Nobuhito Taniki

Prominent Steatosis with Hypermetabolism of the Cell Line Permissive for Years of Infection with Hepatitis C Virus

Most of experiments for HCV infection have been done using lytic infection systems, in which HCV-infected cells inevitably die. Here, to elucidate metabolic alteration in HCV-infected cells in a more stable condition, we established an HCV-persistently-infected cell line, designated as HPI cells. This cell line has displayed prominent steatosis and supported HCV infection for more than 2 years, which is the longest ever reported. It enabled us to analyze metabolism in the HCV-infected cells integrally combining metabolomics and expression arrays. It revealed that rate-limiting enzymes for biosynthesis of cholesterol and fatty acids were up-regulated with actual increase in cholesterol, desmosterol (cholesterol precursor) and pool of fatty acids. Notably, the pentose phosphate pathway was facilitated with marked up-regulation of glucose-6-phosphate dehydrogenase, a rete-limiting enzyme, with actual increase in NADPH. In its downstream, enzymes for purine synthesis were also up-regulated resulting in increase of purine. Contrary to common cancers, the TCA cycle was preferentially facilitated comparing to glycolysis pathway with a marked increase of most of amino acids. Interestingly, some genes controlled by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of antioxidation and metabolism, were constitutively up-regulated in HPI cells. Knockdown of Nrf2 markedly reduced steatosis and HCV infection, indicating that Nrf2 and its target genes play important roles in metabolic alteration and HCV infection. In conclusion, HPI cell is a bona fide HCV-persistently-infected cell line supporting HCV infection for years. This cell line sustained prominent steatosis in a hypermetabolic status producing various metabolites. Therefore, HPI cell is a potent research tool not only for persistent HCV infection but also for liver metabolism, overcoming drawbacks of the lytic infection systems.

On-treatment decrease of NKG2D correlates to early emergence of clinically evident hepatocellular carcinoma after interferon-free therapy for chronic hepatitis C

Background and aims Interferon (IFN)- free direct antiviral agents (DAAs) with rapid HCV eradication might evoke immunological reconstitutions, and some early recurrences of HCC after IFN-free DAAs have been reported. This study aimed to investigate whether natural killer group 2, member D (NKG2D) predicts early emergence of HCC after IFN-free DAAs. Methods We conducted a clinical practice-based observational study of 101 patients infected with genotype 1 HCV who received IFN-free (DAAs), and stratified them into those who did or did not develop early (i.e., during the 6-month surveillance period following treatment.) recurrence or occurrence of clinically evident HCC. We also analyzed the peripheral blood mononuclear cells, both before treatment and at end of treatment (EOT), of 24 of the patients who received IFN-free DAAs, and 16 who received IFN-combined protease inhibitor. Results We found early emergence of clinically evident HCC after IFN-free DAAs in 12 (12%) patients. Higher pre-treatment NKG2D expression, higher FIB-4 score, previous HCC history and failure to achieve sustained viral response were significant factors correlating to early HCC emergence. After IFN-free DAAs, a rapid decrease of NKG2D at EOT correlated with early HCC emergence in the IFN-free DAA-treated patients, but not in patients treated with the IFN-combined regimen. The decrease of NKG2D until EOT was predictive of early HCC emergence at a cut-off of -52% (AUC = 0.92). Conclusions On-treatment decrease of NKG2D may be a useful predictor of early emerging HCC in patients treated with IFN-free DAAs.

Commensal Lactobacillus Controls Immune Tolerance during Acute Liver Injury in Mice

Gut-derived microbial antigens trigger the innate immune system during acute liver injury. During recovery, regulatory immunity plays a role in suppressing inflammation; however, the precise mechanism underlying this process remains obscure. Here, we find that recruitment of immune-regulatory classical dendritic cells (cDCs) is crucial for liver tolerance in concanavalin A-induced acute liver injury. Acute liver injury resulted in enrichment of commensal Lactobacillus in the gut. Notably, Lactobacillus activated IL-22 production by gut innate lymphoid cells and raised systemic IL-22 levels. Gut-derived IL-22 enhanced mucosal barrier function and promoted the recruitment of regulatory cDCs to the liver. These cDCs produced IL-10 and TGF-β through TLR9 activation, preventing further liver inflammation. Collectively, our results indicate that beneficial gut microbes influence tolerogenic immune responses in the liver. Therefore, modulation of the gut microbiota might be a potential option to regulate liver tolerance.

Bone marrow-derived macrophages distinct from tissue-resident macrophages play a pivotal role in Concanavalin A-induced murine liver injury via CCR9 axis

The fundamental mechanism how heterogeneous hepatic macrophage (Mφ) subsets fulfill diverse functions in health and disease has not been elucidated. We recently reported that CCR9+ inflammatory Mφs play a critical role in the course of acute liver injury. To clarify the origin and differentiation of CCR9+Mφs, we used a unique partial bone marrow (BM) chimera model with liver shielding for maintaining hepatic resident Mφs. First, irradiated mice developed less liver injury with less Mφs accumulation by Concanavalin A (Con A) regardless of liver shielding. In mice receiving further BM transplantation, CD11blowF4/80high hepatic-resident Mφs were not replaced by transplanted donors under steady state, while under inflammatory state by Con A, CCR9+Mφs were firmly replaced by donors, indicating that CCR9+Mφs originate from BM, but not from hepatic-resident cells. Regarding the mechanism of differentiation and proliferation, EdU+CCR9+Mφs with a proliferative potential were detected specifically in the inflamed liver, and in vitro study revealed that BM-derived CD11b+ cells co-cultured with hepatic stellate cells (HSCs) or stimulated with retinoic acids could acquire CCR9 with antigen-presenting ability. Collectively, our study demonstrates that inflammatory Mφs originate from BM and became locally differentiated and proliferated by interaction with HSCs via CCR9 axis during acute liver injury.

Simeprevir/pegylated interferon/ribavirin triple therapy for recurrent hepatitis C after living donor liver transplantation.

Simeprevir (SMV) is a protease inhibitor which demonstrates good tolerability and high antiviral response in patients with hepatitis C. The clinical outcomes of triple therapy using simeprevir, pegylated interferon and ribavirin (SMV/PEG IFN/RBV) for recurrent hepatitis C after living donor liver transplantation (LDLT) have not been well reported. In this study, we assessed the outcomes of patients with recurrent hepatitis C (genotype 1) after LDLT who received triple therapy at our hospital.

Education and imaging. Gastrointestinal: capsule endoscopy assists in the complete deworming of parasites.

A 67-year-old man presented with the complaint of ribbon-like substances in the feces. In the beginning of August 2010, he was referred to our hospital for treatment of parasitic worm infection. The patient did not have symptoms such as diarrhea, anemia, or weight loss; the results of physical and hematological examinations and of thoracoabdominal radiography were normal. Although he did not have a history of parasitic worm infection and had never traveled abroad, he had eaten raw salmon 1 month ago. On the first day of the hospital visit, the patient was diagnosed with diphyllobothriasis after examination of the helminth eggs found in the feces. After diatrizoic acid swallow on the second day, the presence of the worm body was confirmed by colonoscopy, and the worm body was extirpated from the anus by using grasping forceps. Although approximately 1 m of the worm body together with most of the neck portion was successfully extirpated, removal of the scolex was not confirmed. On the fourth day, after obtaining the patient’s consent, we performed capsule endoscopy because of possible incomplete deworming. Capsule endoscopy confirmed parasitization by a cestode and detected the scolex attached to the jejunal mucosa (Figure 1). Parasite-specific drugs, i.e., praziquantel and magnesium citrate, were administered, and complete deworming was subsequently confirmed by microscopic identification of the scolex (Figure 2). The worm body was pathologically confirmed as Diphyllobothrium nihonkaiense by performing trichrome and carmine staining (Figure 3). For the successful treatment of diphyllobothriasis, it is essential to remove the scolex of the parasite. The use of capsule endoscopy now allows for (1) easy capture of images of parasites, such as that of the scolex of Diphyllobothrium nihonkaiense, in the small intestine, which was previously considered difficult; and (2) provides information critical for therapeutic decision making before administering anthelmintics.

Gastrointestinal: Capsule endoscopy assists in the complete deworming of parasites

A 67-year-old man presented with the complaint of ribbon-like substances in the feces. In the beginning of August 2010, he was referred to our hospital for treatment of parasitic worm infection. The patient did not have symptoms such as diarrhea, anemia, or weight loss; the results of physical and hematological examinations and of thoracoabdominal radiography were normal. Although he did not have a history of parasitic worm infection and had never traveled abroad, he had eaten raw salmon 1 month ago. On the first day of the hospital visit, the patient was diagnosed with diphyllobothriasis after examination of the helminth eggs found in the feces. After diatrizoic acid swallow on the second day, the presence of the worm body was confirmed by colonoscopy, and the worm body was extirpated from the anus by using grasping forceps. Although approximately 1 m of the worm body together with most of the neck portion was successfully extirpated, removal of the scolex was not confirmed. On the fourth day, after obtaining the patient’s consent, we performed capsule endoscopy because of possible incomplete deworming. Capsule endoscopy confirmed parasitization by a cestode and detected the scolex attached to the jejunal mucosa (Figure 1). Parasite-specific drugs, i.e., praziquantel and magnesium citrate, were administered, and complete deworming was subsequently confirmed by microscopic identification of the scolex (Figure 2). The worm body was pathologically confirmed as Diphyllobothrium nihonkaiense by performing trichrome and carmine staining (Figure 3). For the successful treatment of diphyllobothriasis, it is essential to remove the scolex of the parasite. The use of capsule endoscopy now allows for (1) easy capture of images of parasites, such as that of the scolex of Diphyllobothrium nihonkaiense, in the small intestine, which was previously considered difficult; and (2) provides information critical for therapeutic decision making before administering anthelmintics.

Slow-metabolizing ADH1B and inactive heterozygous ALDH2 increase vulnerability to fatty liver in Japanese men with alcohol dependence

BackgroundGenetic polymorphisms of alcohol dehydrogenase-1B (ADH1B; rs1229984, His48Arg) and aldehyde dehydrogenase-2 (ALDH2; rs671, Glu504Lys) affect body weight, body fat, and lipid metabolism in individuals with alcohol dependence, and the aim of this study was to identify their determinants in relation to the development of fatty liver.MethodsWe evaluated associations between the presence of fatty liver and ADH1B and ALDH2 genotypes and other factors in 1604 Japanese men who had been admitted for treatment of alcohol dependence.ResultsFatty liver was diagnosed when ultrasonography showed both hepatorenal contrast and liver brightness. Age-adjusted usual alcohol intake did not differ according to ADH1B or ALDH2 genotypes. A multivariate analysis showed that the adjusted odds ratio (OR, 95% confidence interval) of slow-metabolizing ADH1B Arg/Arg carriers was 1.61 (1.27–2.03) for fatty liver and 1.82 (1.37–2.41) for fatty liver with deep attenuation in comparison with the ADH1B His/Arg or His/His carriers, and that the OR of inactive heterozygous ALDH2 Glu/Lys carriers was 1.43 (1.08–1.91) for fatty liver and 1.84 (1.31–2.59) for fatty liver with deep attenuation in comparison with the ALDH2 Glu/Glu carriers. Younger age, shorter interval between the last drink and the ultrasound examination, larger body mass index, and absence of cirrhosis were identified as other positive determinants for fatty liver.ConclusionsThe ADH1B Arg/Arg genotype and the ALDH2 Glu/Lys genotype were positive determinants of fatty liver in the subjects. These results suggest that slow ethanol and acetaldehyde metabolism accelerates the development of alcoholic fatty liver in heavy drinkers.

Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/Ribavirin Therapy in Chronic Hepatitis C.

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.

Intestinal barrier regulates immune responses in the liver via IL-10–producing macrophages

The gut-liver axis is of clinical importance as a potential therapeutic target in a wide range of liver diseases; however, the mechanisms underlying interactions between microbial products and immune responses in the liver remain unknown. In this study, we demonstrated that IL-10-producing macrophages contribute to immune tolerance in the inflamed liver under intestinal barrier disruption in a murine tandem model of dextran sulfate sodium (DSS) colitis and concanavalin A (Con A) hepatitis. Intestinal barrier disruption protected mice from subsequent liver injury, and the severity of colitis directly affected susceptibility to such injury. The protective effect of DSS-Con A was canceled in gut-sterilized mice, suggesting that gut microbiota play a substantial role in this process. Altered gut microbiota and their metabolites, along with a disrupted intestinal barrier, directly gave rise to immunological permissiveness in the inflamed liver. We identified 1-methylnicotinamide (1-MNA) as a candidate metabolite capable of suppressing liver injury with the potential to induce IL-10-producing macrophages. Consistently, expression of nicotinamide N-methyltransferase, which converts nicotinamide to 1-MNA, was upregulated in the liver of DSS-Con A mice, and this effect was abrogated by gut sterilization. Collectively, our results provide a mechanistic insight into the regulation of immunological balance in the liver via the gut-liver axis.