Hiroyoshi Doi

Dysfunctional B‐cell activation in cirrhosis resulting from hepatitis C infection associated with disappearance of CD27‐Positive B‐cell population

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC). Both advanced solid tumors and HCV have previously been associated with memory B‐cell dysfunction. In this study, we sought to dissect the effect of viral infection, cirrhosis, and liver cancer on memory B‐cell frequency and function in the spectrum of HCV disease. Peripheral blood from healthy donors, HCV‐infected patients with F1‐F2 liver fibrosis, HCV‐infected patients with cirrhosis, patients with HCV‐related HCC, and non‐HCV‐infected cirrhotics were assessed for B‐cell phenotype by flow cytometry. Isolated B cells were stimulated with anti–cluster of differentiation (CD)40 antibodies and Toll‐like receptor (TLR)9 agonist for assessment of costimulation marker expression, cytokine production, immunoglobulin (Ig) production, and CD4+ T‐cell allostimulatory capacity. CD27+ memory B cells and, more specifically, CD27+IgM+ B cells were markedly less frequent in cirrhotic patients independent of HCV infection. Circulating B cells in cirrhotics were hyporesponsive to CD40/TLR9 activation, as characterized by CD70 up‐regulation, tumor necrosis factor beta secretion, IgG production, and T‐cell allostimulation. Last, blockade of TLR4 and TLR9 signaling abrogated the activation of healthy donor B cells by cirrhotic plasma, suggesting a role for bacterial translocation in driving B‐cell changes in cirrhosis. Conclusion: Profound abnormalities in B‐cell phenotype and function occur in cirrhosis independent of HCV infection. These B‐cell defects may explain, in part, the vaccine hyporesponsiveness and susceptibility to bacterial infection in this population. (HEPATOLOGY 2012)

Peripheral CD27−CD21− B-cells represent an exhausted lymphocyte population in hepatitis C cirrhosis

UNLABELLED Hepatitis C cirrhosis is associated with a profound disappearance of memory B-cells. We sought to determine if this loss is associated with the expansion of the CD27(-)CD21(-) tissue-like memory B-cells with features of B-cell exhaustion. To this end, we quantified the frequency of CD27(-)CD21(-) B-cells in healthy, non-cirrhotic HCV-infected, and cirrhotic patients. We examined the expression of putative inhibitory receptors, the proliferative and immunoglobulin-secreting capacity of CD27/CD21-defined B-cell subsets upon B-cell receptor and/or CD40 stimulation. We found that CD27(-)CD21(-) B-cells are significantly increased in frequency relative to healthy donors in HCV-infected patients. CD27(-)CD21(-) B-cells were hypoproliferative relative to naïve and resting memory B-cells upon agonistic stimulation, but retained similar capacity for antibody secretion. CONCLUSION CD27(-)CD21(-) tissue-like memory B-cells with exhausted proliferation circulate at increased frequency in cirrhotic and non-cirrhotic HCV-infected patients. This B-cell subset does not appear anergic, exhibiting immunoglobulin-secreting capacity on CD40 agonism indistinguishable from other CD27/CD21-defined B-cell subsets.

Cyclooxygenase‐2 gene promoter polymorphisms affect susceptibility to hepatitis C virus infection and disease progression

Aim:  Because polymorphisms of cyclooxygenase‐2 (COX‐2) and osteopontin (OPN) promoter regions and a promoter/enhancer region of forkhead box protein 3 (FOXP3) gene are known to affect immune responses, we examined whether these polymorphisms can influence susceptibility to hepatitis C virus (HCV) infection and progression of liver disease.

Immune Response of Cytotoxic T Lymphocytes and Possibility of Vaccine Development for Hepatitis C Virus Infection

Immune responses of cytotoxic T lymphocytes (CTLs) are implicated in viral eradication and the pathogenesis of hepatitis C. Weak CTL response against hepatitis C virus (HCV) may lead to a persistent infection. HCV infection impairs the function of HCV-specific CTLs; HCV proteins are thought to actively suppress host immune responses, including CTLs. Induction of a strong HCV-specific CTL response in HCV-infected patients can facilitate complete HCV clearance. Thus, the development of a vaccine that can induce potent CTL response against HCV is strongly expected. We investigated HCV-specific CTL responses by enzyme-linked immuno-spot assay and/or synthetic peptides and identified over 40 novel CTL epitopes in the HCV protein. Our findings may contribute to the development of the HCV vaccine. In this paper, we describe the CTL responses in HCV infection and the attempts at vaccine development based on recent scientific articles.

Interleukin-4 and CpG oligonucleotide therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses.

Because IL-4 and CpG oligodeoxynucleotides (CpG-ODNs) are immune stimulants, we evaluated the antitumor effects of IL-4 gene therapy and CpG-ODN treatment in a poorly immunogenic murine cancer model. We used a murine colorectal cancer MC38 cell line overexpressing the IL-4 gene (MC38-IL4). Incubation with MC38-IL4 and CpG-ODN enhanced bone marrow-derived dendritic cell (DC) maturation in vitro. In addition, interferon (IFN)-γ production was significantly increased in naïve splenocytes after they were coincubated with MC38-IL4 and CpG-ODN. When mice bearing MC38 wild-type tumors were inoculated subcutaneously with MC38-IL4 cells and CpG-ODN, the outgrowth of established parental tumors was significantly suppressed compared to those in the MC38-IL4-treated group (IL-4 vs. IL-4 + CpG-ODN, p=0.015). A marked infiltration of CD8+ cells in the established parental tumors of mice treated with MC38-IL4 and CpG-ODN was confirmed by immunohistochemical analyses (MC38-IL4, 2.8 ± 1.9 cells/field vs. MC38-IL4 + CpG-ODN, 20.7 ± 15.3 cells/field, p=0.027). Significant tumor-specific cytolysis was detected when splenocytes of MC38-IL4 + CpG-ODN-treated mice were stimulated by γ-irradiated MC38-IL4 cells and CpG-ODN twice weekly in vitro and used as effector cells in a chromium-release assay (32.2 ± 3.5% for MC38 cells vs. 3.2 ± 1.1% for YAC-1 cells; at an effector to target ratio of 40). These results suggest that IL-4 and CpG-ODN treatment promotes potent Th1-type antitumor immune responses. Therefore, the combination of IL-4 gene therapy and CpG-ODN treatment for cancer should be evaluated in clinical trials.

Impact of oral silymarin on virus- and non-virus-specific T-cell responses in chronic hepatitis C infection.

Silymarin displays anti‐inflammatory effects on T lymphocytes in vitro. The immunomodulatory properties of oral silymarin in vivo in humans with chronic hepatitis C have not previously been characterized. We hypothesized that silymarin would suppress T‐cell proliferation and pro‐inflammatory cytokine production of virus‐ and non‐virus‐specific T cells while increasing anti‐inflammatory IL‐10 production in vivo. Patients from one site of the SyNCH‐HCV double‐masked, placebo‐controlled study of oral silymarin in prior interferon nonresponders with chronic hepatitis C provided blood samples at baseline and treatment week 20. Mononuclear cells were stimulated with recombinant HCV proteins and controls in 3H‐thymidine proliferation assays, IFNγ ELISPOT and IL‐10 ELISPOT. The frequency of CD4+CD25hi and CD4+foxp3+ regulatory T cells, serum cytokine levels, serum IP‐10 and lymphocyte interferon‐stimulated gene expression were also quantified at baseline and week 20. Thirty‐two patients were recruited (10; placebo, 11; 420 mg three times a day, 11; 700 mg three times a day). Serum ALT and HCV RNA titres did not change in any group. HCV‐specific CD4+ T‐cell proliferation and the frequency of IFNγ‐ and IL‐10‐producing T cells were not significantly changed in silymarin‐treated subjects. However, C. albicans‐induced T‐cell IFNγ and phytohaemagglutinin‐induced T‐cell proliferation were suppressed by silymarin therapy. A trend towards augmentation of interferon‐induced ISG15 expression was present in the high‐dose silymarin group. While no effect on HCV‐specific T cells was identified, these data confirm that high‐dose oral silymarin exerts modest nonspecific immunomodulatory effects in vivo. The impact of this anti‐inflammatory effect on long‐term liver health in chronic hepatitis C merits future clinical investigation.

Magnitude of CD8+ T‐cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)‐specific CD8+ T‐cell responses and the clinical course of acute HCV infection.

Dendritic cells stimulated with cytidine‐phosphate‐guanosine oligodeoxynucleotides and interferon‐α‐expressing tumor cells effectively reduce outgrowth of established tumors in vivo

Dendritic cells (DC) are potent antigen‐presenting cells that elicit immune responses to foreign antigens. We have previously demonstrated the synergistic effects of cytidine‐phosphate‐guanosine (CpG) oligodeoxynucleotides (ODN) and interferon (IFN)‐α on DC maturation in vitro. In the present study, the antitumor effects of DC preincubated with IFN‐α gene‐overexpressing murine colorectal cancer MC38 cells (MC38‐IFN‐α) and CpG ODN were evaluated in a poorly immunogenic murine cancer system. When we injected DC preincubated with MC38‐IFNα and CpG ODN subcutaneously to mice bearing MC38 wild‐type tumors, the outgrowth of the established parental tumors was suppressed significantly compared with that following administration of DC with MC38‐IFN‐α (P = 0.008). All mice injected with DC preincubated with MC38‐IFN‐α and CpG ODN rejected a subsequent parental tumor challenge. Immunohistochemical and flow cytometric analyses showed that CD4+, CD8+, and NK1.1+ cells markedly infiltrated the established tumors of mice treated with DC preincubated with MC38‐IFN‐α and CpG ODN. From the results in immune cell‐depleted mice, CD4+ and asialo‐GM‐1+ cells seemed to contribute to the antitumor effects induced by the combination DC therapy. Furthermore, non‐specific cytolysis was detected when splenocytes of mice inoculated with DC preincubated with MC38‐IFNα and CpG ODN were used as effector cells. Using an interleukin (IL)‐12‐neutralizing antibody it was suggested that IL‐12 stimulates natural killer cells and contributes in part to the antitumor effects induced by DC incubated with CpG ODN and IFN‐α. As DC‐based immunotherapy with CpG ODN and IFN‐α‐expressing tumor cells induces a potent antitumor immune response, it should be considered for clinical application. (Cancer Sci 2008; 99: 1663–1669)

Tumor necrosis factor‐α‐mediated hepatocyte apoptosis stimulates fibrosis in the steatotic liver in mice

Hepatocyte apoptosis has been implicated in the progression of nonalcoholic steatohepatitis. However, it is unclear whether the induction of tumor necrosis factor (TNF)‐α‐mediated hepatocyte apoptosis in the simple fatty liver triggers liver fibrosis. To address this question, high‐fat diet‐fed mice were repeatedly administered D‐galactosamine, which increases the sensitivity of hepatocytes to TNF‐α‐mediated apoptosis. In mice treated with a high‐fat diet plus D‐galactosamine, hepatocyte apoptosis and liver fibrosis were induced, whereas both apoptosis and fibrosis were inhibited in these mice following gut sterilization with antimicrobials or knockout of TNF‐α. Furthermore, liver fibrosis was diminished when hepatocyte apoptosis was inhibited by expressing a constitutively active inhibitor of nuclear factor κB kinase subunit β. Thus, hepatocyte apoptosis induced by intestinal dysbiosis or TNF‐α up‐regulation in the steatotic liver caused fibrosis. Organ fibrosis, including liver fibrosis, involves the interaction of cyclic adenosine monophosphate‐response element‐binding protein‐binding protein (CBP) and β‐catenin. Here, hepatocyte‐specific CBP‐knockout mice showed reduced liver fibrosis accompanied by hepatocyte apoptosis diminution; notably, liver fibrosis was also decreased in mice in which CBP was specifically knocked out in collagen‐producing cells because the activation of these cells was now suppressed. Conclusion: TNF‐α‐mediated hepatocyte apoptosis induced fibrosis in the steatotic liver, and inhibition of CBP/β‐catenin signaling attenuated the liver fibrosis due to the reduction of hepatocyte apoptosis and suppression of the activation of collagen‐producing cells. Thus, targeting CBP/β‐catenin may represent a new therapeutic strategy for treating fibrosis in nonalcoholic steatohepatitis. (Hepatology Communications 2018;2:407‐420)

Pro-angiogenic TIE-2-expressing monocytes/TEMs as a biomarker of the effect of sorafenib in patients with advanced hepatocellular carcinoma

Sorafenib, a multi‐kinase inhibitor, inhibits tumor angiogenesis and is the first‐line systemic therapy for patients with advanced hepatocellular carcinoma (HCC). However, due to its limited effects and frequent occurrence of side effects, biomarkers are needed to predict the effects of sorafenib. We considered the possibility of using TIE‐2‐expressing monocytes (TEMs) to predict the response in sorafenib‐treated patients with advanced HCC. TEMs serve as a diagnostic marker of HCC and are related to angiogenesis. We analyzed 25 advanced HCC patients and prospectively evaluated TEMs before (Pre TEMs) and at 1 month after initial therapy (T1m TEMs). The radiologic response was evaluated by modified Response Evaluation Criteria in Solid Tumors (mRECIST). Median survival time (MST) was significantly longer in the partial response/stable disease (PR/SD) group (21.8 months) than in the PD group (8.7 months). ΔTEMs (changes of T1m TEMs compared to Pre TEMs) were significantly lower in the PR/SD group than in the PD group. MST of the ΔTEMs low group (14.2 months) was significantly longer than that of the high group (8.7 months). Univariate and multivariate Cox regression analyses showed that ΔTEMs [hazard ratio (HR) = 8.53, 95% confidence interval (CI) = 1.51–48.16, p = 0.015] and Child‐Pugh class (HR = 5.59, 95% CI = 1.06–29.63, p = 0.043) were independently associated with overall survival. Our results suggest that ΔTEMs could serve as a biomarker for predicting radiologic response and overall survival in sorafenib‐treated patients with advanced HCC.