Kiminori Kimura

Interleukin-18 Inhibits Hepatitis B Virus Replication in the Livers of Transgenic Mice

ABSTRACT Interleukin-18 (IL-18) produced by activated antigen-presenting cells stimulates natural killer (NK) cells, natural killer T (NKT) cells, and T cells to secrete gamma interferon (IFN-γ). In this study, injection of a single 10-μg dose of recombinant murine IL-18 rapidly, reversibly, and noncytopathically inhibited hepatitis B virus (HBV) replication in the livers of HBV transgenic mice. Furthermore, HBV replication was inhibited by as little as 1 μg of IL-18 injected repetitively, and also by a single 0.1-μg dose of IL-18 injected together with 1 ng of IL-12, neither of which inhibited HBV replication individually, demonstrating synergy between these cytokines in this system. The antiviral effect of IL-18 was mediated by its ability to activate resident intrahepatic NK cells and NKT cells to produce IFN-γ and by its ability to induce IFN-α/β production in the liver. These results suggest that IL-18 has the potential to contribute to the control of HBV replication during self-limited infection and that it may have therapeutic value for the treatment of patients with chronic hepatitis.

Hot spots in prion protein for pathogenic conversion

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrPC) to the scrapie form (PrPSc) is still unknown. Here we discovered a chemical chaperone to stabilize the PrPC conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a “pocket” created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrPSc. Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrPC, will open the way to the development of novel anti-prion drugs.

Activated Intrahepatic Antigen-Presenting Cells Inhibit Hepatitis B Virus Replication in the Liver of Transgenic Mice

In this study we evaluated the ability of activated intrahepatic APCs to inhibit hepatitis B virus (HBV) replication in transgenic mice. Intrahepatic APCs were activated by administration of an anti-CD40 agonistic mAb (αCD40). We showed that a single i.v. injection of αCD40 was sufficient to inhibit HBV replication noncytopathically by a process associated with the recruitment of dendritic cells, macrophages, T cells, and NK cells into the liver and the induction of inflammatory cytokines. The antiviral effect depended on the production of IL-12 and TNF-α by activated APCs; however, it was mediated primarily by IFN-γ produced by NK cells, and possibly T cells, that were activated by IL-12. Collectively, these results suggest that activated APCs can directly produce antiviral cytokines (IL-12, TNF-α) and trigger the production of other cytokines (i.e., IFN-γ) by other cells (e.g., NK cells and T cells) that do not express CD40. These results provide insight into a hitherto unsuspected antiviral function of intrahepatic APCs, and they suggest that therapeutic activation of APCs may represent a new strategy for the treatment of chronic HBV infection.

Leflunomide protects from T‐cell–mediated liver injury in mice through inhibition of nuclear factor κB

Leflunomide is a novel immunosuppressive and anti‐inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T‐cell–dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF‐α) and interleukin 2 concentrations, and also reduced TNF‐α mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF‐α. Activation of nuclear factor κB (NF‐κB), which regulates TNF‐α production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF‐α production from lymphocytes infiltrating the liver. In conclusion, leflunomide is capable of regulating T‐cell–mediated liver injury in vivo and that this event may depend on the decrease of TNF‐α production in the liver through inhibition of NF‐κB activation caused by leflunomide. (HEPATOLOGY 2004.)

Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-α-deficient mice

Abstract: Aims/Background: Tumor necrosis factor‐α (TNF‐α) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF‐α also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF‐α influences liver cell apoptosis and regeneration following PH in TNF‐α‐deficient (TNF‐α−/−) mice.

Critical role of CD44 in hepatotoxin-mediated liver injury

BACKGROUND/AIMS Blocking of adhesion molecules is considered to be one of the therapeutic strategies inflammatory diseases, although it remains unclear whether this strategy is beneficial. METHODS We used CD44-deficient mice to assess whether inhibition of CD44 could control liver injury caused by carbon tetrachloride (CCl(4)). RESULTS CD44-deficient mice exhibited suppressed liver inflammation during the early phase (within 6h) after CCl(4) injection due to reduced inflammatory cell infiltration and cytokine production, but showed severe liver inflammation with increased numbers of apoptotic hepatocytes at the late phase (after 12h). The induction of hepatocyte apoptosis was triggered by reduced NF-kappaB activity, which was induced by the low inflammatory cytokine concentrations. Furthermore, macrophages contributed to the induction of hepatocyte apoptosis, since neutralization by an anti-CD11b antibody significantly protected against hepatocyte apoptosis. Finally, we found that blocking of MIP-2 and TNF-alpha reduced hepatocyte apoptosis with decreased numbers of intrahepatic leukocytes and reduced inflammatory cytokine production. CONCLUSIONS These findings suggest that targeting of CD44 as a therapeutic approach for inflammatory liver diseases may require caution for particular immune systems in the liver.

Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice.

ABSTRACT Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.

Elevated intracellular IFN-γ levels in circulating CD8+ lymphocytes in patients with fulminant hepatitis

BACKGROUND/AIMS Fulminant hepatitis usually takes a rapidly progressive course, terminating in death within a short period. Experimental studies have demonstrated that immunological mechanisms play an important role, especially those involving virus-specific CD8+ cytotoxic T lymphocytes and their production of interferon-gamma (IFN-gamma). However, there are no immunological markers for prediction of the development of fulminant hepatitis in man. METHODS Peripheral blood lymphocytes from four patients with fulminant hepatitis, six with acute hepatitis and 11 healthy volunteers as normal controls were analyzed. Intracellular IFN-gamma production in both CD8 positive and negative T lymphocytes was assessed by flow cytometry. RESULTS Populations of CD8+ IFN-gamma+ T lymphocytes were significantly increased in patients with fulminant hepatitis, as compared with those with acute hepatitis and normal controls. Production of IFN-gamma in CD8+ T lymphocytes of patients with fulminant hepatitis was also elevated, furthermore significantly correlating with the prothrombin time (r=-0.64, p<0.01). CONCLUSIONS The capacity for IFN-gamma production by CD8+ lymphocytes is up-regulated in fulminant hepatitis, and this may be important for the development of fulminant hepatitis.

Pivotal role of nuclear factor κB signaling in anti‐CD40–induced liver injury in mice

Nuclear factor κB (NF‐κB) has a central role in coordinating the expression of a wide variety of genes that control immune responses and is also recognized as an antiapoptotic transcription factor. Here, we focused on the role of the NF‐κB signaling pathway in the interaction between inflammatory cells and hepatocytes in liver inflammation. We found that pretreatment of mice with adenoviruses expressing a mutant form of the inhibitor κB superrepressor (Ad5IκB), a NF‐κB inhibitor, reduced the migration of inflammatory cells and cytokine and chemokine expression in the liver 12 hours after a single intravenous injection of an anti‐CD40 antibody (αCD40) compared with mice infected with control adenoviruses (Ad5LacZ). We also confirmed reductions in cytokine production by macrophages, T cells, and natural killer (NK) cells in the liver of Ad5IκB‐treated mice by FACS analysis. However, αCD40 treatment in Ad5IκB‐infected mice induced elevation of serum alanine aminotransferase at 24 hours, and the liver injury was associated with massive hepatocyte apoptosis. Furthermore, interferon gamma (IFN‐γ) production by NK cells and T cells was increased and stimulated tumor necrosis factor alpha (TNF‐α) production by macrophages in the Ad5IκB‐infected liver. Moreover, the liver injury was completely suppressed by the administration of anti–IFN‐γ and anti–TNF‐α. These results suggest that inhibition of NF‐κB activity suppressed αCD40‐induced liver inflammation at an early phase, resulting in a reduction in cytokine and chemokine production, whereas it sensitized hepatocytes to TNF‐α–induced apoptosis and exacerbated liver injury at the late phase. In conclusion, NF‐κB exerts pivotal activities at inflammatory sites, and caution should be exercised in NF‐κB–targeted therapy of liver disease. (HEPATOLOGY 2004;40:1180–1189.)

Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice

Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.