Keigo Kawashima

Background: /Aims : Direct-acting antiviral agents (DAA) effectively cure chronic hepatitis C (CHC) whereas curative therapy for chronic hepatitis B (CHB) is rare. We aimed to compare HCC incidence between suppressed CHB versus cured CHC patients with cirrhosis. Methods: We analyzed 5773 cirrhosis patients from 43 centers in 9 countries: 1877 with treated and suppressed CHB and 3896 with DAA-cured CHC using inverse probability treatment weight (IPTW) and competing risk analysis via Fine and Gray method. Results: After IPTW (on age, sex, ethnicity, study location, albumin, total bilirubin, creatinine, platelet, tobacco use, alcohol use, diabetes mellitus, hypertension, hyperlipidemia, cardiovascular disease, steatotic liver disease, obesity, and follow-up years), two study groups became similar in relevant characteristics. The 5-year cumulative HCC incidence was significantly higher in suppressed CHB compared to cured CHC (18.1% vs. 7.4%, P P P P P =0.954). On multivariable analysis, CHB suppression P P =0.04) were associated with higher HCC risk. Conclusion: HCC risk remains high in both treated and suppressed CHB cirrhosis and cured CHC cirrhosis. However, with prolonged CHB suppression, risk of HCC can be reduced, highlighting benefits of early treatment and viral suppression in patients with CHB.

The liver's unique characteristics have a profound impact on the priming and maintenance of adaptive immunity. This review delves into the cellular circuits that regulate adaptive immune responses in the liver, with a specific focus on hepatitis B virus infection as an illustrative example. A key aspect highlighted is the liver's specialized role in priming CD8⁺ T cells, leading to a distinct state of immune hyporesponsiveness. Additionally, the influence of the liver's hemodynamics and anatomical features, particularly during liver fibrosis and cirrhosis, on the differentiation and function of adaptive immune cells is discussed. While the primary emphasis is on CD8⁺ T cells, recent findings regarding the involvement of B cells and CD4⁺ T cells in hepatic immunity are also reviewed. Furthermore, we address the challenges ahead and propose integrating cutting-edge techniques, such as spatial biology, and combining mouse models with human sample analyses to gain comprehensive insights into the liver's adaptive immunity. This understanding could pave the way for novel therapeutic strategies targeting infectious diseases, malignancies, and inflammatory liver conditions like metabolic dysfunction-associated steatohepatitis and autoimmune hepatitis.

CD8 ⁺ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8 ⁺ T cell dysfunction showed that interleukin-2 (IL-2)–based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2’s effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (T _reg ) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8 ⁺ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8 ⁺ T cells in the liver without substantially altering T _reg or NK cell counts. These expanded CD8 ⁺ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen–positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8 ⁺ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8 ⁺ T cells without affecting NK or T _reg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.

Background & Aims: We recently analysed and reported the features of the micro biome under hepatitis C virus (HCV) infection, but the effect of HCV infection on bile acid (BA) metabolism in the gut‐liver axis remains poorly understood. The aim of this study was to clarify the characteristics of the gut‐liver axis in HCV‐infected patients.Methods: The faecal BAs composition and gut microbiota from 100 chronic hepatitis C (CHC) patients were compared with those from 23 healthy individuals. For transcriptional analysis of the liver, 22 mild CHC (fibrosis stages [F] 0‐2) and 42 advanced CHC (F3‐4) cases were compared with 12 healthy individuals. The findings were confirmed using chimeric mice with human hepatocytes infected with HCV HCR6.Results: Chronic hepatitis C patients, even at earlier disease stages, showed BA profiles distinct from healthy individuals, in which faecal deoxycholic acid (DCA) was significantly reduced and lithocholic acid or ursodeoxycholic acid became dominant. The decrease in faecal DCA was correlated with reduction in commensal Clostridiales and increase in oral Lactobacillales. Impaired biosynthesis of cholic acid (CA) was observed as a reduction in the transcription level of cytochrome P450 8B1 (CYP8B1), a key enzyme in CA biosynthesis. The reductions in faecal DCA and liver CYP8B1 were also observed in HCV‐infected chimeric mice.Conclusions: Chronic hepatitis C alters the intestinal BA profile, in association with the imbalance of BA biosynthesis, which differs from the pattern in NAFLD. These imbalances appear to drive disease progression through the gut‐microbiome‐liver axis.

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.

Hepatitis B virus–specific (HBV-specific) CD8 ⁺ T cells fail to acquire effector functions after priming in the liver, but the molecular basis for the dysfunction is poorly understood. By comparing the gene expression profile of intrahepatically primed, dysfunctional HBV-specific CD8 ⁺ T cells with that of systemically primed, functional effector counterparts, we found that the expression of interferon-stimulated genes (ISGs) is selectively suppressed in the dysfunctional CD8 ⁺ T cells. The ISG suppression was associated with impaired phosphorylation of STAT1 in response to IFN-α treatment. Importantly, a strong induction of type I interferons (IFN-Is) in the liver facilitated the functional differentiation of intrahepatically primed HBV-specific CD8 ⁺ T cells in association with the restoration of ISGs’ expression in the T cells. These results suggest that intrahepatic priming suppresses IFN-I signaling in CD8 ⁺ T cells, which may contribute to the dysfunction. The data also suggest a therapeutic value of the robust induction of intrahepatic IFN-Is for the treatment of chronic HBV infection.

Virus infection, such as hepatitis B virus (HBV), often causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR in HBV infection is poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism by which IFNs selectively induce cell death in virus-infected cells. This study also identifies UPR as a potential target for regulating ER stress-associated cell death. Author summary Hepatitis B virus (HBV) causes acute and chronic infections that kill over 600,000 people every year from severe hepatitis, liver cirrhosis, and cancer. Mechanisms of chronic liver injury remain largely unknown. Both type 1 and type 2 interferons (IFNs) have been implicated in hepatic flares during chronic HBV infection, although HBV per se is a poor IFN inducer. In addition, while IFNα, a type 1 IFN, used to be the first-line treatment for chronic hepatitis B (CHB) patients, adverse side effects, including hepatic flares, severely limit their therapeutic effectiveness. These clinical observations suggest a pathogenic role of IFNs in HBV infection. Here, we demonstrate that IFN-1s cause severe and moderate hepatitis in transgenic mice expressing hepatitis B surface antigen (HBs-Tg mice) and human hepatocyte chimeric mice infected with HBV, respectively. HBsAg accumulation appears to cause ER stress because a counteractive response to ER stress, namely, unfolded protein response (UPR), was induced in both HBs-Tg mice and HBV infected chimeric mice. Our results indicate that IFN-1s suppress UPR before causing liver injury. UPR was also suppressed by IFNγ. Induction of UPR in HBs-Tg mice before treatment with IFNα and IFNγ significantly alleviated liver injury. We suggest that IFNs exert cytotoxicity to ER stress accumulating cells by suppressing UPR.

Background: We recently identified 39 human microRNAs, which effectively suppress hepatitis B virus (HBV) replication in hepatocytes. Chronic HBV infection often results in active, hepatitis-related liver fibrosis; hence, we assessed whether any of these microRNAs have anti-fibrotic potential and predicted that miR-6133-5p may target several fibrosis-related genes. Methods: The hepatic stellate cell line LX-2 was transfected with an miR-6133-5p mimic and subsequently treated with Transforming growth factor (TGF)-β. The mRNA and protein products of the COL1A1 gene, encoding collagen, and the ACTA2 gene, an activation marker of hepatic stellate cells, were quantified. Results: The expression of COL1A1 and ACTA2 was markedly reduced in LX-2 cells treated with miR-6133-5p. Interestingly, phosphorylation of c-Jun N-terminal kinase (JNK) was also significantly decreased by miR-6133-5p treatment. The expression of several predicted target genes of miR-6133-5p, including TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), was also reduced in miR-6133-5p-treated cells. The knockdown of TGFBR2 by the corresponding small interfering RNA greatly suppressed the expression of COL1A1 and ACTA2. Treatment with the JNK inhibitor, SP600125, also suppressed COL1A1 and ACTA2 expression, indicating that TGFBR2 and JNK mediate the anti-fibrotic effect of miR-6133-5p. The downregulation of FGFR1 may result in a decrease of phosphorylated Akt, ERK (extracellular signal-regulated kinase), and JNK. Conclusion: miR-6133-5p has a strong anti-fibrotic effect, mediated by inactivation of TGFBR2, Akt, and JNK.