Tomonori Aoyama

Toll-Like Receptor 9 Promotes Steatohepatitis by Induction of Interleukin-1β in Mice

BACKGROUND & AIMS Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)-containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1beta (IL-1beta) in steatohepatitis, fibrosis, and insulin resistance. METHODS Wild-type (WT), TLR9(-/-), IL-1 receptor (IL-1R)(-/-), and MyD88(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. RESULTS The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9(-/-) mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R(-/-) mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9(-/-), IL-1R(-/-), and MyD88(-/-) mice had less insulin resistance than WT mice on the CDAA diet. CONCLUSIONS In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.

BACKGROUND & AIMS Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice. METHODS Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice). RESULTS In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis. CONCLUSIONS IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis.

Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis

TGF-β–activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1ΔHEP) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase in Experimental Liver Fibrosis: GKT137831 as a Novel Potential Therapeutic Agent

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex, including Ras‐related botulinum toxin substrate 1 (Rac1). Superoxide dismutase 1 (SOD1) interacts with the NOX‐Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. To induce liver fibrosis, wild‐type (WT) and SOD1mu mice were treated with CCl4 or bile duct ligation (BDL). Then, to address the role of NOX‐SOD1‐mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of thiobarbituric acid reactive substances and NOX‐related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor, GKT137831, attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as messenger RNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and tumor growth factor beta up‐regulated NOX4, but Ang II required NOX1. Conclusions: SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 up‐regulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis. (HEPATOLOGY 2012)

CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice.

Chronic liver disease is associated with hepatocyte injury, inflammation, and fibrosis. Chemokines and chemokine receptors are key factors for the migration of inflammatory cells such as macrophages and noninflammatory cells such as hepatic stellate cells (HSCs). The expression of CX3CR1 and its ligand, CX3CL1, is up‐regulated in chronic liver diseases such as chronic hepatitis C. However, the precise role of CX3CR1 in the liver is still unclear. Here we investigated the role of the CX3CL1‐CX3CR1 interaction in a carbon tetrachloride (CCl4)–induced liver inflammation and fibrosis model. CX3CR1 was dominantly expressed in Kupffer cells in the liver. In contrast, the main source of CX3CL1 was HSCs. Mice deficient in CX3CR1 showed significant increases in inflammatory cell recruitment and cytokine production [including tumor necrosis factor α (TNF‐α); monocyte chemoattractant protein 1; macrophage inflammatory protein 1β; and regulated upon activation, normal T cell expressed, and secreted (RANTES)] after CCl4 treatment versus wild‐type (WT) mice. This suggested that CX3CR1 signaling prevented liver inflammation. Kupffer cells in CX3CR1‐deficient mice after CCl4 treatment showed increased expression of TNF‐α and transforming growth factor β and reduced expression of the anti‐inflammatory markers interleukin‐10 (IL‐10) and arginase‐1. Coculture experiments showed that HSCs experienced significantly greater activation by Kupffer cells from CCl4‐treated CX3CR1‐deficient mice versus WT mice. Indeed, augmented fibrosis was observed in CX3CR1‐deficient mice versus WT mice after CCl4 treatment. Finally, CX3CL1 treatment induced the expression of IL‐10 and arginase‐1 in WT cultured Kupffer cells through CX3CR1, which in turn suppressed HSC activation. Conclusion: The CX3CL1‐CX3CR1 interaction inhibits inflammatory properties in Kupffer cells/macrophages and results in decreased liver inflammation and fibrosis. (Hepatology 2010)

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Role of NADPH Oxidases in Liver Fibrosis

SIGNIFICANCE Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. RECENT ADVANCES In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. CRITICAL ISSUES Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. FUTURE DIRECTIONS A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antio

Toll-like receptor signaling and liver fibrosis.

Liver fibrosis occurs as a wound-healing scar response following acute and chronic liver inflammation including alcoholic liver disease, non-alcoholic steatohepatitis, hepatitis B and C, and autoimmune hepatitis. Myofibroblasts, mainly transdifferentiated from hepatic stellate cells, are pivotal cell types that produce fibrillar collagen. The activation of inflammatory cells, including Kupffer cells, is a crucial step for activating hepatic stellate cells. Toll-like receptors (TLRs) are pattern recognition receptors that sense pathogen-associated molecular patterns (PAMPs), which discriminate the products of microorganisms from the host. TLRs are expressed on Kupffer cells, endothelial cells, dendritic cells, biliary epithelial cells, hepatic stellate cells, and hepatocytes in the liver. TLR signaling induces potent innate immune responses in these cell types. The liver is constantly exposed to PAMPs, such as LPS and bacterial DNA through bacterial translocation because there is a unique anatomical link, the portal vein system between liver and intestine. Recent evidence demonstrates the role of TLRs in the activation of hepatic immune cells and stellate cells during liver fibrosis. Moreover, crosstalk between TLR4 signaling and TGF-β signaling in hepatic stellate cells has been reported. This paper highlights the role of TLR signaling in stellate cell activation and the progression of liver fibrosis.

Abnormality of autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease

Recent evidences indicate that hepatic steatosis suppresses autophagic proteolysis. The present study evaluated the correlation between autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease (NAFLD).

Role of apoptosis in acetaminophen hepatotoxicity

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen‐induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen‐induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 μmol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP‐generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP‐depletion‐dependent necrosis or caspase‐dependent apoptosis as determined, in part, by ATP availability from glycolysis.