Yoon Seok Roh

Toll‐like receptors in alcoholic liver disease, non‐alcoholic steatohepatitis and carcinogenesis

Activation of innate immune systems including Toll-like receptor (TLR) signaling is a key in chronic liver disease. Recent studies suggest that gut microflora-derived bacterial products (i.e. lipopolysaccharide [LPS], bacterial DNA) and endogenous substances (i.e. high-mobility group protein B1 [HMGB1], free fatty acids) released from damaged cells activate hepatic TLRs that contribute to the development of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) and liver fibrosis. The crucial role of TLR4, a receptor for LPS, has been implicated in the development of ASH, NASH, liver fibrosis, and hepatocellular carcinoma. However, the role of other TLRs, such as TLR2 and TLR9 in chronic liver disease remains less clear. In this review, we will discuss the role of TLR2, 4, and 9 in Kupffer cells and hepatic stellate cells in the development of ASH, NASH, and hepatocarcinogenesis.

Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice

Transforming growth factor beta (TGF‐β) signaling activates Smad‐ and TGF‐β‐activated kinase 1 (TAK1)‐dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF‐β signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild‐type (WT) and hepatocyte‐specific TGF‐β receptor type II‐deficient (Tgfbr2ΔHEP) mice were fed a choline‐deficient amino acid (CDAA)‐defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2ΔHEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitate‐induced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF‐β‐mediated death. TGF‐β‐mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2ΔHEP mice was accompanied by the increased expression of genes involved in β‐oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF‐β signaling promoted lipid accumulation with induction of lipogenesis‐related genes and suppression of β‐oxidation‐related genes in hepatocytes. Silencing Smad2 decreased TGF‐β‐mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3. Conclusions: TGF‐β signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH. (Hepatology 2014;59:483–495)

Transforming Growth Factor–β Signaling in Hepatocytes Promotes Hepatic Fibrosis and Carcinogenesis in Mice With Hepatocyte-Specific Deletion of TAK1

BACKGROUND & AIMS Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is activated in different cytokine signaling pathways. Deletion of Tak1 from hepatocytes results in spontaneous development of hepatocellular carcinoma (HCC), liver inflammation, and fibrosis. TGF-β activates TAK1 and Smad signaling, which regulate cell death, proliferation, and carcinogenesis. However, it is not clear whether TGF-β signaling in hepatocytes, via TGF-β receptor-2 (Tgfbr2), promotes HCC and liver fibrosis. METHODS We generated mice with hepatocyte-specific deletion of Tak1 (Tak1ΔHep), as well as Tak1/Tgfbr2DHep and Tak1/Smad4ΔHep mice. Tak1flox/flox, Tgfbr2ΔHep, and Smad4ΔHep mice were used as controls, respectively. We assessed development of liver injury, inflammation, fibrosis, and HCC. Primary hepatocytes isolated from these mice were used to assess TGF-β-mediated signaling. RESULTS Levels of TGF-β, TGF-βR2, and phospho-Smad2/3 were increased in HCCs from Tak1ΔHep mice, which developed liver fibrosis and inflammation by 1 month and HCC by 9 months. However, Tak1/Tgfbr2ΔHep mice did not have this phenotype, and their hepatocytes did not undergo spontaneous cell death or compensatory proliferation. Hepatocytes from Tak1ΔHep mice incubated with TGF-β did not activate p38, c-Jun N-terminal kinase, or nuclear factor-κB; conversely, TGF-β-mediated cell death and phosphorylation of Smad2/3 were increased, compared with control hepatocytes. Blocking the Smad pathway inhibited TGF-β-mediated death of Tak1-/- hepatocytes. Accordingly, disruption of Smad4 reduced the spontaneous liver injury, inflammation, fibrosis, and HCC that develops in Tak1ΔHep mice. Levels of the anti-apoptotic protein Bcl-xL, β-catenin, connective tissue growth factor, and vascular endothelial growth factor were increased in HCC from Tak1ΔHep mice, but not in HCCs from Tak1/Tgfbr2ΔHep mice. Injection of N-nitrosodiethylamine induced HCC formation in wild-type mice, but less in Tgfbr2ΔHep mice. CONCLUSIONS TGF-β promotes development of HCC in Tak1ΔHep mice by inducing hepatocyte apoptosis and compensatory proliferation during early phases of tumorigenesis, and inducing expression of anti-apoptotic, pro-oncogenic, and angiogenic factors during tumor progression.

TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis

Progressive liver fibrosis is characterized by the deposition of collagen by activated hepatic stellate cells (HSCs). Activation of HSCs is a multiple receptor-driven process in which profibrotic signals are enhanced, and anti-fibrotic pathways are suppressed. Here we report the discovery of a novel signaling platform comprised of G protein subunit, Gαi and GIV, its guanine exchange factor (GEF), which serves as a central hub within the fibrogenic signalling network initiated by diverse classes of receptors. GIV is expressed in the liver after fibrogenic injury and is required for HSC activation. Once expressed, GIV enhances the profibrotic (PI3K-Akt-FoxO1 and TGFβ-SMAD) and inhibits the anti-fibrotic (cAMP-PKA-pCREB) pathways to skew the signalling network in favor of fibrosis, all via activation of Gαi. We also provide evidence that GIV may serve as a biomarker for progression of fibrosis after liver injury and a therapeutic target for arresting and/or reversing HSC activation during liver fibrosis.

TAK1 regulates hepatic cell survival and carcinogenesis

TGF-β-activated kinase 1 (TAK1 or MAP3K7) is an intracellular hub molecule that regulates both nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways that play key roles in development, cell survival, immune response, metabolism, and carcinogenesis. TAK1 activity is tightly regulated by its binding proteins, TAB1 and TAB2/TAB3, as well as by post-translational modification including ubiquitination and phosphorylation. Accumulating evidence demonstrates that TAK1 plays a role in tumor initiation, progression, and metastasis as a tumor prompter or tumor suppressor. An understanding of the role of TAK1 in liver physiology and diseases is required for the development of therapeutic agencies targeting TAK1. In this review, we highlight the activation mechanism and pathophysiological roles of TAK1 in the liver.

TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

Toll-like receptor 7-mediated Type I Interferon signaling prevents cholestasis- and hepatotoxin-induced liver fibrosis

Toll‐like receptor 7 (TLR7) signaling predominantly regulates production of type I interferons (IFNs), which has been suggested in clinical studies to be antifibrotic. However, the mechanistic role of the TLR7‐type I IFN axis in liver fibrosis has not been elucidated. In the present study, liver fibrosis was induced in wild‐type (WT), TLR7‐deficient, and IFN‐α/β receptor‐1 (IFNAR1)‐deficient mice and TLR7‐mediated signaling was assessed in liver cells isolated from these mice. TLR7‐deficient and IFNAR1‐deficient mice were more susceptible to liver fibrosis than WT mice, indicating that TLR7‐type I IFN signaling exerts a protective effect against liver fibrosis. Notably, the hepatic expression of interleukin‐1 receptor antagonist (IL‐1ra) was suppressed in TLR7‐ or IFNAR1‐deficient mice compared with respective WT mice, and treatment with recombinant IL‐1ra reduced liver fibrosis. In vivo activation of TLR7 significantly increased IFNa4 and IL‐1ra expression in the liver. Interestingly, each cytokine had a different cellular source, showing that dendritic cells (DCs) are the responsible cell type for production of type I IFN, while Kupffer cells (KCs) mainly produce IL‐1ra in response to type I IFN. Furthermore, TLR7 activation by R848 injection suppressed liver fibrosis and production of proinflammatory cytokines, and these effects were dependent on type I IFN signaling. Consistent with in vivo data, IFN‐α significantly induced IL‐1ra production in primary KCs. Conclusion: TLR7 signaling activates DCs to produce type I IFN, which in turn induces antifibrogenic IL‐1ra production in KCs. Thus, manipulation of the TLR7‐type I IFN‐IL‐1ra axis may be a new therapeutic strategy for the treatment of liver fibrosis. (Hepatology 2014;60:237–249)

Protective effects of red ginseng extract against vaginal herpes simplex virus infection

Numerous studies have suggested that Korean red ginseng (KRG) extract has various immune modulatory activities both in vivo and in vitro. In this study, we used a mouse model to examine the effects of orally administered KRG extract on immunity against herpes simplex virus (HSV). Balb/c mice were administered with 100, 200, and 400 mg/kg oral doses of KRG extract for 10 d and then vaginally infected with HSV. We found that KRG extract rendered recipients more resistant against HSV vaginal infection and further systemic infection, including decreased clinical severity, increased survival rate, and accelerated viral clearance. Such results appeared to be mediated by increased vaginal IFN-γ secretion. Moreover, increased mRNA expression of IFN-γ, granzyme B, and Fas-ligand was identified in the iliac lymph node and vaginal tracts of KRG extract treated groups (200 and 400 mg/kg). These results suggest that the activities of local natural killer cells were promoted by KRG extract consumption and that KRG may be an attractive immune stimulator for helping hosts overcome HSV infection.

TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

Background & Aims Toll-like receptor 4 (TLR4) signaling is activated through 2 adaptor proteins: MyD88 and TIR-domain containing adaptor-inducing interferon-β (TRIF). TLR4 and MyD88 are crucial in nonalcoholic steatohepatitis (NASH) and fibrosis. However, the role of TRIF in TLR4-mediated NASH and fibrosis has been elusive. This study investigated the differential roles of TRIF in hepatic steatosis and inflammation/fibrosis. Methods A choline-deficient amino acid defined (CDAA) diet was used for the mouse NASH model. On this diet, the mice develop hepatic steatosis, inflammation, and fibrosis. TLR4 wild-type and TLR4-/- bone marrow chimeric mice and TRIF-/- mice were fed CDAA or a control diet for 22 weeks. Hepatic steatosis, inflammation, and fibrosis were examined. Results In the CDAA diet–induced NASH, the mice with wild-type bone marrow had higher alanine aminotransferase and hepatic tumor necrosis factor levels than the mice with TLR4-/- bone marrow. The nonalcoholic fatty liver disease activity score showed that both wild-type and TLR4-/- bone marrow chimeras had reduced hepatic steatosis, and that both types of chimeras had similar levels of inflammation and hepatocyte ballooning to whole-body wild-type mice. Notably, wild-type recipients showed more liver fibrosis than TLR4-/- recipients. Although TRIF-/- mice showed reduced hepatic steatosis, these mice showed more liver injury, inflammation, and fibrosis than wild-type mice. TRIF-/- stellate cells and hepatocytes produced more C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand than wild-type cells in response to lipopolysaccharide. Consistently, TRIF-/- mice showed increased CXCL1 and CCL3 expression along with neutrophil and macrophage infiltration, which promotes liver inflammation and injury. Conclusions In TLR4-mediated NASH, different liver cells have distinct roles in hepatic steatosis, inflammation, and fibrosis. TRIF promotes hepatic steatosis but it inhibits injury, inflammation, and fibrosis.