Impaired ability of interferon-alpha-primed dendritic cells to stimulate Th1-type CD4 T-cell response in chronic hepatitis C virus infection

In interferon-alpha (IFN-alpha)/ribavirin combination therapy for chronic hepatitis C (CHC), an enhanced T helper 1 (Th1) response is essential for the eradication of hepatitis C virus (HCV). We aimed to elucidate the role of IFN-alpha or IFN-alpha/ribavirin in dendritic cell (DC) ability to induce Th1 response in HCV infection. We generated monocyte-derived DC from 20 CHC patients and 15 normal subjects driven by granulocyte-macrophage colony-stimulating factor and interleukin 4 (IL-4) without IFN-alpha (GM/4-DC), with IFN-alpha (IFN-DC), with ribavirin (R-DC) or with IFN-alpha/ribavirin (IFN/R-DC) and compared their phenotypes and functions between the groups. We also compared them in 14 CHC patients between who subsequently attained sustained virological response (SVR) and who did not (non-SVR) by 24 weeks of IFN-alpha/ribavirin therapy. Compared with GM/4-DC, IFN-DC displayed higher CD86 expression, but lesser ability to secrete IL-10 and were more potent to prime CD4(+) T cells to secrete IFN-gamma and IL-2. Such differences were more significant in healthy subjects than in CHC patients. No additive effect of ribavirin was observed in DC phenotypes and functions in vitro either which was used alone or in combined with IFN-alpha. However, in the SVR patients, an ability of IFN/R-DC to prime T cells to secrete IFN-gamma and IL-2 was higher than those of IFN-DC and those of IFN/R-DC in the non-SVR group, respectively. In conclusion, DC from CHC patients are impaired in the ability to drive Th1 in response to IFN-alpha. Such DC impairment is restored in vitro by the addition of ribavirin in not all but some patients who cleared HCV by the combination therapy.     

Infection and dysfunction of circulating blood dendritic cells and their subsets in chronic hepatitis C virus infection

Background To understand interactions between dendritic cells (DCs) and viruses, in vitro-cultured monocyte-derived DCs are usually used for functional analyses. However, several recent studies indicate that circulating blood DCs are different from monocyte-derived DCs, both phenotypically and functionally. Indeed, circulating DCs act as functional antigen-presenting cells in vivo. This study was conducted to evaluate the function of circulating blood DCs in patients with chronic hepatitis C and to examine whether circulating DCs from these patients were infected by hepatitis C virus (HCV).Methods The phenotypes and biological functions of circulating DCs from patients with chronic hepatitis C (n = 27), patients with non-HCV chronic liver disease (n = 7), and normal volunteers (n = 13) were analyzed. The presence of the HCV genome sequence in circulating blood DCs and in subsets of circulating DCs (myeloid DCs and plasmacytoid DCs) in patients with chronic hepatitis C was assessed.Results The stimulatory capacity of circulating DCs was significantly reduced in patients with chronic hepatitis C compared to patients with non-HCV chronic liver diseases and normal controls (P < 0.01). HCV RNA was identified in the overall population of circulating DCs, and in myeloid DCs and plasmacytoid DCs. Nucleotide sequences of the 5 non-coding region of HCV RNA showed marked differences between paired samples of circulating DCs and sera from the same patients.Conclusions Our results indicate the dysfunction and infection of circulatory blood DCs in chronic HCV infection. This may compromise the capacity of patients with hepatitis C to induce an effective antiviral immune response.     

Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

Summary. Studies assessing the function of monocyte derived dendritic cells (MD‐DC) in individuals with hepatitis C virus (HCV) infection have shown conflicting results. Impaired MD‐DC function in chronic HCV infection would have important implications both for understanding the pathogenesis of HCV infection and in the use of autologous MD‐DC in vaccination strategies. We determined the allostimulatory capacity of MD‐DC in the same patient before and after HCV infection. Next, the phenotype, cytokine production and allostimulatory function of immature and mature MD‐DC in individuals with persistent HCV infection were compared directly with MD‐DC from healthy individuals. Finally, we assessed the ability of MD‐DC to prime autologous naïve peptide specific CD8+ T cells using HLA‐A2 class‐I tetramers. DCs retained the same allostimulatory capacity before and following the establishment of persistent HCV infection. The surface phenotype and the amount of interleukin (IL)‐10 and IL‐12(p70) produced during DC maturation did not differ between HCV‐infected individuals and healthy controls. Mature DCs from HCV‐infected individuals performed comparably in an allogeneic MLR compared with healthy individuals. Mature MD‐DC from HCV‐infected individuals stimulated the expansion of peptide specific naïve CD8+ T cells. MD‐DC from HCV‐infected and healthy individuals are phenotypically indistinguishable and perform comparably in functional assays.     

DC-SIGN在慢性乙型肝炎病毒感染时树突状细胞成熟活化中的作用

目的研究慢性HBV感染时,DC-SIGN在树突状细胞（DC）成熟和活化中介导的作用。方法将α-甘露糖苷酶抑制剂-基夫碱作用于HepG2.2.15细胞,收获上清中的高甘露糖型HBV颗粒,并于第5 d加入到外周血单核细胞衍生的DC培养基中,培养至第7 d,采用流式细胞仪检测DC表面CDla、CD83、CD80、CD86、HLA-DR分子的表达,MTT法检测DC刺激淋巴细胞增殖的能力,ELISA法检测DC分泌IL-12的水平。结果高甘露糖型HBV组,与天然的HBV组相比,DC表面CDla、CD83、CD80、CD86、HLA-DR分子的表达增加,分泌IL-12的水平升高,刺激同种异体淋巴细胞增殖的能力亦明显增强,且上述效应均可被DC-SIGN特异性抗体所阻断。结论 DC-SIGN识别高甘露糖型HBV后可以促进DC的成熟和活化,天然的HBV可能利用α-甘露糖苷酶参与的去甘露聚糖修饰来逃避DC-SIGN的识别,从而诱导DC功能的缺陷。     

Impaired cytotoxic T lymphocyte inductivity by dendritic cells derived from patients with hepatitis C virus-positive hepatocellular carcinoma

Aim: Peptide-based therapeutic vaccines are being developed. The aim of this study was to determine the feasibility of immunotherapy to hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) by assessing the inductivity of peptide-specific cytotoxic T lymphocyte (CTL) by dendritic cells. Methods: The inductivity of CTL was characterized in six patients with HCV-positive HCC, and compared to seven healthy volunteers and six patients with chronic HCV hepatitis (control). Results: Peptide-specific CTL was comparably induced in controls, but not induced in any patients with HCC. To characterize this, the cytokine profile and the expression of surface molecules interacting between dendritic and T cells were evaluated. Among the cytokines, production of interferon (IFN)-gamma was found to be impaired and closely related to the results of CTL assays, while the expression of surface molecules showed no significant changes. Conclusions: In HCV-positive HCC patients, CTL inductivity by dendritic cells is impaired. This may be related to the impaired production of IFN-gamma.     

树突状细胞与丙型病毒性肝炎

树突状细胞（DC）是目前已知体内功能最强的抗原递呈细胞,在丙型肝炎病毒（HCV）感染的患者,DC功能减弱可促进病毒的持续感染,造成疾病的慢性化。目前DC成为HCV疫苗的研究重点,可能成为治疗慢性丙型肝炎（CHC）的有效免疫学方法,本文就DC与丙型肝炎的关系及其在治疗中的应用前景作一综述。     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

前列腺素E1抑制慢性乙型重型肝炎树突状细胞成熟及CD4~+/CD8~+T细胞活性的观察

目的研究前列腺素E1对慢性乙型重型肝炎树突状细胞成熟及CD4+/CD8+T细胞活性的影响。方法抽取前列腺素E1治疗10 d前后的慢性乙型重型肝炎患者外周静脉血,密度梯度离心法分离淋巴细胞,贴壁培养获得PBMC,经重组人白细胞介素4(rhIL-4)、重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)、加或不加前列腺素E1刺激培养8 d获得树突细胞(DC)。经流式细胞仪检测DC表达的HLA-DR、CD83、CD86,ELISA测定分泌的IFNγ、IL-12p70。悬浮T细胞部分检测CD4+/CD8+T细胞比例及其细胞表面CD69、HLA-DR表达;部分经IL-2培养,用于检测DC体外诱导的T淋巴细胞毒活性。结果前列腺素E1治疗慢性乙型重型肝炎,对患者CD4+/CD8+T细胞比例无明显影响,可抑制CD4+/CD8+T细胞活化分子CD69、HLA-DR的表达;前列腺素E1体外培养的DC低表达HLA-DR、CD83、CD86,分泌IFNγ、IL-12p70均下降,体外诱导的自体淋巴细胞毒活性减弱,与未加用前列腺素E1比较差异有统计学意义(P<0.01)。结论前列腺素E1治疗慢性乙型重型肝炎,可抑制CD4+/CD8+T细胞活化;体外培养可抑制DC成熟,诱导减弱自体淋巴细胞毒活性。     

Regulatory T cells in hepatitis C virus infection

Hepatitis C virus (HCV) is a highly mutable RNA virus with a high propensity for chronic infection, affecting over 3% of the world's population. Persistent infection is associated with chronic hepatitis that may progress to cirrhosis and hepatocellular carcinoma over many years of infection. While cellular immune response plays a key role in viral infection, HCV persistence is associated with antiviral effector T-cell dysfunction with increased CD4+ CD25+ Tregs and interleukin-10+ Tr1cells, raising the possibility that the balance between antiviral effector and regulatory T-cell subsets contributes to the outcome of HCV infection.     

CD4+CXCR5+ T cells activate CD27+IgG+ B cells via IL-21 in patients with hepatitis C virus infection

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG+ B cells in patients with chronic hepa-titis C (CHC) infection have not been thoroughly elucidated. CD4+CXCR5+ follicularhelperT(Tfh)cells,viainterleukin (IL)-21 secretion, activate B cells. However, the role of CD4+CXCR5+T cellsintheactivationof IgG+ BcellsinCHCpatientsis not clear.
METHODS: The frequency of IgG+ B cells, including CD27?IgG+B and CD27+IgG+ B cells,the expression of the activation markers (CD86 and CD95) in IgG+ B cells, and the percentage of circu-lating CD4+CXCR5+ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The con-centrations of serum IL-21 were analyzed using ELISA. The role of CD4+CXCR5+ T cells in the activation of IgG+ B cells was investigated using a co-culture system.
RESULTS: A significantly lower proportion of CD27+IgG+ B cells with increased expression of CD86 and CD95 was observed in CHC patients.The expression of CD95 was negatively correlated with the percentage of CD27+IgG+ B cells, and it contributed to CD27+IgG+ B cell apoptosis. Circulating CD4+CXCR5+ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4+CXCR5+ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27+IgG+B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27+IgG+ B cells.
CONCLUSIONS: HCV infection increased the frequency of CD4+CXCR5+ T cells and decreased the frequency of CD27+IgG+B cells. CD4+CXCR5+ T cells activated CD27+IgG+ B cells via the secretion of IL-21.     

Importance of age in chronic hepatitis C virus infection

This study was designed to investigate the value of liver biopsy in the management of patients with chronic hepatitis C virus infection and to identify risk factors for fibrosis. It was a prospective audit of clinical, biochemical, virological and radiological features for predicting liver fibrosis in 140 consecutive patients seropositive for antibody against hepatitis C virus. Seventy-five per cent of patients were asymptomatic and 69% had no clinical signs of chronic liver disease. Serum transaminase levels were normal in 44% of patients, less than twice normal in 35% and more than twice normal in 21%. Ultrasound scan was unremarkable in 85%. Sixty-nine per cent of patients were viraemic at the time of liver biopsy. Liver histology revealed that fibrosis was absent in only 10% of patients (stage 0) while cirrhosis was evident in 7% (stage 5). Liver fibrosis was detected in 90% of patients (stage 1, 11%; stage 2, 41%; stage 3, 21%; and stage 4, 10%). Univariate analysis showed that increasing age, clinical signs of chronic liver disease and abnormal ultrasound scan findings were associated with liver fibrosis ( P <0.05); however, multiple linear regression analysis of all the clinical features did not reveal a useful model (sensitivity 42% and specificity 23%) for predicting liver fibrosis. Hence, no combination of clinical, biochemical, virological or radiological data was reliable in discriminating the stage of liver fibrosis in patients with chronic hepatitis C virus infection. However, older patients, especially those with clinical signs of chronic liver disease and abnormal ultrasound scan findings, were more likely to have advanced fibrosis. We recommend liver biopsy as the single most important investigation in detecting liver fibrosis.     

Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection

AIM: To identify the property of dendritic cells (DCs) of peripheral blood monocytes (PBMC) in patients with chronic HBV infection. METHODS: Twenty patients with persistent HBV infection were included in this study, 10 healthy subjects being used as a control group. The peripheral blood mononuclear cells (PBMC) of T cell-depleted populations were incubated and induced into mature dendritic cells in the RPMI-1640 medium in the presence of cytokines GM-CSF, IL-4, FLt-3,TNF-alpha and 100mL.L(-1 )of fetal calf serum for a total of 10-12 days. The expressions of surface markers on DCs were evaluated using flow cytometric analysis. ELISA method was used to determine the cytokine levels of interleukin-12 (IL-12) and IL-10 in the supernatant produced by DCs. For detection of the stimulatory capacity of DCs to T cell proliferation, mytomycin C-treated DC were incubated with allogenic T cells. RESULTS: A typical morphology of mature DCs from healthy subjects and HBV-infected patients was induced in in vitro incubation, but the proliferation ability and cellular number of DCs from HBV-infected patients significantly decreased compared with healthy individuals. In particular, the expression levels of HLA-DR, CD80 (B7-1) and CD86 (B7-2) on DC surface from patients were also lower than that from healthy individuals (0.46 vs 0.92 for HLA-DR, 0.44 vs 0.88 for CD80 and 0.44 vs 0.84 for CD86,P<0.05). The stimulatory capacity and production of IL-12 of DCs from patients in allogenic mixed lymphocyte reaction (AMLR) significantly decreased, but the production level of nitric oxide (NO) by DCs simultaneously increased compared with healthy subjects (86 +/- 15 vs 170 +/- 22 micromol.L(-1), P <0.05). CONCLUSION: The patients with chronic HBV infection have the defective function and immature phenotype of dendritic cells, which may be associated with the inability of efficient presentation of HBV antigens to host immune system for the clearance of HBV.     

Immune regulation in chronic hepatitis C virus infection

The immunological result of infection with Hepatitis C virus (HCV) depends on the delicate balance between a vigorous immune response that may clear the infection, but with a risk of unspecific inflammation and, or a less inflammatory response that leads to chronic infection. In general, exhaustion and impairment of cytotoxic function of HCV-specific T cells and NK cells are found in patients with chronic HCV infection. In contrast, an increase in immune regulatory functions is found primarily in form of increased IL-10 production possibly due to increased level and function of anti-inflammatory Tregs. Thus, the major immune players during chronic HCV infection are characterized by a decrease of cytotoxic function and increase of inhibitory functions. This may be an approach to diminish intrahepatic and systemic inflammation. Finally, there has been increasing awareness of regulatory functions of epigenetic changes in chronic HCV infection. A vast amount of studies have revealed the complexity of immune regulation in chronic HCV infection, but the interplay between immune regulation in virus and host remains incompletely understood. This review provides an overview of regulatory functions of HCV-specific T cells, NK cells, Tregs, IL-10, and TGF-β, as well as epigenetic changes in the setting of chronic HCV infection.     

Sarcoidosis in untreated chronic hepatitis C virus infection

Chronic hepatitis C has been implicated in the pathogenesis of sarcoidosis in several cases of patients treated with interferon‐α. On the other hand, only in a few cases previously has a possible link between sarcoidosis and untreated chronic hepatitis C virus infection been demonstrated. We report on a patient with chronic hepatitis C who developed cutaneous sarcoidosis without prior interferon‐α treatment. We hypothesize that viral persistence seen in chronic hepatitis C virus infection might be one of the potential factors that trigger cellular immune response in granulomatous reactions as seen in sarcoidosis, in genetically predisposed patients.     

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).     

Distribution of different hepatitis C virus genotypes in patients with hepatitis C virus infection

AIM:To investigate the presence of mixed infection and discrepancy between hepatitis C virus(HCV) genotypes in plasma,peripheral blood mononuclear cells(PBMCs),and liver biopsy specimens.METHODS:From September 2008 up to April 2009,133 patients with chronic hepatitis C referred to Firouzgar Hospital for initiation of an antiviral therapy were recruited in the study.Five milliliters of peripheral blood was collected from each patient and liver biopsy was performed in those who gave consent or had indications...     

Selective functional deficit in dendritic cell--T cell interaction is a crucial mechanism in chronic hepatitis B virus infection

Summary. A defect in specific T cell immunity has long been assumed to be the central mechanism of persistent Hepatitis B virus (HBV) infection. Recent studies on HBV transgenic mice have suggested, however, that functional deficit of dendritic cells (DC) was an underlying cause for the T cell dysfunction. The functions of monocyte‐derived DC were determined by studying 75 subjects that included chronic hepatitis B patients with low or high HBV load; antibody to hepatitis B surface antigen (anti‐HBs) positive individuals who had recovered completely from previous acute HBV infection; healthy donors who had received hepatitis B vaccination and were anti‐HBs positive; and immunologically naïve to HBV or the vaccine individual. Impaired interactions between monocyte‐derived DC and T cells were shown in chronic HBV infection patients, especially in those with active virus replication. The dysfunctions included: (i) failure of DC to increase human leukocyte antigen (HLA‐II), B7 expression and interleukin‐12 secretion in responses to hepatitis B surface antigen (HBsAg), (ii) defective induction of T cell proliferative response to HBsAg, (iii) failure to activate T cells to produce cytokines and (iv) deficit in the induction of antigen specific cytotoxic T lymphocytes (CTLs). In vitro treatment of DC with tumour necrosis factor‐α improved HLA‐II and B7 expression, as well as Th cell and CTL responses. It is concluded that defective DC‐T cell interactions may account for the specific T cell immune defects in chronic HBV infection. Immunotherapy that aims at restoring DC functions could offer a new opportunity for effectively managing persistent HBV infections.