Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

Ambiguous Pathogenic Roles of Macrophages in Alcohol-Associated Liver Diseases.

Alcohol-associated liver disease (ALD) represents a major public health issue worldwide and is a leading etiology of liver cirrhosis. Alcohol-related liver injuries include a range of manifestations including alcoholic hepatitis (AH), simple steatosis, steatohepatitis, hepatic fibrosis, cirrhosis and liver cancer. Liver disease occurs from several pathological disturbances such as the metabolism of ethanol, which generates reactive oxygen species (ROS) in hepatocytes, alterations in the gut microbiota, and the immune response to these changes. A common hallmark of these liver affections is the establishment of an inflammatory environment, and some (broad) anti-inflammatory approaches are used to treat AH (eg, corticosteroids). Macrophages, which represent the main innate immune cells in the liver, respond to a wide variety of (pathogenic) stimuli and adopt a large spectrum of phenotypes. This translates to a diversity of functions including pathogen and debris clearance, recruitment of other immune cells, activation of fibroblasts, or tissue repair. Thus, macrophage populations play a crucial role in the course of ALD, but the underlying mechanisms driving macrophage polarization and their functionality in ALD are complex. In this review, we explore the various populations of hepatic macrophages in alcohol-associated liver disease and the underlying mechanisms driving their polarization. Additionally, we summarize the crosstalk between hepatic macrophages and other hepatic cell types in ALD, in order to support the exploration of targeted therapeutics by modulating macrophage polarization.

Interleukin-17 programs liver progenitor cell transformation into cancer stem cells through miR-122 downregulation with increased risk of primary liver cancer initiation.

Chronic inflammation is a key component in the development of virtually all types of primary liver cancers. However, how chronic inflammation potentiates or even may initiate liver parenchymal cell transformation remains unclear. Cancer stem cells (CSCs) represent an exciting target for novel anticancer therapeutic strategies in several types of cancers and were also described in primary liver cancers as tumor initiating cells. Recently, we reported a key role of Interleukin (IL)-17 in Liver Progenitor Cell (LPC) accumulation in preneoplastic cirrhotic livers. In this study, we evidenced in vitro, that long-term stimulation of LPCs with IL-17 led to their transformation into CSCs. Indeed, they acquired CSC-marker expression, and self-renewal properties, showed by their increased capacity to form spheroids. The miRNome analysis revealed that long-term IL-17 treatment of LPCs led to a 90% decrease in miR-122 expression. In a model using immunodeficient mice, ectopic engraftment of LPCs in an IL-17-enriched environment led to tumor occurrence with an aggressive phenotype. Contrastingly, in a murine model of hepatocellular carcinoma induced by a unique injection of diethyl-nitrosamine associated with chronic administration of carbon tetrachloride, IL-17-deficiency or anti-IL-17 therapy protected mice from liver tumor growth. In conclusion, we showed that a chronic exposure of LPCs to IL-17 cytokine promotes their transformation into CSCs. In addition, we demonstrated that IL-17-neutralizing strategies limit CSC occurrence and liver tumor progression through miR-122 restored-expression.

Immune Profiling of Combined Hepatocellular- Cholangiocarcinoma Reveals Distinct Subtypes and Activation of Gene Signatures Predictive of Response to Immunotherapy.

PURPOSE: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare malignancy associated with an overall poor prognosis. We aimed to investigate the immune profile of cHCC-CCA and determine its impact on disease outcome. EXPERIMENTAL DESIGN: We performed a multicenter study of 96 patients with cHCC-CCA. Gene expression profile was analyzed using nCounter PanCancer IO 360 Panel. Densities of main immune cells subsets were quantified from digital slides of IHC stainings. Genetic alterations were investigated using targeted next-generation sequencing. RESULTS: Two main immune subtypes of cHCC-CCA were identified by clustering analysis: an "immune-high" (IH) subtype (57% of the cases) and an "immune-low" (IL) subtype (43% of the cases). Tumors classified as IH showed overexpression of genes related to immune cells recruitment, adaptive and innate immunity, antigen presentation, cytotoxicity, immune suppression, and inflammation (P < 0.0001). IH cHCC-CCAs also displayed activation of gene signatures recently shown to be associated with response to immunotherapy in patients with HCC. Quantification of immunostainings confirmed that IH tumors were also characterized by higher densities of immune cells. Immune subtypes were not associated with any genetic alterations. Finally, multivariate analysis showed that the IH subtype was an independent predictor of improved overall survival. CONCLUSIONS: We have identified a subgroup of cHCC-CCA that displays features of an ongoing intratumor immune response, along with an activation of gene signatures predictive of response to immunotherapy in HCC. This tumor subclass is associated with an improved clinical outcome. These findings suggest that a subset of patients with cHCC-CCA may benefit from immunomodulating therapeutic approaches.

Autofluorescence imaging within the liver: a promising tool for the detection and characterization of primary liver tumors.

OBJECTIVES: To assess the performance of 405 nm-induced autofluorescence for the characterization of primary liver nodules on ex vivo resected specimens. MATERIALS AND METHODS: Forty resected liver specimens bearing 53 primary liver nodules were included in this IRB-approved prospective study. Intratissular spectroscopic measurements were performed using a 25-G fibered-needle on all ex vivo specimens: 5 autofluorescence measurements were performed in both nodules and adjacent parenchyma. The spectra derivatives of the 635 and 670 nm autofluorescence peaks observed in nodules and in adjacent liver parenchyma were compared (Kruskal-Wallis and Mann-Whitney when appropriate). RESULTS: A total of 42 potentially evolutive primary liver nodules-34 hepatocellular carcinomas, 4 intrahepatic cholangiocarcinomas, 4 hepatocellular adenomas-and 11 benign nodules-5 focal nodular hyperplasias, 6 regenerative nodules-were included. Both 635 and 670 nm Δderivatives were significantly higher in benign as compared to potentially evolutive (PEV) nodules (respectively 32.9 ± 4.5 vs 15.3 ± 1.4; p < 0.0001 and 5.7 ± 0.6 vs 2.5 ± 0.1; p < 0.0001) with respective sensitivity and specificity of 78% and 91% for distinguishing PEV from benign nodules. CONCLUSION: 405 nm-induced autofluorescence enables the discrimination of benign from PEV primary liver nodules, suggesting that autofluorescence imaging could be used to optimize US targeted liver biopsies. KEY POINTS: • 405 nm-induced autofluorescence can distinguish liver tumors from the adjacent liver parenchyma. • The analysis of autofluorescence imaging observed within primary liver tumors can discriminate benign tumors from those requiring follow-up or targeted liver biopsy. • In current practice, autofluorescence imaging could be embedded within biopsy needle, to enable, in addition to ultrasound guidance, optimal targeting of liver nodules which could optimize tissue sampling.

Kupffer cell restoration after partial hepatectomy is mainly driven by local cell proliferation in IL-6-dependent autocrine and paracrine manners.

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.

Immunopathobiology and therapeutic targets related to cytokines in liver diseases.

Chronic liver injury with any etiology can progress to fibrosis and the end-stage diseases cirrhosis and hepatocellular carcinoma. The progression of liver disease is controlled by a variety of factors, including liver injury, inflammatory cells, inflammatory mediators, cytokines, and the gut microbiome. In the current review, we discuss recent data on a large number of cytokines that play important roles in regulating liver injury, inflammation, fibrosis, and regeneration, with a focus on interferons and T helper (Th) 1, Th2, Th9, Th17, interleukin (IL)-1 family, IL-6 family, and IL-20 family cytokines. Hepatocytes can also produce certain cytokines (such as IL-7, IL-11, and IL-33), and the functions of these cytokines in the liver are briefly summarized. Several cytokines have great therapeutic potential, and some are currently being tested as therapeutic targets in clinical trials for the treatment of liver diseases, which are also described.

Early Hepatic Lesions Display Immature Tertiary Lymphoid Structures and Show Elevated Expression of Immune Inhibitory and Immunosuppressive Molecules.

PURPOSE: The impact of tertiary lymphoid structures (TLS) in hepatocellular carcinoma (HCC) progression is being extensively investigated. However, their presence during the early steps of human liver carcinogenesis remains unknown. We thus aimed to determine whether TLS are induced in preneoplastic/early hepatic lesions (EHL), and whether they are associated with a particular immune profile. EXPERIMENTAL DESIGN: A series of 127 EHLs (low/high-grade dysplastic nodules, early HCC, and small and progressed HCC) was included in the study. TLSs were investigated by pathologic reviewing. Densities of immune cells were assessed using IHC. A subset of lesions was microdissected and gene expression profiling was performed with a custom NanoString panel. RESULTS: Compared with surrounding cirrhotic nodules, EHL of all stages displayed increased densities of T cells, B cells, and dendritic cells. Immature TLSs were identified in 24% of EHL. Gene expression profiling identified a subset of EHL with elevated mRNA levels of various cytokines involved in immune cells' recruitment and TLS induction. This subgroup of EHL also showed overexpression of genes related to T- and B-cells' activation and antigen presentation, as well as those related to immunosuppression and immune exhaustion. CONCLUSIONS: Local immune activation occurs in the very early steps of liver carcinogenesis; however, it may not be fully efficient and paradoxically favor immune evasion and progression to full-blown HCC. These results have implications for the development of anti-HCC chemopreventive strategies in cirrhotic patients.

Intrahepatic immune changes after hepatitis c virus eradication by direct-acting antiviral therapy.

BACKGROUND & AIMS: The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. METHODS: Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. RESULTS: Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. CONCLUSION: In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

Angiogenesis and immune checkpoint inhibitors as therapies for hepatocellular carcinoma: current knowledge and future research directions.

Hepatocellular carcinoma (HCC) is the second deadliest cancer worldwide, due to its high incidence and poor prognosis. Frequent initial presentation at advanced stages along with impaired liver function limit the use of a broad therapeutic arsenal in patients with HCC. Although main HCC oncogenic drivers have been deciphered in recent years (TERT, TP53, CTNNB1 mutations, miR122 and CDKN2A silencing), therapeutic applications derived from this molecular knowledge are still limited. Given its high vascularization and immunogenicity, antiangiogenics and immune checkpoint inhibitors (ICI), respectively, are two therapeutic approaches that have shown efficacy in HCC. Depending on HCC immune profile, combinations of these therapies aim to modify the protumoral/antitumoral immune balance, and to reactivate and favor the intratumoral trafficking of cytotoxic T cells. Combination therapies involving antiangiogenics and ICI may be synergistic, because vascular endothelial growth factor A inhibition increases intratumoral infiltration and survival of cytotoxic T lymphocytes and decreases regulatory T lymphocyte recruitment, resulting in a more favorable immune microenvironment for ICI antitumoral activity. First results from clinical trials evaluating combinations of these therapies are encouraging with response rates never observed before in patients with HCC. A better understanding of the balance and interactions between protumoral and antitumoral immune cells will help to ensure the success of future therapeutic trials. Here, we present an overview of the current state of clinical development of antitumoral therapies in HCC and the biological rationale for their use. Moreover, translational studies on tumor tissue and blood, prior to and during treatment, will help to identify biomarkers and immune signatures with predictive value for both clinical outcome and response to combination therapies.

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

Interleukins-17 and 27 promote liver regeneration by sequentially inducing progenitor cell expansion and differentiation.

Liver progenitor cells (LPCs)/ductular reactions (DRs) are associated with inflammation and implicated in the pathogenesis of chronic liver diseases. However, how inflammation regulates LPCs/DRs remains largely unknown. Identification of inflammatory processes that involve LPC activation and expansion represent a key step in understanding the pathogenesis of liver diseases. In the current study, we found that diverse types of chronic liver diseases are associated with elevation of infiltrated interleukin (IL)-17-positive (+) cells and cytokeratin 19 (CK19)+ LPCs, and both cell types colocalized and their numbers positively correlated with each other. The role of IL-17 in the induction of LPCs was examined in a mouse model fed a choline-deficient and ethionine-supplemented (CDE) diet. Feeding of wild-type mice with the CDE diet markedly elevated CK19+Ki67+ proliferating LPCs and hepatic inflammation. Disruption of the IL-17 gene or IL-27 receptor, alpha subunit (WSX-1) gene abolished CDE diet-induced LPC expansion and inflammation. In vitro treatment with IL-17 promoted proliferation of bipotential murine oval liver cells (a liver progenitor cell line) and markedly up-regulated IL-27 expression in macrophages. Treatment with IL-27 favored the differentiation of bipotential murine oval liver cells and freshly isolated LPCs into hepatocytes. Conclusion: The current data provide evidence for a collaborative role between IL-17 and IL-27 in promoting LPC expansion and differentiation, respectively, thereby contributing to liver regeneration. (Hepatology Communications 2018;2:329-343).

Programmed death ligand 1 expression in hepatocellular carcinoma: Relationship With clinical and pathological features.

The prognosis of hepatocellular carcinoma (HCC) remains poor, with only one third of patients eligible for curative treatments and very limited survival benefits with the use of sorafenib, the current standard of care for advanced disease. Recently, agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 (PD-1) immune checkpoint were shown to display impressive antitumor activity in various solid or hematological malignancies, including HCC. PD-L1 immunohistochemical expression is thought to represent a biomarker predictive of drug sensitivity. Here, we investigated PD-L1 expression in a series of 217 HCCs and correlated our results with clinical and histological features and immunohistochemical markers (PD-1, cytokeratin 19, glutamine synthetase, and ß-catenin expression). PD-L1 expression by neoplastic cells was significantly associated with common markers of tumor aggressiveness (high serum alpha-fetoprotein levels, P = 0.038; satellite nodules, P < 0.001; macrovascular invasion, P < 0.001; microvascular invasion, P < 0.001; poor differentiation, P < 0.001) and with the progenitor subtype of HCC (cytokeratin 19 expression, P = 0.031). High PD-L1 expression by inflammatory cells from the tumor microenvironment also correlated with high serum alpha-fetoprotein levels (P < 0.001), macrovascular invasion (P = 0.001), poor differentiation (P = 0.001), high PD-1 expression (P < 0.001), and the so-called lymphoepithelioma-like histological subtype of HCC (P = 0.003). CONCLUSION: PD-L1 expression by either neoplastic or intratumoral inflammatory cells is related to tumor aggressiveness and suggests that the response to treatments targeting the PD-L1/PD-1 immune checkpoint could be restricted to particular HCC variants; thus, enrichment of these tumor subtypes in future clinical trials should be considered. (Hepatology 2016;64:2038-2046).

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.

M2 Kupffer cells promote M1 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease.

UNLABELLED: Alcoholic and nonalcoholic fatty liver disease (ALD and NAFLD) are the predominant causes of liver-related mortality in Western countries. We have shown that limiting classical (M1) Kupffer cell (KC) polarization reduces alcohol-induced liver injury. Herein, we investigated whether favoring alternatively activated M2 KCs may protect against ALD and NAFLD. Ongoing alcohol drinkers and morbidly obese patients, with minimal hepatic injury and steatosis, displayed higher hepatic expression of M2 genes, as compared to patients with more severe liver lesions; individuals with limited liver lesions showed negligible hepatocyte apoptosis but significant macrophage apoptosis. Experiments in mouse models of ALD or NAFLD further showed that BALB/c or resveratrol-treated mice fed alcohol or a high-fat diet displayed preponderant M2 KC polarization, M1 KC apoptosis, and resistance to hepatocyte steatosis and apoptosis, as compared to control C57BL6/J mice. In vitro experiments in isolated KC, peritoneal, and Raw264.7 macrophages demonstrated that M1 macrophage apoptosis was promoted by conditioned medium from macrophages polarized into an M2 phenotype by either interleukin (IL)4, resveratrol, or adiponectin. Mechanistically, IL10 released from M2 cells promoted M1 death, and anti-IL10 antibodies blunted the proapoptic effects of M2-conditioned media. IL10 secreted by M2 KCs promoted selective M1 death by a mechanism involving activation of arginase in high inducible nitric oxide synthase-expressing M1 KCs. In alcohol-exposed mice, neutralization of IL10 impaired M1 apoptosis. CONCLUSION: These data uncover a novel mechanism regulating the M1/M2 balance that relies on apoptotic effects of M2 KCs towards their M1 counterparts. They suggest that promoting M2-induced M1 KC apoptosis might prove a relevant strategy to limit alcohol- and high fat-induced inflammation and hepatocyte injury.

Cannabinoid receptor 2 counteracts interleukin-17-induced immune and fibrogenic responses in mouse liver.

UNLABELLED: Interleukin (IL)-17 is a proinflammatory and fibrogenic cytokine mainly produced by T-helper (Th)17 lymphocytes, together with the hepatoprotective and antifibrogenic cytokine, IL-22. Cannabinoid receptor 2 (CB2) is predominantly expressed in immune cells and displays anti-inflammatory and antifibrogenic effects. In the present study, we further investigated the mechanism underlying antifibrogenic properties of CB2 receptor and explored its effect on the profibrogenic properties of IL-17. After bile duct ligation (BDL), the hepatic expression of Th17 markers and IL-17 production were enhanced in CB2(-/-) mice, as compared to wild-type (WT) counterparts, and correlated with increased fibrosis in these animals. In contrast, IL-22-induced expression was similar in both animal groups. Inhibition of Th17 differentiation by digoxin lowered Th17 marker gene expression and IL-17 production and strongly reduced liver fibrosis in CB2(-/-) BDL mice. In vitro, differentiation of CD4(+) naïve T cells into Th17 lymphocytes was decreased by the CB2 agonist, JWH-133, and was associated with reduced Th17 marker messenger RNA expression and IL-17 production, without modification of IL-22 release. The inhibitory effect of JWH-133 on IL-17 production relied on signal transducer and activator of transcription (STAT)5 phosphorylation. Indeed, STAT5 phosphorylation and translocation into the nucleus was enhanced in JWH133-treated Th17 lymphocytes, and the addition of a STAT5 inhibitor reversed the inhibitory effect of the CB2 agonist on IL-17 production, without affecting IL-22 levels. Finally, in vitro studies also demonstrated that CB2 receptor activation in macrophages and hepatic myofibroblasts blunts IL-17-induced proinflammatory gene expression. CONCLUSION: These data demonstrate that CB2 receptor activation decreases liver fibrosis by selectively reducing IL-17 production by Th17 lymphocytes via a STAT5-dependent pathway, and by blunting the proinflammatory effects of IL-17 on its target cells, while preserving IL-22 production.

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.

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

UNLABELLED: 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(-/-) STAT3(Hep-/-) 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.

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.