Hisafumi Yamagata

CD1d-restricted natural killer T cells contribute to hepatic inflammation and fibrogenesis in mice

BACKGROUND & AIMS Several lines of evidence suggest that innate immunity plays a key role in hepatic fibrogenesis. To clarify the role of natural killer (NK) T cells in hepatic inflammation and fibrogenesis, we here investigated xenobiotics-induced liver injury and subsequent fibrogenesis in mice lacking mature NKT cells caused by genetic disruption of the CD1d molecule. METHODS Male CD1d-knockout (KO) and wild-type (WT) mice were given repeated intraperitoneal injections of thioacetamide (TAA, 3times/week; 0.1-0.2mg/g BW) for up to 9 weeks, or a single intraperitoneal injection of CCl(4) (1 μl/g). Liver histology was evaluated, and expression levels of cytokines and matrix-related genes in the liver were quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS Mortality following repeated injections of TAA was prevented almost completely in CD1d-KO mice. TAA-induced inflammatory responses and hepatocellular damage were markedly ameliorated in CD1d-KO mice. TAA-induced expression of smooth muscle α-actin (SMA) and transforming growth factor (TGF)β1 mRNA in the liver were also prevented largely in CD1d-KO mice. In fact, CD1d-KO mice developed minimal hepatic fibrosis after 9-weeks of administration of TAA, which caused overt bridging fibrosis in WT mice. Indeed, TAA-induced increases in α1(I)procollagen (COL1A1) and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA were blunted significantly in CD1d-KO mice. Similarly, acute CCl(4)-induced hepatic injury and subsequent profibrogenic responses were also reduced significantly in CD1d-KO mice. CONCLUSIONS These findings clearly indicated that CD1d-restricted NKT cells contribute to xenobiotics-induced hepatic inflammation, hepatocellular damage, and subsequent profibrogenic responses in the liver.

Innate immune responses involving natural killer and natural killer T cells promote liver regeneration after partial hepatectomy in mice

To clarify the roles of innate immune cells in liver regeneration, here, we investigated the alteration in regenerative responses after partial hepatectomy (PH) under selective depletion of natural killer (NK) and/or NKT cells. Male, wild-type (WT; C57Bl/6), and CD1d-knockout (KO) mice were injected with anti-NK1.1 or anti-asialo ganglio-N-tetraosylceramide (GM1) antibody and then underwent the 70% PH. Regenerative responses after PH were evaluated, and hepatic expression levels of cytokines and growth factors were measured by real-time RT-PCR and ELISA. Phosphorylation of STAT3 was detected by Western blotting. Depletion of both NK and NKT cells with an anti-NK1.1 antibody in WT mice caused drastic decreases in bromodeoxyuridine uptake, expression of proliferating cell nuclear antigen, and cyclin D1, 48 h after PH. In mice given NK1.1 antibody, increases in hepatic TNF-α, IL-6/phospho-STAT3, and hepatocyte growth factor (HGF) levels following PH were also blunted significantly, whereas IFN-γ mRNA levels were not different. CD1d-KO mice per se showed normal liver regeneration; however, pretreatment with an antiasialo GM1 antibody to CD1d-KO mice, resulting in depletion of both NK and NKT cells, also blunted regenerative responses. Collectively, these observations clearly indicated that depletion of both NK and NKT cells by two different ways results in impaired liver regeneration. NK and NKT cells most likely upregulate TNF-α, IL-6/STAT3, and HGF in a coordinate fashion, thus promoting normal regenerative responses in the liver.

Radiofrequency ablation of hepatocellular carcinoma: The feasibility of magnetic resonance imaging with gadolinium ethoxybenzyl diethylene triamine pentaacetic acid for evaluating the ablative margin

Aim:  The aim of this study was to evaluate the feasibility of gadolinium ethoxybenzyl diethylene triamine pentaacetic acid (Gd‐EOB‐DTPA) in magnetic resonance imaging (MRI) to assess the ablative margin of radiofrequency (RF) ablation to hepatocellular carcinoma (HCC).

Altered expression and function of hepatic natural killer T cells in obese and diabetic KK-Ay mice

Aim:  To evaluate the role of natural killer (NK)T cells in the pathogenesis of non‐alcoholic steatohepatitis (NASH), here we investigated the expression and function of hepatic NKT cells in KK‐Ay mice, an animal model of metabolic syndrome.

103 CD1D-RESTRICTED NATURAL KILLER T CELLS CONTRIBUTE TO THIOACETAMIDE-INDUCED HEPATIC INFLAMMATION AND FIBROGENESIS IN MICE

T-cells were similarly decreased in all fibrotic-groups. NK-cells decreased following fibrosis in control groups AD however, both treated groups BC as compared to WT. In-vitro, increased pGEM7-concentrations elevated hepatic-stellate-cell (HSC) proliferation, however, T-cell proliferation decreased. Conclusions: TLR9 activation had inversed effects on lymphocytes and HSCs. The net balance of TLR9 activation in WT, displayed significant anti-fibrotic activity, accompanied by CD8 suppression and increased NK-cells. TLR9 fibrosis model had decreased fibrosis (due to direct HSC block of activation) but increased ALT levels (due to increased lymphocyte proliferation and hepatocyte injury).

S1833 Ursolic Acid Ameliorates Dietary Steatohepatitis in Obese Diabetic Kk-aY Mice

Background and aims: Metabolic syndrome-related NASH is one of the emerging healththreatening problems in industrialized countries worldwide. Despite of numerous experimental/clinical challenges, therapeutic strategies for NASH have not been well established. In this study, we investigated the therapeutic effect of ursolic acid, a pentacyclic triterpene acid, on high-fat diet (HFD)-induced steatohepatitis in obese, diabetic KK-Ay mice.Methods: Male, 8-week old KK-Aymice were fed a diet containing 32% fat for 4 weeks, and subsequently given daily intra-gastric injections of UA (100 mg/kg BW, suspended in 0.5% metyl-cellulose solution) or vehicle alone in combination with continuous HFD-feeding for up to 4 weeks. Liver histology was assessed by H-E staining. Serum aminotransferases and tissue triglyceride levels were determined colorimetrically by standard enzymatic methods. Steady state mRNA levels for SREBP-1c, fatty acid synthase (FAS), enoyl-CoA hydratase (ECHD) and acyl-CoA oxidase 1 (ACOX1) in the liver were analyzed quantitatively by real-time RT-PCR. Results: All KK-Ay mice gained body weight constantly during 4-week pre-feeding period, and the control mice continued gaining body weight over 10% during the subsequent 4-week treatment period; however, mice given UA lost body weight nearly 7% (P<0.001 vs. control group) despite of the equivalent dietary consumption. Except for body weight loss, mice treated with UA appeared to be healthy. KK-Ay mice developed remarkable steatohepatitis following 4-week pre-feeding with HFD as expected; however, treatment with UA dramatically improved liver pathology within 1 week, and the effect was sustained for 4-week treatment period. Indeed, serum transaminases levels, as well as hepatic triglyceride contents, were decreased significantly in mice treated with UA. Interestingly, mice treated with UA showed significantly decreased expression of hepatic SREBP-1c and FAS mRNA levels, indicating that fatty acid synthesis is down-regulated by UA. Further, hepatic mRNA levels of β-oxidation enzymes such as ECHD and ACOX1 were also significantly lower in UAtreated mice.Conclusions: These findings clearly demonstrated that ursolic acid significantly improves HFD-induced severe steatohepatitis in KK-Ay mice without dietary restriction. The mechanisms underlying this therapeutic effect of UA most likely involve decreased fatty acid synthesis and radical-generating β-oxidation in the liver. Since this chemical is an active ingredient of herbal medicine, it is postulated that UA is useful for a new preventive/ therapeutic reagent for NASH.