Ken Takahashi

A Toll-like receptor–independent antiviral response induced by double-stranded B-form DNA

The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-β through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-κB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-β and NF-κB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.*Note: In the version of this article initially published, the GEO database accession number is missing. This should be the final subsection of Methods, as follows: code. GEO: microarray data, GSE4171. The error has been corrected in the PDF version of the article.

Essential role of IPS-1 in innate immune responses against RNA viruses

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

Roles of caspase-8 and caspase-10 in innate immune responses to double-stranded RNA.

Upon viral infection, host cells trigger antiviral immune responses by inducing type I IFN and inflammatory cytokines. dsRNA generated during viral replication is recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, which interact with an adaptor, IFN-β promoter stimulator-1, to activate the transcription factors NF-κB and IFN regulatory factor 3. In this article we demonstrate that caspase-8 and caspase-10 are involved in these pathways. Both caspases were cleaved during dsRNA stimulation, and overexpression of a cleaved form of these caspases activated NF-κB. Knockdown of caspase-10 or caspase-8 in a human cell line resulted in the reduction of inflammatory cytokine production. Cells derived from caspase-8-deficient mice also showed reduced expression of inflammatory cytokines as well as NF-κB activation. Furthermore, the Fas-associated death domain protein interacted with these two caspases and IFN-β promoter stimulator 1. These results indicate that caspase-8 and caspase-10 are essential components that mediate NF-κB-dependent inflammatory responses in antiviral signaling.

Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing.

Background and Aims The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. Methods Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy. Results Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents. Conclusion Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.

NLRC5 Deficiency Does Not Influence Cytokine Induction by Virus and Bacteria Infections

Nucleotide-binding domain and leucine rich repeat containing gene family receptors (NLRs) are cytosolic proteins that respond to a variety of pathogen and host components to induce inflammatory cytokines. NLRC5 is a recently identified member of the NLR family that has been implicated in positive and negative regulation of antiviral innate immune responses. To clarify whether NLRC5 controls antiviral innate immunity in vivo, we generated NLRC5-deficient mice. Macrophages and dendritic cells derived from NLRC5-deficient mice induced relatively normal levels of IFN-β, IL-6, and TNF-α after treatment with RNA viruses, DNA viruses, and bacteria. The serum cytokine levels after polyinosinic-polycytidylic acid infection were also comparable between control and NLRC5-deficient mice. NLRC5 overexpression promoted IL-1β production via caspase-1, suggesting that NLRC5 constitutes an inflammasome. However, there was no reduction of IL-1β in NLRC5-deficient cells in response to known inflammasome activators, suggesting that NLRC5 controls IL-1β production through an unidentified pathway. These findings indicate that NLRC5 is dispensable for cytokine induction in virus and bacterial infections under physiologic conditions.

Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

Background and Aims Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. Methods To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. Results Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. Conclusion Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

Mouse models of hepatitis B virus infection comprising host-virus immunologic interactions.

Hepatitis B virus (HBV) infection is one of the most prevalent infectious diseases associated with various human liver diseases, including acute, fulminant and chronic hepatitis; liver cirrhosis; and hepatocellular carcinoma. Despite the availability of an HBV vaccine and the development of antiviral therapies, there are still more than 350 million chronically infected people worldwide, approximately 5% of the world population. To understand the virus biology and pathogenesis in HBV-infected patients, several animal models have been developed to mimic hepatic HBV infection and the immune response against HBV, but the narrow host range of HBV infection and lack of a full immune response spectrum in animal models remain significant limitations. Accumulating evidence obtained from studies using a variety of mouse models that recapitulate hepatic HBV infection provides several clues for understanding host-virus immunologic interactions during HBV infection, whereas the determinants of the immune response required for HBV clearance are poorly defined. Therefore, adequate mouse models are urgently needed to elucidate the mechanism of HBV elimination and identify novel targets for antiviral therapies.

Hepatitis C Treatment with Sofosbuvir and Ledipasvir Accompanied by Immediate Improvement in Hemoglobin A1c

Background/Aims: Direct-acting antiviral agents (DAAs) have increased the sustained viral response rate with minimal adverse effects and short treatment duration. In addition, recent data suggest the possibility that hepatitis C virus (HCV) clearance results in rapid improvement in metabolic pathways. The aim of the present study was to evaluate whether the DAA treatment without ribavirin lowers hemoglobin A1c (HbA1c) at 12 weeks after therapy completion. Methods: We performed an observational study to assess the effect of sofosbuvir and ledipasvir (SOF/LED) treatment on glycemic control. We compared HbA1c levels before and after treatment with SOF/LED, considering that anemia is not a side effect of these drugs. Results: In the 36 patients with HCV eradication, HbA1c levels decreased significantly after treatment (pre-treatment 5.85% vs. post-treatment 5.65%, p < 0.01). Conclusion: This pilot study shows the possibility that HCV eradication by SOF/LED was accompanied by an improvement of glucose metabolism in the population with or without diabetes, and suggests further investigation.

Sonazoid-enhanced ultrasonography guidance improves the quality of pathological diagnosis in the biopsy of focal hepatic lesions.

Background/aim Contrast-enhanced ultrasonography (US) has improved the detection and characterization of focal hepatic lesions. Recently, the importance of obtaining high-quality samples in the biopsy of hepatic lesions has been increasing not only in the field of pathological diagnosis but also in molecular analysis for predicting the effectiveness of anticancer agents and molecular targeted drugs. We evaluated the utility of Sonazoid-enhanced ultrasonography (SEUS) in guiding percutaneous biopsy of focal hepatic lesions by comparing the results of histopathological diagnosis between B-mode US and SEUS guidance. Methods and materials This retrospective study examined 121 focal hepatic lesions in 108 patients (mean age: 63.8 years) referred for US-guided percutaneous biopsy. The technical success rate was defined as the percentage of the lesions diagnosed clearly at the initial biopsy. Results Among 121 lesions, 56 lesions were subjected to biopsy with B-mode US guidance whereas 65 were subjected to SEUS guidance. The technical success rate was significantly higher under SEUS guidance than under B-mode US guidance (92.3 vs. 76.8%, respectively, P<0.05). When biopsies were performed to diagnose or rule out malignancy in indeterminate lesions, the technical success rate was also significantly higher under SEUS guidance than under B-mode US guidance (100 vs. 73.9%, respectively, P<0.05). SEUS guidance resulted in a significantly higher rate of successful single-puncture attempts compared with B-mode US guidance (55.4 vs. 35.7%, respectively, P<0.05). Conclusion SEUS guidance is recommended for more accurate localization of suitable hepatic lesion biopsy areas as it increases conspicuity and differentiates viable areas from denaturalization or necrosis.

Activation of TNF-α-AID axis and co-inhibitory signals in coordination with Th1-type immunity in a mouse model recapitulating hepatitis B

&NA; Hepatitis B virus (HBV) infection evokes host immune responses that primarily determine the outcome of HBV infection and the clinical features of HBV‐associated liver disease. The precise mechanisms by which host factors restrict HBV replication, however, are poorly understood due to the lack of useful animal models that recapitulate immune responses to HBV. Here, we performed comprehensive immunologic gene expression profiling of the liver of a mouse model recapitulating anti‐HBV immune response using a high sensitivity direct digital counting system. Anti‐HBV cellular immunity with liver inflammation was elicited in mice hydrodynamically injected with a CpG‐depleted plasmid encoding hepatitis B surface antigen (HBsAg) gene after preimmunization with HBsAg vaccine. Comprehensive expression analyses revealed the upregulation of Th1‐associated genes including tumor necrosis factor (Tnf) and negative regulators of T cell function in the inflamed liver. Interestingly, activation‐induced cytidine deaminase (Aicda, termed AID in humans), which reportedly suppresses HBV infection in vitro, was upregulated in hepatocytes in the course of anti‐HBV immunity. Hepatocytic expression of Aicda in a Tnf‐dependent manner was confirmed by the administration of Tnf antagonist into Aicda‐tdTomato mice with anti‐HBV immunity. Our findings suggest that activation of Tnf–Aicda axis and co‐inhibitory signals to T cells in coordination with Th1‐type immunity has critical roles in the immune response against HBV infection. HighlightsA mouse model recapitulating liver inflammation associated with anti‐HBV immunity was developed by hydrodynamic injection.In this model, nonspecific immune response potentially caused by injection was avoided by using a CpG‐free plasmid.Comprehensive analysis showed the upregulation of Th1 genes and negative regulators to T cells in the inflamed liver.Aicda (AID), which contributes to noncytolytic immunity against HBV was also upregulated in hepatocytes via Tnf signaling.