Michiyo Inoue

Reduced Numbers and Impaired Ability of Myeloid and Plasmacytoid Dendritic Cells to Polarize T Helper Cells in Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Immunohistochemical evaluation of alpha-catenin expression in human gastric cancer

E-cadherin (E-cad) plays a major role in the maintenance of cell-cell adhesion in epithelial tissues, and impaired E-cad expression correlates with tumour invasion and metastasis. Alpha-catenin (α-cat), an undercoat protein of adherens junctions, binds to the cytoplasmic domain of E-cad and is essential for linking E-cad to actin-based cytoskeleton. We investigated E-cad and α-cat expression in 60 human gastric cancers immunohistochemically. The 60 gastric cancers were classified into 18 (30%) in which α-cat expression was preserved, and 42 (70%) reduced cases. The reduction of α-cat expression was significantly related to dedifferentiation, depth of invasion, infiltrative growth and lymph node metastasis. We also examined the co-expression of α-cat and E-cad. Seventeen (28%) tumours preserved both molecules [α-cat(+)/E-cad(+)] and 33 (55%) tumours reduced both [α-cat(−)/E-cad(−)], whereas 9 (15%) tumours exhibited α-cat(−)/E-cad(+). The frequency of lymph node metastasis in α-cat(−)/E-cad(+) tumour (67%) was significantly higher than that in α-cat(+)/E-cad(+) tumours (24%) and was close to that in α-cat(−)/E-cad(−) tumours (82%). The frequency of haematogenous liver metastasis in α-cat(−)/E-cad(+) tumours (44%) was significantly higher than that in α-cat(+)/E-cad(+) tumours (6%) or α-cat(−)/E-cad(−) tumours (9%). Thus, in all E-cad(+) tumours, the frequency of lymph node and liver metastasis was higher in α-cat(−) tumours than in α-cat(+) tumours. α-Cat expression is apparently better at predicting tumour invasion and metastasis than E-cad expression.

Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I.

Dendritic cells utilize various sets of Toll‐like receptors (TLR) or cytosolic sensors to detect pathogens and evoke immune responses. In patients with hepatitis C virus (HCV) infection, a higher prevalence of various infectious diseases is reported; suggesting that innate immunity against pathogens is impaired. The aim of this study was to clarify whether the TLR and retinoic acid inducible gene‐I (RIG‐I) system in myeloid dendritic cells is preserved or not in chronic HCV infection. The expression of TLRs, RIG‐I and its relatives were compared in myeloid dendritic cells between 39 patients and 52 healthy volunteers. The induction of type‐I interferon (IFN) and inflammatory cytokines was examined in response to agonists for TLR2 (palmitoyl‐3‐cysteine‐serine‐lysine‐4), TLR3/RIG‐I (polyinosine–polycytidylic acid) or TLR4 (lipopolysaccharide). The relative expressions of TLR2, TLR4, RIG‐I, and LGP2 from the patients were significantly higher than those from the volunteers, whereas TLR3 and MDA‐5 expressions did not differ. In search for factors regulating TLR/RIG‐I expression, it was shown that IFN‐α, polyinosine–polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG‐I, but TNF‐α, HCV core or HCV non‐structural proteins did not. For the functional analyses, myeloid dendritic cells from the patients induced significantly less amounts of IFN‐β, TNF‐α and IL‐12p70 in response to polyinosine–polycytidylic acid or lipopolysaccharide. It is noteworthy that the expression of TRIF and TRAF6, which are essential adaptor molecules transmitting TLR3 or TLR4‐dependent signals, is reduced in the patients. Thus, innate cytokine responses in myeloid dendritic cells are impaired regardless of enhanced expressions of TLR2, TLR4, and RIG‐I in HCV infection. J. Med. Virol. 80:980–988, 2008.

Impaired Function of Dendritic Cells Circulating in Patients Infected with Hepatitis C Virus Who Have Persistently Normal Alanine Aminotransferase Levels

Hepatitis C virus (HCV) induces chronic liver disease in hosts which can eventually progresses to liver cirrhosis and hepatocellular carcinoma. However, progression of liver disease is slower in patients with persistently normal levels of alanine aminotransferase (ALT) than in those with active hepatitis. Although distinct immune responses against HCV have been proposed in asymptomatic infection, the role of circulating dendritic cells (DC) in the pathogenesis of these patients remains obscure. To address this issue, we compared the number and function of myeloid DC (MDC) and plasmacytoid DC (PDC) between uninfected individuals and HCV-infected patients with or without elevated ALT levels. Numbers of DC and DC progenitors were significantly lower in patients with chronic active hepatitis than in control subjects. However, no differences were found in the number of DC between normal controls and HCV-infected patients with persistently normal ALT levels. MDC from patients with active hepatitis were less able to polarize naive CD4 T cells into the Th1 phenotype, while their MDC and PDC primed more CD4 T cells producing IL-10 than those from normal controls. Such dysfunction of DC was also observed in patients with persistently normal ALT levels. In conclusion, circulating DC decrease in number predominantly in HCV-infected patients with active hepatitis, and the function of DC is impaired even in those with normal ALT levels.

Enhanced ability of regulatory T cells in chronic hepatitis C patients with persistently normal alanine aminotransferase levels than those with active hepatitis

Summary.  In hepatitis C virus (HCV) infection, the Th1‐type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV‐infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally‐occurring Treg (N‐Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real‐time reverse transcriptase‐polymerase chain reaction. Bulk or CD25‐depleted CD4+ T cells cultured with HCV‐NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127–CD25–FOXP3+ cells as distinct subsets other than CD25+ N‐Treg. The frequencies of N‐Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV‐specific T cell responses, showing that co‐existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127–CD25–FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV‐infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis.

Quick generation of fully mature dendritic cells from monocytes with OK432, low-dose prostanoid, and interferon-alpha as potent immune enhancers.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-α (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

Comparative analyses of regulatory T cell subsets in patients with hepatocellular carcinoma: A crucial role of CD25−FOXP3− T cells

Regulatory T cells (Tregs) play pivotal role in cancer‐induced immunoediting. Increment of CD25high+FOXP3+ natural Tregs has been reported in patients with hepatocellular carcinoma (HCC); however, the involvement of other type of Tregs remain elusive. We aimed to clarify whether FOXP3− Tregs are increased and functionally suppressive or not in patients with HCC. We enrolled 184 hepatitis C‐infected patients with chronic liver diseases or HCC, 57 healthy subjects and 27 HCC patients with other etiology. Distinct Treg subsets were phenotypically identified by the expression of CD4, CD25, CD127 and forkhead/winged helix transcription factor (FOXP3). Their gene profiles, frequency and suppressor functions against T cell proliferation were compared among the subjects. To examine the molecules involving in Treg differentiation, we cultured naive CD4+ T cells in the presence of HCC cells and dendritic cells. We determined two types of CD4+CD127− T cells with comparable regulatory ability; one is CD25high+ cells expressing FOXP3 (CD25high+FOXP3+ Tregs) and the other is CD25− cells without FOXP3− expression (CD25−FOXP3− cells). The peripheral or intrahepatic frequency of CD25−FOXP3− Tregs in HCC patients is higher than those in other groups, of which significance is more than CD25high+FOXP3+ cells. Of importance, CD25−FOXP3− Tregs, but not CD25high+FOXP3+ cells, dynamically change in patients accompanied by the ablation or the recurrence of HCC. CD25−FOXP3− T cells with CD127−IL‐10+ phenotype are inducible in vitro from naive CD4+ T cells, in which programmed cell death 1 ligand 1, immunoglobulin‐like transcript 4 and human leukocyte antigen G are involved.. In conclusion, CD25−FOXP3− Tregs with suppressive capacity are increased in patients with HCC, suggesting their distinct roles from CD25+FOXP3+ Tregs.

644 Pseudotype hepatitis C virus infects immature myeloid dendritic cells through the interaction with lectin

Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca 2+ chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca 2+ -independent manner.

Immunohistochemical Evaluation of E-Cadherin and α-Catenin Expression in Human Esophageal Cancer

Tumor metastasis is initiated by disaggregation of invasive cells from the primary tumor - a step that requires a breakdown of intercellular adhesion. To investigate the mechanism of dysfunction in cell-cell adhesion of cancerous tissues, we evaluated E-cadherin (E-cad) and α-catenin (α-cat) expression of human esophageal cancer immunohistochemically. Eighty percent of the tumors showed reduced expression of both of the molecules. The frequency of α-cat negative tumors was observed in 47%, but none of them were E-cad negative. These results suggest that esophageal cancers lose α-cat expression more frequently than E-cad, and the loss of α-cat mights cause dysfunction of E-cad-mediated intercellular adhesion.

Immunohistochemical Evaluation of α-Catenin, Cadherin-Associated Intercellular Protein, Expression in Human Gastric Cancer

Immunohistochemical study of α-catenin, cadherin associated intercellular protein, was performed in 36 human gastric cancer tissues. All normal epithelium strongly expressed α-catenin as well as E- cadherin, while 67% of the tumor had reduced expression of α-catenin. Compared to E-cadherin expression, α-catenin expression was similar in 26 cases (72%), but weaker in 9 cases (25%). The reduction of α-catenin expression was associated with dedifferentiation, and infiltrative growth. These results indicated that the reduction of α-catenin expression may play a crucial role for cancer invasion through down-regulation of E-cadherin function.