Tomohide Tatsumi

Critical role of MHC class I-related chain A and B expression on IFN-α-stimulated dendritic cells in NK cell activation: Impairment in chronic hepatitis C virus infection

Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-γ production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-α or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-α-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-α stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-α. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-α stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-α activates NK cells; this impairment might affect IFN-α responsiveness in hepatitis C virus infection.

Expression and role of MICA and MICB in human hepatocellular carcinomas and their regulation by retinoic acid

Natural killer (NK) cells are important effector cells for the first line of defense against tumor, but the mechanisms by which they recognize and kill human hepatocellular carcinoma (HCC) remains to be elucidated. Distant MHC class I homologs MICA and MICB are recently identified human ligands for NK cell activating receptor NKG2D. In our present study, MICA or MICB transcript was detected in 6 of 10 human hepatocellular carcinoma tissues, but not in the surrounding non‐cancerous tissues. Immunohistochemical analysis showed that MICA/B were expressed in the tumor cells of the cancerous tissues. Huh7 and HepG2 hepatoma cells, but not Hep3B cells, substantially expressed MICA/B on their cell surface. MICA/B expressed on hepatoma cells contributed to their NK sensitivity, because Huh7 and HepG2 were less susceptible to NK cytolysis when MAb against MICA/B was added during the cytolysis assay. Of interest is the finding that retinoic acid upregulated expression of MICA/B in Huh7 and HepG2 cells. Retinoic acid‐treated hepatoma cells induced IFNγ production from cocultured NK cells and rendered themselves more susceptible to NK cells. This was clearly dependent on upregulation of MICA/B, because both the enhanced IFNγ production and NK cytolysis were completely abolished by MAb‐mediated masking of MICA/B. These results suggest that MICA/B, expressed on a subset of human HCCs, may play an important role in their susceptibility to NK cells. Furthermore, retinoic acid can function as a modulator of MICA/B expression and thereby further activate NK cells.

Negative Regulation of NK Cell Activities by Inhibitory Receptor CD94/NKG2A Leads to Altered NK Cell-Induced Modulation of Dendritic Cell Functions in Chronic Hepatitis C Virus Infection

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFβ when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFβ. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.

Autocrine/Paracrine IL-15 That Is Required for Type I IFN-Mediated Dendritic Cell Expression of MHC Class I-Related Chain A and B Is Impaired in Hepatitis C Virus Infection

We previously reported that monocyte-derived dendritic cells activate resting NK cells by expressing MHC class I-related chain A and B (MICA/B), ligands for NKG2D, in response to IFN-α, but the MICA/B expression was severely impaired in patients with chronic hepatitis C virus (HCV) infection. In the present study, we examined induction of MICA/B on DCs by various innate cytokines and found that DCs from either healthy donors or HCV-infected individuals, upon IL-15 stimulation, express MICA/B and can activate NK cells, which is solely dependent on MICA/B-NKG2D interaction. Of interest is the finding that IL-15- and type I IFN-mediated induction of MICA/B in healthy donors is completely inhibited when DCs are incubated in the presence of anti-IFN-α/βR or anti-IL-15Rα, respectively, suggesting interdependent roles of these cytokines in MICA/B expression. Indeed, DCs produced IL-15 in response to type I IFN, whereas they directly produced IFN-β, in response to IL-15, which was followed by the production of IFN-α. In HCV-infected individuals, type I IFN-mediated production of IL-15 was virtually absent, but IL-15-mediated production of type I IFN was not compromised, which is consistent with the distinct ability of these cytokines to induce MICA/B in these patients. The present study demonstrates that IL-15 and type I IFN lead to DC expression of MICA/B and subsequent DC activation of NK cells, which is critically dependent on each other’s autocrine/paracrine effect, and suggests that impaired IL-15 production is one of the mechanisms of the aberrant response of DC to type I IFN in HCV-infected patients.

Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease‐stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self‐digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3‐I to LC3‐II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3‐II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP‐GFP‐LC3 fluorescence change in sorafenib‐treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3‐II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA‐mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib†

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease‐stabilizing and cytostatic rather than one of tumor regression. Bcl‐xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma‐2 (Bcl‐2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl‐xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT‐737, a small molecule that specifically inhibits Bcl‐xL but not myeloid cell leukemia‐1 (Mcl‐1), could control HCC progression, especially when used with sorafenib. Administration of ABT‐737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT‐737 caused the levels of Mcl‐1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT‐737, because decreasing Mcl‐1 expression levels to the baseline by a small interfering RNA–mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT‐737 to Mcl‐1 knockout mice induced severe liver apoptosis, suggesting that tumor‐specific inhibition of Mcl‐1 is required for therapeutic purposes. Sorafenib transcriptionally down‐regulated Mcl‐1 expression specifically in tumor cells and abolished Mcl‐1 up‐regulation induced by ABT‐737. Sorafenib, not alone but in combination with ABT‐737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. Conclusion: Bcl‐xL inactivation by ABT‐737 in combination with sorafenib was found to be safe and effective for anti‐HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC. (HEPATOLOGY 2010)

Thrombocytopenia exacerbates cholestasis-induced liver fibrosis in mice.

BACKGROUND & AIMS Circulating platelet counts gradually decrease in parallel with progression of chronic liver disease. Thrombocytopenia is a common complication of advanced liver fibrosis and is thought to be a consequence of the destruction of circulating platelets that occurs during secondary portal hypertension or hypersplenism. It is not clear whether thrombocytopenia itself affects liver fibrosis. METHODS Thrombocytopenic mice were generated by disruption of Bcl-xL, which regulates platelet life span, specifically in thrombocytes. Liver fibrosis was examined in thrombocytopenic mice upon bile duct ligation. Effect of platelets on hepatic stellate cells (HSCs) was investigated in vitro. RESULTS Thrombocytopenic mice developed exacerbated liver fibrosis, with increased expression of type I collagen alpha1 and alpha2, during cholestasis. In vitro experiments revealed that, upon exposure to HSCs, platelets became activated, released hepatocyte growth factor (HGF), and then inhibited HSC expression of the type I collagen genes in a Met signal-dependent manner. In contrast to the wild-type mice, the thrombocytopenic mice did not accumulate hepatic platelets or phosphorylate Met in the liver following bile duct ligation. Administration of recombinant HGF to thrombocytopenic mice reduced liver fibrosis to the levels observed in wild-type mice and attenuated hepatic expression of the type I collagen genes. CONCLUSIONS Thrombocytopenia exacerbates liver fibrosis; platelets have a previously unrecognized, antifibrotic role in suppressing type I collagen expression via the HGF-Met signaling pathway.

Alterations in microRNA expression profile in HCV-infected hepatoma cells: involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway.

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

Polarized type-1 dendritic cells (DC1) producing high levels of IL-12 family members rescue patient TH1-type antimelanoma CD4+ T cell responses in vitro.

Deficiencies in TH1-type immunity in patients with cancer may facilitate tumor progression and limit the effectiveness of current immunotherapy approaches. We hypothesized that Type-1 polarized dendritic cells (DC1) might be able to recondition patient antitumor CD4+ T cell responses toward the TH1-type in vitro. Although DC1 have been previously demonstrated to prime TH1 responses from naive CD4+ T cells, their impact on antigen-experienced TH responses remains unknown. We confirmed our own earlier observations that patient CD4+ T cell reactivity against melanoma-associated antigens (MAA) was weaker and less Type-1–polarized than their corresponding antiviral responses. Stimulation of patient CD4+ T cells with peptide-pulsed DC1 (producing multiple IL-12 family member cytokines, including IL-12p70, IL-23, and IL-27) promoted robust TH1-type, epitope-specific T cell responses. Addition of exogenous IL-12 family member cytokines alone, or in combination, to nonpolarized DC was insufficient to equate to the benefits associated with DC1-based stimulation; however, IL-27 and IL-12p70 blockade neutralized the ability of DC1 cells to enhance TH1-type antitumor immunity in vitro. Notably, DC1-based stimulation seemed capable of “revitalizing” defective TH1-type responses within the CD45RO+ subset of antigen-experienced CD4+ T cells in melanoma patients. In addition to promoting elevated levels of IFN-γ from responder CD4+ T cells, DC1-based stimulation also led to increased levels of IL-12Rβ2 and t-bet expression by TH cells. These results suggest that preexisting CD4+ T cell immunity to cancer is not relegated to Type-1 insufficiency and may be corrected via the application of DC1-based vaccination protocols.