Kazumoto Murata

Hepatitis B virus-induced lipid alterations contribute to natural killer T cell-dependent protective immunity

In most adult humans, hepatitis B is a self-limiting disease leading to life-long protective immunity, which is the consequence of a robust adaptive immune response occurring weeks after hepatitis B virus (HBV) infection. Notably, HBV-specific T cells can be detected shortly after infection, but the mechanisms underlying this early immune priming and its consequences for subsequent control of viral replication are poorly understood. Using primary human and mouse hepatocytes and mouse models of transgenic and adenoviral HBV expression, we show that HBV-expressing hepatocytes produce endoplasmic reticulum (ER)-associated endogenous antigenic lipids including lysophospholipids that are generated by HBV-induced secretory phospholipases and that lead to activation of natural killer T (NKT) cells. The absence of NKT cells or CD1d or a defect in ER-associated transfer of lipids onto CD1d results in diminished HBV-specific T and B cell responses and delayed viral control in mice. NKT cells may therefore contribute to control of HBV infection through sensing of HBV-induced modified self-lipids.

Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection.

We have recently demonstrated that immunization with hepatitis C virus-like particles (HCV-LPs) generated in insect cells can elicit both humoral and cellular immune responses in BALB/c mice. Here, we evaluate the immunogenicity of HCV-LPs in HLA2.1 transgenic (AAD) mice in comparison to DNA immunization. HCV-LP immunization elicited a significantly stronger humoral immune response than DNA immunization. HCV-LP-immunized mice also developed stronger HCV-specific cellular immune responses than DNA-immunized mice as determined by using quantitative enzyme-linked immunospot (ELISpot) assay and intracellular cytokine staining. In BALB/c mice, immunization with HCV-LPs resulted in a >5 log10 reduction in vaccinia titer when challenged with a recombinant vaccinia expressing the HCV structural proteins (vvHCV.S), as compared to 1 log10 decrease in DNA immunization. In HLA2.1 transgenic mice, a 1–2 log10 reduction resulted from HCV-LP immunization, whereas no reduction was seen from DNA immunization. Adoptive transfer of lymphocytes from HCV-LP-immunized mice to naive mice provided protection against vvHCV.S challenge, and this transferred immunity can be abrogated by either CD4 or CD8 depletion. Our results suggest that HCV-LPs can induce humoral and cellular immune responses that are protective in a surrogate HCV challenge model and that a strong cellular immunity provided by both CD4 and CD8 effector lymphocytes may be important for protection from HCV infection.

Cellular FLICE/Caspase-8–Inhibitory Protein as a Principal Regulator of Cell Death and Survival in Human Hepatocellular Carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8–inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R–mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-κB and cFLIP down-regulation attenuated NF-κB activation induced by TNF-α or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-κB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-α, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor–mediated apoptosis but also by regulating NF-κB activation in human HCCs.

Complications of Partial Splenic Embolization in Cirrhotic Patients

In recent years, partial splenic embolization (PSE) has been widely used in patients with cirrhosis and hypersplenism caused by portal hypertension. We investigated the complications associated with PSE cases seen in our hospital. Seventeen cases of liver cirrhosis that had undergone PSE were examined to investigate the complications associated with it. Mean infarcted area of the spleen was 66.2%. Leukocyte and platelet counts in 16 of 17 patients were seen to improve after PSE and persisted for at least one year. The most frequent side effects were abdominal pain (82.4%) and fever (94.1%). Severe side effects were seen in two of those 17 patients. One patient died from acute on chronic liver failure. The other patients contracted bacterial peritonitis and splenic abscess and needed drainage of splenic abscess before recovery. These two cases were in Child-Pugh class B. In conclusions, PSE is a useful treatment for patients with cirrhosis and hypersplenism caused by portal hypertension. However, the possibility of severe complications, especially in patients with noncompensated cirrhosis, should be kept in mind.

The rs8099917 Polymorphism, When Determined by a Suitable Genotyping Method, Is a Better Predictor for Response to Pegylated Alpha Interferon/Ribavirin Therapy in Japanese Patients than Other Single Nucleotide Polymorphisms Associated with Interleukin-28B

ABSTRACT We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.

Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID).

Background: Cytokines and host factors triggering innate immunity against hepatitis B virus (HBV) are not well understood. Results: IL-1 and TNFα induced cytidine deaminase AID, an anti-HBV host factor, and reduced HBV infection into hepatocytes. Conclusion: IL-1/TNFα reduced host susceptibility to HBV infection through AID up-regulation. Significance: Proinflammatory cytokines modulate HBV infection through a novel innate immune pathway involving AID. Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

Human Blood Dendritic Cell Antigen 3 (BDCA3)^+ Dendritic Cells Are a Potent Producer of Interferon-λ in Response to Hepatitis C Virus

The polymorphisms in the interleukin (IL)‐28B (interferon‐lambda [IFN]‐λ3) gene are strongly associated with the efficacy of hepatitis C virus (HCV) clearance. Dendritic cells (DCs) sense HCV and produce IFNs, thereby playing some cooperative roles with HCV‐infected hepatocytes in the induction of interferon‐stimulated genes (ISGs). Blood dendritic cell antigen 3 (BDCA3)+ DCs were discovered as a producer of IFN‐λ upon Toll‐like receptor 3 (TLR3) stimulation. We thus aimed to clarify the roles of BDCA3+ DCs in anti‐HCV innate immunity. Seventy healthy subjects and 20 patients with liver tumors were enrolled. BDCA3+ DCs, in comparison with plasmacytoid DCs and myeloid DCs, were stimulated with TLR agonists, cell‐cultured HCV (HCVcc), or Huh7.5.1 cells transfected with HCV/JFH‐1. BDCA3+ DCs were treated with anti‐CD81 antibody, inhibitors of endosome acidification, TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐specific inhibitor, or ultraviolet‐irradiated HCVcc. The amounts of IL‐29/IFN‐λ1, IL‐28A/IFN‐λ2, and IL‐28B were quantified by subtype‐specific enzyme‐linked immunosorbent assay (ELISA). The frequency of BDCA3+ DCs in peripheral blood mononuclear cell (PBMC) was extremely low but higher in the liver. BDCA3+ DCs recovered from PBMC or the liver released large amounts of IFN‐λs, when stimulated with HCVcc or HCV‐transfected Huh7.5.1. BDCA3+ DCs were able to induce ISGs in the coexisting JFH‐1‐positive Huh7.5.1 cells. The treatments of BDCA3+ DCs with anti‐CD81 antibody, cloroquine, or bafilomycin A1 reduced HCVcc‐induced IL‐28B release, whereas BDCA3+ DCs comparably produced IL‐28B upon replication‐defective HCVcc. The TRIF‐specific inhibitor reduced IL‐28B release from HCVcc‐stimulated BDCA3+ DCs. In response to HCVcc or JFH‐1‐Huh7.5.1, BDCA3+ DCs in healthy subjects with IL‐28B major (rs8099917, TT) released more IL‐28B than those with IL‐28B minor genotype (TG). Conclusion: Human BDCA3+ DCs, having a tendency to accumulate in the liver, recognize HCV in a CD81‐, endosome‐, and TRIF‐dependent manner and produce substantial amounts of IL‐28B/IFN‐λ3, the ability of which is superior in subjects with IL‐28B major genotype. (HEPATOLOGY 2013)

Hepatitis C and B virus infections in populations at low or high risk in Ho Chi Minh and Hanoi, Vietnam

Abstract Inhabitants and patients of two cities in Vietnam were tested for antibodies to hepatitis C virus (anti‐HCV), hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti‐HBs). Anti‐HCV was detected in 43 (9%) of 491 individuals without liver disease in Ho Chi Minh, more frequently (P < 0.001) than in 18 (4%) of 511 in Hanoi. There was no apparent age‐specific distribution of anti‐HCV. Among inhabitants of both cities, HBsAg and anti‐HBs were frequent, detected in 10‐14% and 35‐37%, respectively; the prevalence of anti‐HBs increased in parallel with age. Among individuals at high risk, the prevalence of anti‐HCV was particularly high in drug users (58/67 or 87%) and patients on maintenance haemodialysis (15/28 or 54%) or with haemophilia (7/24 or 29%) in Ho Chi Minh, and in drug users in Hanoi (61/200 or 31%). Prevalence of HBsAg and anti‐HBs in high‐risk groups was not different from those in the general population. Screening of anti‐HCV in blood donors in Vietnam is of urgent necessity because blood supply is dependent on commercial blood donors, many of whom are drug users at high risk.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.

15-Deoxy-Δ-12-14-PGJ2 regulates apoptosis induction and nuclear factor-κB activation via a peroxisome proliferator-activated receptor-γ-independent mechanism in hepatocellular carcinoma

The peroxisome proliferator-activated receptor-γ (PPARγ) high-affinity ligand, 15-deoxy-Δ-12,14-PGJ2 (15d-PGJ2), is toxic to malignant cells through cell cycle arrest and apoptosis induction. In this study, we investigated the effects of 15d-PGJ2 on apoptosis induction and expression of apoptosis-related proteins in hepatocellular carcinoma (HCC) cells. 15d-PGJ2 induced apoptosis in SK-Hep1 and HepG2 cells at a 50 μm concentration. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (2-VAD-fmk), only partially blocked apoptosis induced by 40 μm 15d-PGJ2. This indicated that 15d-PGJ2 induction of apoptosis was associated with a caspase-3–independent pathway. 15d-PGJ2 also induced down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bclx, and apoptotic protease-activating factor-1 in SK-Hep1 cells but not in HepG2 cells. However, 15d-PGJ2 sensitized both HCC cell lines to TNF-related apoptosis-induced ligand–induced apoptosis. In SK-Hep1 cells, cell toxicity, nuclear factor-κB (NF-κB) suppression, and XIAP down-regulation were induced by 15d-PGJ2 treatment under conditions in which PPARγ was down-regulated. These results suggest that the effect of 15d-PGJ2 was through a PPARγ-independent mechanism. Although cell toxicity was induced when PPARγ was down-regulated in HepG2 cells, NF-κB suppression and XIAP down-regulation were not induced. In conclusion, 15d-PGJ2 induces apoptosis of HCC cell lines via caspase-dependent and -independent pathways. In SK-Hep1 cells, the ability of 15d-PGJ2 to induce cell toxicity, NF-κB suppression, or XIAP down-regulation seemed to occur via a PPARγ-independent mechanism, but in HepG2 cells, NF-κB suppression by 15d-PGJ2 was dependent on PPARγ.