HFD feeding. An additional deletion of IL‐6 or hepatic STAT3 restored steatosis and hepatocellular damage but further enhanced liver inflammatory response in IL‐10−/− mice. In addition, the hepatic expression of sterol regulatory element‐binding protein 1 and key downstream lipogenic proteins and enzymes in fatty acid synthesis were down‐regulated in IL‐10−/− mice. Conversely, IL‐10−/− mice displayed enhanced levels of phosphorylated adenosine monophosphate‐activated protein kinase and its downstream targets including phosphorylated acetyl‐coenzyme A carboxylase and carnitine palmitoyltransferase 1 in the liver. Such dysregulations were corrected in IL‐10−/−IL‐6−/− or IL‐10−/−STAT3Hep−/− double knockout mice. Conclusion: IL‐10−/− mice are prone to liver inflammatory response but are resistant to steatosis and hepatocellular damage induced by ethanol or HFD feeding. Resistance to steatosis in these mice is attributable to elevation of inflammation‐associated hepatic IL‐6/STAT3 activation that subsequently down‐regulates lipogenic genes but up‐regulates fatty acid oxidation‐associated genes in the liver. (HEPATOLOGY 2011; 54:846–856)

Hyperactivation of anandamide synthesis and regulation of cell-cycle progression via cannabinoid type 1 (CB1) receptors in the regenerating liver

The mammalian liver regenerates upon tissue loss, which induces quiescent hepatocytes to enter the cell cycle and undergo limited replication under the control of multiple hormones, growth factors, and cytokines. Endocannabinoids acting via cannabinoid type 1 receptors (CB1R) promote neural progenitor cell proliferation, and in the liver they promote lipogenesis. These findings suggest the involvement of CB1R in the control of liver regeneration. Here we report that mice lacking CB1R globally or in hepatocytes only and wild-type mice treated with a CB1R antagonist have a delayed proliferative response to two-thirds partial hepatectomy (PHX). In wild-type mice, PHX leads to increased hepatic expression of CB1R and hyperactivation of the biosynthesis of the endocannabinoid anandamide in the liver via an in vivo pathway involving conjugation of arachidonic acid and ethanolamine by fatty-acid amide hydrolase. In wild-type but not CB1R−/− mice, PHX induces robust up-regulation of key cell-cycle proteins involved in mitotic progression, including cyclin-dependent kinase 1 (Cdk1), cyclin B2, and their transcriptional regulator forkhead box protein M1 (FoxM1), as revealed by ultrahigh-throughput RNA sequencing and pathway analysis and confirmed by real-time PCR and Western blot analyses. Treatment of wild-type mice with anandamide induces similar changes mediated via activation of the PI3K/Akt pathway. We conclude that activation of hepatic CB1R by newly synthesized anandamide promotes liver regeneration by controlling the expression of cell-cycle regulators that drive M phase progression.

Interleukin-22 ameliorates cerulein-induced pancreatitis in mice by inhibiting the autophagic pathway.

Pancreatitis occurs when digestive enzymes are activated in the pancreas. Severe pancreatitis has a 10-30% mortality rate. No specific treatments for pancreatitis exist now. Here, we discovered that interleukin-22 (IL-22) may have therapeutic potential in treating acute and chronic pancreatitis. Wild-type and IL-22 knockout mice were equally susceptible to cerulein-induced acute and chronic pancreatitis, whereas liver-specific IL-22 transgenic mice were completely resistant to cerulein-induced elevation of serum digestive enzymes, pancreatic necrosis and apoptosis, and inflammatory cell infiltration. Treatment of wild-type mice with recombinant IL-22 or adenovirus IL-22 markedly attenuated the severity of cerulein-induced acute and chronic pancreatitis. Mechanistically, we show that the protective effect of IL-22 on pancreatitis was mediated via the induction of Bcl-2 and Bcl-XL, which bind to Beclin-1 and subsequently inhibit autophagosome formation to ameliorate pancreatitis. In conclusion, IL-22 ameliorates cerulein-induced pancreatitis by inhibiting the autophagic pathway. IL-22 could be a promising therapeutic drug to treat pancreatitis.

Invariant NKT cell activation induces neutrophil accumulation and hepatitis: Opposite regulation by IL‐4 and IFN‐γ

Alpha‐Galactosylceramide (α‐Galcer), a specific agonist for invariant natural killer T (iNKT) cells, is being evaluated in clinical trials for the treatment of viral hepatitis and liver cancer. However, the results from α‐Galcer treatment are mixed, partially because of the variety of cytokines produced by activated iNKT cells that have an unknown synergistic effect on the progression of liver disease. It is well documented that injection of α‐Galcer induces mild hepatitis with a rapid elevation in the levels of interleukin (IL)−4 and a delayed elevation in the levels of interferon‐gamma (IFN‐γ), and both of these cytokines are thought to mediate many functions of iNKT cells. Surprisingly, genetic deletion of both IL‐4 and IFN‐γ aggravated, rather than abolished, α‐Galcer‐induced iNKT hepatitis. Moreover, genetic ablation of IL‐4, the IL‐4 receptor, or its downstream signaling molecule signal transducer and activator of transcription (STAT)6 ameliorated α‐Galcer‐induced neutrophil infiltration, liver injury, and hepatitis. In contrast, genetic deletion of IFN‐γ, the IFN‐γ receptor, or its downstream signaling molecule STAT1 enhanced liver neutrophil accumulation, thereby exacerbating liver injury and hepatitis. Moreover, depletion of neutrophils eradicated α‐Galcer‐induced liver injury in wild‐type, STAT1 knockout, and IFN‐γ knockout mice. Conclusion: Our results propose a model in which activated iNKT cells rapidly release IL‐4, which promotes neutrophil survival and hepatitis but also sequentially produce IFN‐γ, which acts in a negative feedback loop to ameliorate iNKT hepatitis by inducing neutrophil apoptosis. Thus, modification of iNKT production of IL‐4 and IFN‐γ may have the potential to improve the efficacy of α‐Galcer in the treatment of liver disease. (Hepatology 2013;58:1474–1485)

Tissue inhibitor of metalloproteinase 1 (TIMP-1) deficiency exacerbates carbon tetrachloride-induced liver injury and fibrosis in mice: involvement of hepatocyte STAT3 in TIMP-1 production

BackgroundTissue inhibitor of metalloproteinase 1 (TIMP-1), which is thought to be produced mainly by activated hepatic stellate cells and Kupffer cells in the liver, plays a pivotal role in matrix remodeling during liver injury and repair; while the effect of TIMP-1 on hepatocellular damage remains obscure.ResultsHepatic expression of TIMP-1 mRNA and protein was up-regulated both in acute and chronic liver injury induced by carbon tetrachloride (CCl4). Compared with wild-type mice, TIMP-1 knockout mice were more susceptible to CCl4-induced acute and chronic liver injury, as shown by higher levels of serum alanine aminotransferase (ALT), greater number of apoptotic hepatocytes, and more extended necroinflammatory foci. TIMP-1 knockout mice also displayed greater degree of liver fibrosis after chronic CCl4 injection when compared with wild-type mice. In vitro treatment with TIMP-1 inhibited cycloheximide-induced cell death of primary mouse hepatocytes. Finally, up-regulation of TIMP-1 in the liver and serum after chronic CCl4 treatment was markedly diminished in hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. In vitro treatment with interleukin-6 stimulated TIMP-1 production in primary mouse hepatocytes, but to a lesser extent in STAT3-deficient hepatocytes.ConclusionsTIMP-1 plays an important role in protecting against acute and chronic liver injury and subsequently inhibiting liver fibrosis induced by CCl4. In addition to activated stellate cells and Kupffer cells, hepatocytes are also responsible for TIMP-1 production during liver injury via a STAT3-dependent manner.

Hepatoprotective versus Oncogenic Functions of STAT3 in Liver Tumorigenesis

Aberrantly hyperactivated STAT3 has been found in human liver cancers as an oncogene; however, STAT3 has also been shown to exert hepatoprotective effects during liver injury. The balancing act that STAT3 plays between hepatoprotection and liver tumorigenesis remains poorly defined. In this study, the diethylnitrosamine (DEN)-induced liver tumor model and the chronic carbon tetrachloride (CCl(4))-induced liver fibrosis model were both used to investigate the role of STAT3 in liver tumorigenesis. Hepatocyte-specific STAT3 knockout mice were resistant to liver tumorigenesis induced by a single DEN injection, whose tumorigenesis was associated with minimal chronic liver inflammation, injury, and fibrosis. In contrast, long-term CCl(4) treatment resulted in severe hepatic oxidative damage, inflammation, and fibrosis but rarely induced liver tumor formation in wild-type mice. Despite the oncogenic function of STAT3 in DEN-induced liver tumor, hepatocyte-specific STAT3 knockout mice were more susceptible to liver tumorigenesis after 16 weeks of CCl(4) injection, which was associated with higher levels of liver injury, inflammation, fibrosis, and oxidative DNA damage compared with wild-type mice. These findings suggest that the hepatoprotective feature of STAT3 prevents hepatic damage and fibrosis under the condition of persistent inflammatory stress, consequently suppressing injury-driven liver tumor initiation. Once liver tumor cells have developed, STAT3 likely acts as an oncogenic factor to promote tumor growth.

Enhanced Liver Regeneration in IL-10–Deficient Mice after Partial Hepatectomy via Stimulating Inflammatory Response and Activating Hepatocyte STAT3

Emerging evidence suggests that proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a critical role in the initiation and progression of liver regeneration; however, relatively little is known about the role of anti-inflammatory cytokine IL-10 in liver regeneration after partial hepatectomy (PHx). Here, we examined the role of IL-10 in liver regeneration using a model of PHx in several strains of genetically modified mice. After PHx, expression of IL-10 mRNA in the liver and spleen was significantly elevated. Such elevation was diminished in TLR4 mutant mice. Compared with wild-type mice, IL-10(-/-) mice had higher levels of expression of proinflammatory cytokines (IL-6, TNF-α, and IFN-γ) and inflammatory markers (CCR2 and F4/80) in the liver, as well as higher serum levels of proinflammatory cytokines after PHx. The number of neutrophils and macrophages was also higher in the livers of IL-10(-/-) mice than in wild-type mice after PHx. Liver regeneration as determined by BrdU incorporation after PHx was higher in IL-10(-/-) mice than in wild-type mice, which was associated with higher levels of activation of IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes significantly reduced liver regeneration in IL-10(-/-) mice after PHx. Collectively, IL-10 plays an important role in negatively regulating liver regeneration via limiting inflammatory response and subsequently tempering hepatic STAT3 activation.