Dechun Feng

Interleukin‐22 induces hepatic stellate cell senescence and restricts liver fibrosis in mice

Interleukin (IL)‐22 is known to play a key role in promoting antimicrobial immunity, inflammation, and tissue repair at barrier surfaces by binding to the receptors, IL‐10R2 and IL‐22R1. IL‐22R1 is generally thought to be expressed exclusively in epithelial cells. In this study, we identified high levels of IL‐10R2 and IL‐22R1 expression on hepatic stellate cells (HSCs), the predominant cell type involved in liver fibrogenesis in response to liver damage. In vitro treatment with IL‐22 induced the activation of signal transducer and activator of transcription (STAT) 3 in primary mouse and human HSCs. IL‐22 administration prevented HSC apoptosis in vitro and in vivo, but surprisingly, the overexpression of IL‐22 by either gene targeting (e.g., IL‐22 transgenic mice) or exogenous administration of adenovirus expressing IL‐22 reduced liver fibrosis and accelerated the resolution of liver fibrosis during recovery. Furthermore, IL‐22 overexpression or treatment increased the number of senescence‐associated beta‐galactosidase‐positive HSCs and decreased alpha‐smooth muscle actin expression in fibrotic livers in vivo and cultured HSCs in vitro. Deletion of STAT3 prevented IL‐22‐induced HSC senescence in vitro, whereas the overexpression of a constitutively activated form of STAT3 promoted HSC senescence through p53‐ and p21‐dependent pathways. Finally, IL‐22 treatment up‐regulated the suppressor of cytokine signaling (SOCS) 3 expression in HSCs. Immunoprecipitation analyses revealed that SOCS3 bound p53 and subsequently increased the expression of p53 and its target genes, contributing to IL‐22‐mediated HSC senescence. Conclusion: IL‐22 induces the senescence of HSCs, which express both IL‐10R2 and IL‐22R1, thereby ameliorating liver fibrogenesis. The antifibrotic effect of IL‐22 is likely mediated by the induction of HSC senescence, in addition to the previously discovered hepatoprotective functions of IL‐22. (HEPATOLOGY 2012;56:1150–1159)

Interleukin-22 Promotes Proliferation of Liver Stem/Progenitor Cells in Mice and Patients With Chronic Hepatitis B Virus Infection

BACKGROUND & AIMS Proliferation of liver stem/progenitor cells (LPCs), which can differentiate into hepatocytes or biliary epithelial cells, is often observed in chronically inflamed regions of liver in patients. We investigated how inflammation might promote proliferation of LPCs. METHODS We examined the role of interleukin (IL)-22, a survival factor for hepatocytes, on proliferation of LPCs in patients with chronic hepatitis B virus (HBV) infection and in mice. Proliferation of LPCs in mice was induced by feeding a diet that contained 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). RESULTS Hepatic expression of IL-22 was increased in patients with HBV and correlated with the grade of inflammation and proliferation of LPCs. Mice on the DDC diet that overexpressed an IL-22 transgene specifically in liver (IL-22TG), or that were infected with an IL-22-expressing adenovirus, had increased proliferation of LPCs. Signal transducer and activator of transcription (STAT) 3, a component of the IL-22 signaling pathway, was activated in LPCs isolated from DDC-fed IL-22TG mice. Deletion of STAT3 from livers of IL-22TG mice reduced proliferation of LPCs. In addition, the receptors IL-22R1 and IL-10R2 were detected on epithelial cell adhesion molecule(+)CD45(-) LPCs isolated from DDC-fed wild-type mice. Culture of these cells with IL-22 activated STAT3 and led to cell proliferation, but IL-22 had no effect on proliferation of STAT3-deficient EpCAM(+)CD45(-) LPCs. IL-22 also activated STAT3 and promoted proliferation of cultured BMOL cells (a mouse LPC line). CONCLUSIONS In livers of mice and patients with chronic HBV infection, inflammatory cells produce IL-22, which promotes proliferation of LPCs via STAT3. These findings link inflammation with proliferation of LPCs in patients with HBV infection.

STAT proteins – Key regulators of anti-viral responses, inflammation, and tumorigenesis in the liver

Since its discovery in the early 1990s, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway has been found to play key roles in regulating many key cellular processes such as survival, proliferation, and differentiation. There are seven known mammalian STAT family members: STAT1, 2, 3, 4, 5a, 5b, and 6. In the liver, activation of these STAT proteins is critical for anti-viral defense against hepatitis viral infection and for controlling injury, repair, inflammation, and tumorigenesis. The identification of functions for these STAT proteins has increased our understanding of liver disease pathophysiology and treatments, while also suggesting new therapeutic modalities for managing liver disease.

Liver is the major source of elevated serum lipocalin‐2 levels after bacterial infection or partial hepatectomy: A critical role for IL‐6/STAT3

Lipocalin‐2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase‐associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily responsible for LCN2 production remain unclear. To address these issues, hepatocyte‐specific Lcn2 knockout (Lcn2Hep–/–) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2Hep–/– mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼6,000 ng/mL) postinfection and more than 60% post‐PHx (∼700 ng/mL). Interestingly, both Lcn2Hep–/– and global Lcn2 knockout (Lcn2–/–) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)‐6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte‐specific ablation of the IL‐6 receptor or Stat3, a major downstream effector of IL‐6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL‐6. Conclusion: Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL‐6 activation of the STAT3 signaling pathway. Thus, hepatocyte‐derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration. (Hepatology 2015;61:692‐702)

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)

MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6–p47phox–oxidative stress pathway in neutrophils

Objectives Chronic-plus-binge ethanol feeding activates neutrophils and exacerbates liver injury in mice. This study investigates how recent excessive drinking affects peripheral neutrophils and liver injury in alcoholics, and how miR-223, one of the most abundant microRNAs (miRNAs) in neutrophils, modulates neutrophil function and liver injury in ethanol-fed mice. Designs Three hundred alcoholics with (n=140) or without (n=160) recent excessive drinking and 45 healthy controls were enrolled. Mice were fed an ethanol diet for 10 days followed by a single binge of ethanol. Results Compared with healthy controls or alcoholics without recent drinking, alcoholics with recent excessive drinking had higher levels of circulating neutrophils, which correlated with serum levels of alanine transaminase (ALT) and aspartate transaminase (AST). miRNA array analysis revealed that alcoholics had elevated serum miR-223 levels compared with healthy controls. In chronic-plus-binge ethanol feeding mouse model, the levels of miR-223 were increased in both serum and neutrophils. Genetic deletion of the miR-223 gene exacerbated ethanol-induced hepatic injury, neutrophil infiltration, reactive oxygen species (ROS) and upregulated hepatic expression of interleukin (IL)-6 and phagocytic oxidase (phox) p47phox. Mechanistic studies revealed that miR-223 directly inhibited IL-6 expression and subsequently inhibited p47phox expression in neutrophils. Deletion of the p47phox gene ameliorated ethanol-induced liver injury and ROS production by neutrophils. Finally, miR-223 expression was downregulated, while IL-6 and p47phox expression were upregulated in peripheral blood neutrophils from alcoholics compared with healthy controls. Conclusions miR-223 is an important regulator to block neutrophil infiltration in alcoholic liver disease and could be a novel therapeutic target for the treatment of this malady.

Short- or long-term high-fat diet feeding plus acute ethanol binge synergistically induce acute liver injury in mice: an important role for CXCL1.

Obesity and alcohol consumption often coexist and work synergistically to promote steatohepatitis; however, the underlying mechanisms remain obscure. Here, we demonstrate that feeding mice a high‐fat diet (HFD) for as little as 3 days markedly exacerbated acute ethanol binge–induced liver neutrophil infiltration and injury. Feeding mice with an HFD for 3 months plus a single binge of ethanol induced much more severe steatohepatitis. Moreover, 3‐day or 3‐month HFD‐plus‐ethanol binge (3d‐HFD+ethanol or 3m‐HFD+ethanol) treatment markedly up‐regulated the hepatic expression of several chemokines, including chemokine (C‐X‐C motif) ligand 1 (Cxcl1), which showed the highest fold (approximately 20‐fold and 35‐fold, respectively) induction. Serum CXCL1 protein levels were also markedly elevated after the HFD+ethanol treatment. Blockade of CXCL1 with a CXCL1 neutralizing antibody or genetic deletion of the Cxcl1 gene reduced the HFD+ethanol‐induced hepatic neutrophil infiltration and injury, whereas overexpression of Cxcl1 exacerbated steatohepatitis in HFD‐fed mice. Furthermore, expression of Cxcl1 messenger RNA was up‐regulated in hepatocytes, hepatic stellate cells, and endothelial cells isolated from HFD+ethanol‐fed mice compared to mice that were only given the HFD, with the highest fold induction observed in hepatocytes. In vitro stimulation of hepatocytes with palmitic acid up‐regulated the expression of Cxcl1 messenger RNA, and this up‐regulation was attenuated after treatment with an inhibitor of extracellular signal–regulated kinase 1/2, c‐Jun N‐terminal kinase, or nuclear factor κB. In addition, hepatic or serum levels of free fatty acids were higher in HFD+ethanol‐fed mice than in the control groups. Conclusion: An HFD combined with acute ethanol consumption synergistically induces acute liver inflammation and injury through the elevation of hepatic or serum free fatty acids and subsequent up‐regulation of hepatic CXCL1 expression and promotion of hepatic neutrophil infiltration. (Hepatology 2015;62:1070‐1085)

Hepatoprotective and anti-fibrotic functions of interleukin-22: Therapeutic potential for the treatment of alcoholic liver disease

Interleukin-22 (IL-22) plays a key role in promoting antimicrobial immunity and tissue repair at barrier surfaces by binding to the receptors IL-22R1, which is generally thought to be expressed exclusively in epithelial cells, and IL-10R2. Our laboratory previously demonstrated that IL-22 plays an important role in ameliorating liver injury in many rodent models by targeting hepatocytes that express high levels of IL-22R1 and IL-10R2. Recently, we have identified high expression levels of IL-22R1 and IL-10R2 in liver progenitor cells and hepatic stellate cells (HSCs). Overexpression of IL-22 in vivo or treatment with IL-22 in vitro promotes proliferation of liver progenitor cells via a signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. IL-22 treatment also prevents HSC apoptosis in vitro and in vivo. Surprisingly, overexpression of IL-22, via either gene targeting or exogenous administration of adenovirus expressing IL-22, reduces liver fibrosis and accelerates the resolution of liver fibrosis during recovery. The anti-fibrotic effects of IL-22 are mediated via the activation of STAT3 in HSCs and subsequent induction of suppressor of cytokine signaling 3, which induces HSC senescence. Taken together, the hepatoprotective, mitogenic, and anti-fibrotic effects of IL-22 are beneficial in ameliorating alcoholic liver injury. Importantly, due to the restricted expression of IL-22R1, IL-22 therapy is expected to have few side effects, thus making IL-22 a potential candidate for treatment of alcoholic liver disease.

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.