Sachiyo Yoshio

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)

TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.

Angiogenesis is a critical step in the development and progression of hepatocellular carcinoma (HCC). Myeloid lineage cells, such as macrophages and monocytes, have been reported to regulate angiogenesis in mouse tumor models. TIE2, a receptor of angiopoietins, conveys pro‐angiogenic signals and identifies a monocyte/macrophage subset with pro‐angiogenic activity. Here, we analyzed the occurrence and kinetics of TIE2‐expressing monocytes/macrophages (TEMs) in HCC patients. This study enrolled 168 HCV‐infected patients including 89 with HCC. We examined the frequency of TEMs, as defined as CD14+CD16+TIE2+ cells, in the peripheral blood and liver. The localization of TEMs in the liver was determined by immunofluorescence staining. Micro‐vessel density in the liver was measured by counting CD34+ vascular structures. We found that the frequency of circulating TEMs was significantly higher in HCC than non‐HCC patients, while being higher in the liver than in the blood. In patients who underwent local radio‐ablation or resection of HCC, the frequency of TEMs dynamically changed in the blood in parallel with HCC recurrence. Most TEMs were identified in the perivascular areas of tumor tissue. A significant positive correlation was observed between micro‐vessel density in HCC and frequency of TEMs in the blood or tumors, suggesting that TEMs are involved in HCC angiogenesis. Receiver operating characteristic analyses revealed the superiority of TEM frequency to AFP, PIVKA‐II and ANG‐2 serum levels as diagnostic marker for HCC. Conclusion: TEMs increase in patients with HCC and their frequency changes with the therapeutic response or recurrence. We thus suggest that TEM frequency can be used as a diagnostic marker for HCC, potentially reflecting angiogenesis in the liver. (HEPATOLOGY 2013)

Association of enhanced activity of indoleamine 2,3-dioxygenase in dendritic cells with the induction of regulatory T cells in chronic hepatitis C infection

BackgroundAltered functions of dendritic cells (DCs) and/or increases of regulatory T cells (Tregs) are involved in the pathogenesis of chronic hepatitis C virus (HCV) infection. A tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), is reported to be an inducer of immune tolerance. Our aim was to clarify whether or not IDO is activated in chronic hepatitis C patients and its role in immune responses.MethodsThis study enrolled 176 patients with chronic HCV infection and 37 healthy volunteers. Serum kynurenine concentration was evaluated by high-performance liquid chromatography, and its correlation with clinical parameters was examined. Monocyte-derived DCs were prepared from the subjects and subsequently stimulated with a combination of lipopolysaccharide and interferon-gamma to induce functional IDO (defined as IDO-DCs). The phenotypes, kynurenine or cytokine production, and T-cell responses with IDO-DCs were compared between the patients and healthy volunteers.ResultsThe serum kynurenine level in the patients was significantly higher than that in the healthy volunteers, and the level of serum kynurenine was positively correlated with the histological activity or fibrosis score. IDO activity in IDO-DCs from the patients was significantly higher than that in IDO-DCs from the volunteers. Furthermore, IDO-DCs from the patients induced more Tregs in vitro compared with those from the volunteers, and the frequency of induced Tregs by IDO-DCs was decreased with an IDO-specific inhibitor.ConclusionsSystemic IDO activity is enhanced in chronic hepatitis C patients in correlation with the degree of liver inflammation and fibrosis. In response to inflammatory stimuli, DCs from the patients tend to induce Tregs, with some of this action being dependent on IDO.

Long noncoding RNA #32 contributes to antiviral responses by controlling interferon-stimulated gene expression.

Significance Here, we describe a key feature of the long noncoding RNA (lncRNA) involved in innate immunity. We identified 182 lncRNAs that were highly modulated in poly(I:C)-treated hepatocyte cells. Of these, lncRNA#32 regulated the level of IFN-stimulated genes under both unstimulated and type I IFN-stimulated conditions through interactions with hnRNPU and ATF2, and therefore plays an important role in antiviral immunity. Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses.

Serum YKL-40 as a marker of liver fibrosis in patients with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. YKL-40, chitinase-like protein expressed in multiple tissues including liver, is involved in cell proliferation, inflammation and remodeling of the extracellular matrix. The aim of this study was to assess whether serum YKL-40 levels are associated with liver fibrosis in NAFLD patients. Serum YKL-40 levels were quantified in 111 NAFLD patients and 23 HCC patients with NAFLD. To identify the source of YKL-40, immunofluorescence staining of liver specimens from NAFLD patients was performed. Serum YKL-40 levels in NAFLD patients increased in accordance with the progression of liver fibrosis. Multivariate analysis revealed that YKL-40 was one of the independent factors significantly associated with severe fibrosis (F3-4). We established a new predictive model for fibrosis of NAFLD, using logistic regression analysis: YKL-40 based fibrosis score = −0.0545 + type IV collagen 7s * 0.3456 + YKL-40 * 0.0024. Serum YKL-40 levels of HCC patients with non-cirrhotic NAFLD were significantly higher than those without HCC. Immunofluorescence staining showed that YKL-40 was expressed by macrophages in liver tissue of NAFLD patients. In conclusion, macrophage-derived YKL-40 is a feasible biomarker of liver fibrosis in NAFLD patients.

Indoleamine-2,3-dioxygenase as an effector and an indicator of protective immune responses in patients with acute hepatitis B.

Indoleamine‐2, 3‐dioxygenase (IDO), an interferon‐γ‐inducible enzyme catalyzing tryptophan into kynurenine, exerts dual functions in infectious diseases, acting as a suppressor of intracellular pathogens and as an immune regulator. We explored the roles of IDO in hepatitis B virus (HBV) clearance from infected patients. We examined IDO activity, serum chemokines, and cytokines in 53 HBV‐positive patients (25 acute hepatitis, 14 chronic hepatitis, and 14 hepatic flare) and 14 healthy volunteers. In order to clarify the mechanisms of IDO induction and its impact on HBV replication, we used a culture model consisting of human natural killer cells, plasmacytoid dendritic cells, and HBV‐transfected Huh7 cells in which IDO expression is controlled. A robust activation of IDO with an inverse correlation of alanine aminotransferase at the peak was observed in patients with acute hepatitis B but not in patients with hepatic flare. In acute hepatitis patients who eventually cleared HBV, IDO activity, chemokine (C‐X‐C motif) ligand 9 (CXCL9), CXCL10, and CXCL11 increased at the peak of alanine aminotransferase. In contrast, in patients with hepatic flare, IDO activity remained at lower levels during the observation period, regardless of the surge of CXCL9, CXCL10, and CXCL11 at the alanine aminotransferase peak. Natural killer cells and plasmacytoid dendritic cells synergistically produced interferon‐γ and interferon‐α, thereby enhancing IDO activity and HBV suppression in Huh7 cells. Such suppressor capacity of IDO on HBV was abrogated in IDO‐knockout cells and recovered by the reinduction of IDO in the cells. Conclusion: IDO is an anti‐HBV effector and an indicator of subsequent immune responses operative during the early phase of infection; its activity is boosted by coexisting natural killer cells and plasmacytoid dendritic cells. (Hepatology 2016;63:83–94)

Interleukin-34 as a fibroblast-derived marker of liver fibrosis in patients with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. Activation of macrophages and hepatic stellate cells is a critical step that promotes liver fibrosis. We aimed to explore the feasibility of interleukin-34 (IL-34), a key regulator of macrophages, as a fibrosis marker in patients with NAFLD. We enrolled 197 liver biopsy-proven NAFLD patients. We evaluated the serum levels of IL-34, macrophage-colony stimulating factor (M-CSF), soluble CD163 (sCD163), 40 cytokines/chemokines, hyaluronic acid, type IV collagen 7s, and clinically-approved fibrosis scores. IL-34 increased with the progression of fibrosis and was an independent marker for liver fibrosis. Immunostaining experiments, using resected liver specimens from NAFLD patients, revealed that IL-34 was mainly expressed on liver fibroblasts. IL-34 based fibrosis score (0.0387*IL-34 (pg/ml) + 0.3623*type IV collagen 7s (ng/ml) + 0.0184*age (year)–1.1850) was a practical predictive model of liver fibrosis. Using receiver-operating characteristic analyses, the area under the curve, sensitivity, and specificity of IL-34 based fibrosis score were superior or comparable to the other fibrosis biomarkers and scores. In conclusion, the IL-34 based fibrosis score, including serum IL-34, type IV collagen 7s and age, is a feasible diagnostic marker of liver fibrosis in NAFLD patients.

Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

ABSTRACT Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly135 with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that hydroxylase activity of JMJD5 in the C terminus region is crucial for expression of HNF4A and replication of HBV. Furthermore, a mutant JMJD5 with Gly135 replaced by Glu failed to interact with HBx and to rescue the replication of HBV in JMJD5-knockout cells. Taken together, our data suggest that interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.

Host–virus interactions in hepatitis B and hepatitis C infection

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are among the most endemic pathogens worldwide, with more than 500 million people globally currently infected with these viruses. These pathogens can cause acute and chronic hepatitis that progress to liver cirrhosis or hepatocellular carcinoma. Both viruses utilize multifaceted strategies to evade the host surveillance system and fall below the immunological radar. HBV has developed specific strategies to evade recognition by the innate immune system and is acknowledged to be a stealth virus. However, extensive research has revealed that HBV is recognized by dendritic cells (DCs) and natural killer (NK) cells. Indoleamine-2, 3-dioxygenase is an enforcer of sequential immune reactions in acute hepatitis B, and this molecule has been shown to be induced by the interaction of HBV-infected hepatocytes, DCs, and NK cells. The interleukin-28B genotype has been reported to influence HCV eradication either therapeutically or spontaneously, but the biological function of its gene product, a type-III interferon (IFN-λ3), remains to be elucidated. Human BDCA3+DCs have also been shown to be a potent producer of IFN-λ3 in HCV infection, suggesting the possibility that BDCA3+DCs could play a key role in developing therapeutic HCV vaccine. Here we review the current state of research on immune responses against HBV and HCV infection, with a specific focus on innate immunity. A comprehensive study based on clinical samples is urgently needed to improve our understanding of the immune mechanisms associated with viral control and thus to develop novel immune modulatory therapies to cure chronic HBV and HCV infection.

MicroRNA-125b expression and intrahepatic metastasis are predictors for early recurrence after hepatocellular carcinoma resection

Early hepatocellular carcinoma (HCC) recurrence after curative resection is a known poor prognostic factor. We aimed to identify microRNAs associated with recurrence after curative HCC resection.