Role of A20 in interferon‐α‐mediated functional restoration of myeloid dendritic cells in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon‐α (IFN‐α) ‐based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN‐α treatment are unclear. The ubiquitin‐editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN‐α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV‐infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN‐α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN‐α in vitro. Additionally, A20 expression by polyI:C‐activated mDCs, with or without IFN‐α treatment, negatively correlated with the expression of HLA‐DR, CD86 and CCR7, and the secretion of interleukin‐12 (IL‐12), but positively associated with the production of IL‐10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL‐12 in mDCs of chronically HCV‐infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.     

Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.     

Increased PD‐L1 expression and PD‐L1/CD86 ratio on dendritic cells were associated with impaired dendritic cells function in HCV infection

Impaired hepatitis C virus (HCV)‐specific T cell immunity was associated with the persistence of HCV infection. Dysfunction of dentritic cells (DCs) was believed to be involved in T cell exhaustion, but the mechanisms were rarely understood. In this study, surface costimulatory marker (CD83, CD86, and CD40), coinhibitory marker (PD‐L1) expression and allostimulatory capacity of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were evaluated in HCV‐infected patients. Results showed that the expression of both costimulatory and coinhibitory markers was increased in HCV‐infected patients compared with healthy controls. PD‐L1/CD86 ratio was increased and positively correlated with PD‐L1 expression on DCs in HCV‐infected patients. Allostimulatory capacity of DCs was impaired and inversely correlated with PD‐L1 expression and PD‐L1/CD86 ratio. These findings suggested that the effect of inhibitory marker PD‐L1 overwhelmed the effect of costimulatory markers and down regulated DC‐T activation in HCV‐infected patients. The results will be helpful to understand the mechanism of dysfunction of DCs in HCV infection and shed light on the DC‐based immunotherapeutic strategy. J. Med. Virol. 82: 1152–1159, 2010. © 2010 Wiley‐Liss, Inc.     

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.     

Involvement of dendritic cell frequency and function in virological relapse in pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C patients

A combination of pegylated interferon alpha (PEG-IFNalpha) and ribavirin has been used widely. Enhancement of immune response against hepatitis C virus (HCV) is known to be involved in the efficacy of the combination therapy. The aim of the study was to elucidate whether the frequency or function of immunocompetent blood cells is related to the outcome of the therapy. Twenty-five chronic hepatitis C patients with high viral load of HCV genotype 1 who underwent 48 weeks of PEG-IFNalpha2b and ribavirin therapy were examined. During the treatment, frequencies of dendritic cell subsets, helper T cell subsets, and NK cells were phenotypically determined. In some patients, the ability of dendritic cells to stimulate allogeneic CD4(+)T cells was examined at the end and after the therapy. Among the 25 patients, 11 showed a sustained virological response, 11 a transient response, and 3 no response. In comparison with sustained virological responders, non-sustained virological responders showed impaired dendritic cell function at the end and after the treatment. The transient responders showed a decline of plasmacytoid dendritic cell frequency from Weeks 1-12 and impaired dendritic cell function as well. Even in patients who attained negative serum HCV RNA at Week 12, the transient responders showed a significant decrease of plasmacytoid dendritic cell frequency and impaired dendritic cell function. In conclusion, in PEG-IFNalpha and ribavirin combination therapy for chronic hepatitis C patients, the early-phase plasmacytoid dendritic cell frequency and/or end-of-treatment dendritic cell function are related to the virological outcome of the therapy.     

Therapy with interferon-alpha and ribavirin for chronic hepatitis C virus infection upregulates membrane HLA-ABC, CD86, and CD28 on peripheral blood mononuclear cells

Multiple interferon-stimulated genes (ISGs) involving T-cell activation are upregulated during initial interferon-alpha-based therapy for chronic hepatitis C virus (HCV) infection. However, the long-term impact on therapeutic outcome in patients remains unknown. In this study, the effects of anti-HCV therapy on the surface expression of HLA-ABC, CD86, and CD28 were longitudinally assessed. These proteins are integral membrane receptors of antigen presentation and triggering of costimulatory signals for activating CD8+ T cells. Peripheral blood mononuclear cells were collected at baseline and post-treatment for 1 day, and 2, 4, 12, and 24 weeks, respectively. This treatment led to a time-related elevation of membrane levels of HLA-ABC and CD86 on B-cells and monocytes in patients with a sustained response (n = 23), but not in those without (n = 8). Meanwhile, upregulation of CD28 on CD4+ and CD8+ T cells was comparable in both groups of sustained responders and non-responders. Steady increases in the B cells' surface and intracellular HLA-ABC were observed, thus, the surface-to-intracellular ratios did not alter over the period of treatment. Furthermore, multivariate analysis shows that increased HLA-ABC on monocytes by week 12 correlates significantly with sustained response (P = 0.033). In conclusion, differential modulation of T-cell activation ISGs, such as HLA-ABC and CD86 might correlate with the outcome of interferon-alpha-based therapy in chronic hepatitis C patients.     

Recruitment of immature plasmacytoid dendritic cells (plasmacytoid monocytes) and myeloid dendritic cells in primary cutaneous melanomas

The present study has analysed the distribution and phenotype of dendritic cells (DCs) in primary cutaneous melanomas and sentinel lymph nodes by immunohistochemistry. In primary melanomas, an increase of DCs was found in the epidermis and the peritumoural area. Intraepidermal DCs were mostly CD1a⁺/Langerin⁺ Langerhans cells. Peritumoural DCs included a large population of DC‐SIGN⁺/mannose‐receptor⁺/CD1a^? DCs, a small subset of CD1a⁺ DCs, and, remarkably, plasmacytoid monocytes/plasmacytoid DCs (PM/PDCs). The PM/PDCs, most likely recruited by SDF‐1 secreted by melanoma cells, produced type I interferon (IFN‐I), but the expression of the IFN‐α inducible protein MxA was extremely variable and very limited in the majority of cases. All DC subsets were predominantly immature. The peritumoural area also contained a minor subset of mature CD1a⁺ DCs. However, the small amount of local interleukin (IL)‐12 p40 mRNA and the naïve phenotype of 20–50% of peritumoural T‐lymphocytes are consistent with poor T‐cell stimulation or erroneous recruitment. In sentinel lymph nodes, notable expansion of mature CD1a⁺/Langerin⁺ DCs was observed. The paucity of intratumoural DCs and the predominant immature phenotype of peritumoural dermal DCs indicate defective maturation of primary cutaneous melanoma‐associated DCs, resulting in lack of T‐cell priming. These results may explain why melanoma cells grow despite the presence of infiltrating immune cells. Copyright © 2003 John Wiley & Sons, Ltd.     

Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.     

Decreased dendritic cell numbers but increased TLR9-mediated interferon-alpha production in first degree relatives of type 1 diabetes patients

Objective

Dendritic cells (DCs) play an important role in pathogenesis of autoimmunity, including type 1 diabetes (T1D). In this study, we investigated DC subpopulations and their responses to TLR stimulation in T1D patients and their relatives.

Methods

We analyzed the frequency of myeloid (mDCs) and plasmacytoid DCs (pDCs) in 97 T1D patients (69 onset, 28 long-term), 67 first-degree relatives, and 64 controls. We additionally tested the IFN-alpha production by pDCs upon stimulation with TLR 7, 8 and 9 agonists.

Results

A lower number of mDCs and pDCs were found in T1D patients and their relatives. Of all the tested TLR ligands, only stimulation with CpG 2216 induced IFN-alpha production that was the highest in T1D relatives, except of autoantibody-negative relatives bearing the protective haplotypes.

Conclusion

Our data demonstrate disturbances in DC number and function expressed most significantly in T1D relatives and point to a potential role of TLR9-induced IFN-alpha production in T1D development.     

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.     

Interferon-gamma is produced by CD8 T cells in response to HLA-A24-restricted hepatitis C virus epitopes after sustained virus loss

Differences in cytotoxic T lymphocyte activity in hepatitis C virus infection may account for the outcome of interferon monotherapy. To investigate this hypothesis, we analysed the response of peripheral CD8(+) T cells that recognized epitopes presented by HLA-A*2402. We synthesized HLA/beta2-microglobulin/peptide complexes using two epitopes. Production of interferon-gamma by CD8(+) T cells in response to plastic-bound monomeric HLA/peptide complex was observed frequently in sustained virus responders (SVR) (n = 13) against all the peptides, NS31296-1304 (the percentage of responding patients, 61.5%) and core 129-137 (53.8%), while no interferon-gamma production was observed in non-responders (NR) (n = 13) for any of the peptides. Tetramer-staining showed the presence of CD8(+) T cells specific for all the peptides except NS31296-1304 in two SVR at the end of interferon monotherapy, although hardly any such cells were found in four NR. Specific killing was observed against peptides NS31296-1304 (3/4) and core 129-137 (1/4) in sustained responders but none in non-responders. These results suggest that the responses of cytotoxic T lymphocytes (CTLs) were induced during interferon therapy in these patients and that interferon-gamma production by CD8(+) T lymphocytes against HCV NS31296-1304 and core 129-137 are well maintained in patients with SVR compared with those with NR. These findings emphasize the importance of the CD8(+) T cell response in controlling HCV infection.     

nTreg与基因1型CHC患者抗病毒疗效的关系

目的研究基因1型慢性丙型肝炎（CHC）患者治疗过程中外周血自然调节性T细胞（nTreg）的动态变化及其与抗病毒疗效的关系。方法32例初治的CHC患者,均为基因1型,治疗方案为干扰素联合利巴韦林治疗48周。分别在治疗前、治疗过程中2、4、8、12、24、48周以及治疗结束后12或24周采集外周血标本。用流式的方法检测外周血单个核细胞（PBMC）中nTreg的比率。COBASAmpliprep／COBAS TaqmanHCV试剂盒检测HCV—RNA定量。结果24例患者治疗12周时HCV—RNA转阴,并且随访至治疗结束后24周仍阴性,达到持续病毒学应答（SVR）;8例患者治疗12周时病毒量未转阴,无应答（NVR）,24周时终止治疗。应答组和无应答组治疗前外周血中nTreg的比率差异无统计学意义（P〉0．05）。应答组治疗过程中2、4、12、24、48周及治疗结束后各个时间点PBMC中nTreg水平分别为（0．91±0．22）％、（1．31±0．29）％、（1．78±0．43）％、（1．92±0．44）％、（1．90±0．37）％、（1．14±0．35）％;在治疗2周（P〈0．001）及治疗结束后（P〈0．001）下降明显。无应答组治疗过程中2、4、12、24周及治疗结束后各时间点PBMC中nTreg水平分别为（1．21±0．15）％、（1．24±0．18）％、（1．42±0．11）％、（1．45±0．11）％、（1．14±0．10）％;Treg水平在治疗4周时缓慢上升（P=O．005）,在治疗结束后显著下降,与治疗24周比较差异有统计学意义（P〈0．001）。结论CHC患者外周血nTreg水平与干扰素／N巴韦林抗病毒治疗的疗效密切相关,抗病毒治疗2周时nTreg水平的下降可以预测其疗效。提示nTreg可能在机体清除病毒的早期过程中起着重要作用。     

Synergistic antiviral effect of a combination of mouse interferon-alpha and interferon-gamma on mouse hepatitis virus

Although interferon (IFN)-alpha and IFN-gamma have been reported to exhibit a synergistic antiviral effect through the different signaling pathways in vitro, their therapeutic efficacy is not well defined in vivo. The current study was carried out to investigate the combined antiviral effect in a model of mouse hepatitis virus Type 2 (MHV-2) infection, in which fulminant hepatitis is developed. MHV-2 was injected intraperitoneally into 4-week-old ICR mice, IFN or the vehicle was administered intramuscularly for 5 days, and the antiviral effect was evaluated based on survival periods, liver histology, serum alanine transaminase (ALT) levels, and MHV-2 virus titers in the liver tissues. The animals in the group treated with a combination of IFN-alpha and IFN-gamma survived for longer periods than the groups treated with IFN-alpha alone and IFN-gamma alone (IFN-alpha 10(3) (IU/mouse)/-gamma 10(3) vs. IFN-alpha 10(3), P < 0.005; IFN-alpha 10(3)/-gamma 10(3) vs. IFN-gamma 10(3), P < 0.001). This is consistent with the lower levels of hepatocellular necrosis and serum ALT and the decreased titers of MHV-2 virus in the liver tissues (48 hr, P < 0.001; 72 hr, P < 0.001). These findings indicate that a combination of IFN-alpha and IFN-gamma exhibits a synergistic antiviral effect on MHV-2 infection. The biology of MHV-2 is quite different from that of human hepatitis viruses; however, these results suggest the beneficial combined therapy of IFN-alpha and IFN-gamma for the treatment of human viral hepatitis.     

Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection

There are several lines of evidence suggesting that specific vaccine therapy with a standard hepatitis B virus (HBV) vaccination reduces HBV replication. The aim of this study was to investigate the anti-viral mechanism of vaccine therapy in chronic hepatitis B patients. Nineteen patients were assigned to receive either vaccine therapy (n = 13) or no treatment as a control (n = 6). Vaccinated patients were analyzed for T cell proliferative responses specific for envelope antigen and cytokine production by antigen-specific T cells. ELISPOT and cytotoxicity assays also were carried out for limited blood samples. Serum HBV DNA levels decreased significantly at 3 months after completion of therapy and thereafter as compared to the baseline ones, and were significantly lower in vaccinated patients than in controls at 12 and 18 months after completion of therapy. Vaccination induced antigen-specific CD4+ T cell proliferative responses in four patients (30.8%). The production of high levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by antigen-specific T cells was found in six patients (46.0%) who showed significantly lower HBV DNA levels in serum at 6 (P = 0.04) and 18 months (P = 0.005) after completion of therapy than those without high levels of cytokine production. Vaccination did not induce antigen-specific CD8+ T cells or cytotoxic T cells. These results suggest that envelope-specific CD4+ T cells may control directly HBV replication by producing anti-viral cytokines rather than providing help for cytotoxic T cells in therapeutic vaccination against chronic HBV infection.     

Reduced virus specific T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B - restoration by exogenous interleukin-12

The balance between Type 1 and Type 2 cytokines is important for the outcome of several infectious diseases. As elderly humans show increased morbidity and mortality from infectious diseases, this study tests if ageing is associated with a change towards Type 2 dominance in T cells. Expression of IFN-gamma, and IL-4 was measured in CD4+ and CD8+ T cells by flow cytometry in three groups: young controls (n=28), 81-year-olds (n=22), and centenarians (n=25). The major findings were that the percentage of IFN-gamma+ as well as IL-4+ T cells was increased in aged subjects. Furthermore, after adjusting for decreased lymphocyte counts in the elderly, the concentration in the blood of IFN-gamma+ and IL-4+ CD8+ T cells was still increased in the 81-year-olds. In centenarians, a shift towards a relative dominance of Type 2 cytokine expression was found within CD8+ T cells. Furthermore, the percentage of T cells with cytokine expression was closely correlated to the in vivo expression of CD95 and CD45RO. In conclusion, we found some evidence for an age-related shift towards a Type 2 cytokine profile.     

Circulating dendritic cells and interferon-alpha production in patients with tuberculosis: correlation with clinical outcome and treatment response

Dendritic cells (DC) have been characterized recently as having an important role in the initiation and control of immunological response to Mycobacterium tuberculosis infection. Blood DC have been subdivided into myeloid (mDC) and plasmacytoid (pDC) subsets, on the basis of differences in phenotype markers and function. Little is known about the enumeration and functional evaluation of circulating DC in patients with tuberculosis and their correlation with clinical outcome during the course of anti-tuberculous treatment. We assessed circulating mDC and pDC counts measured by a newly developed single-platform flow cytometric assay based on TruCOUNT, as well as the production of interferon (IFN)-alpha after in vitro stimulation by herpes simplex virus (HSV-1) in 24 patients with active tuberculosis (TB) and 37 healthy donors. Absolute numbers of both DC subsets were decreased significantly in patients with active TB compared to controls. Similarly, the production of IFN-alpha was highly impaired. In 13 patients these parameters were assessed longitudinally, before and after the specific anti-microbial treatment. Most interestingly, in all nine patients with successful anti-tuberculous therapy there was a significant and marked increase of pDC counts and IFN-alpha production. In contrast, no significant longitudinal variations in DC counts and IFN-alpha production were observed in four patients with lack of response to specific treatment. In conclusion, active TB is associated with a defect in blood DC numbers and IFN-alpha production that is restored after bacterial clearance and clinical improvement, as a result of effective anti-tuberculous treatment.     

Monocyte-derived dendritic cell function in chronic hepatitis C is impaired at physiological numbers of dendritic cells

Monocyte-derived dendritic cells (MoDCs) are a promising cellular adjuvant for effector immune responses against tumours and chronic viral infections, including hepatitis C virus (HCV). If autologous DC therapeutic approaches are to be applied in persistent HCV infections in patients, it is important to have an unambiguous understanding of the functional status of the cell type used, namely MoDCs from patients with chronic hepatitis C (CHC) infection. Because of conflicting published reports of either impaired or normal MoDC function in CHC infection, we re-examined the ability of MoDCs from CHC and normal healthy donors (NHD) to mature to an inflammatory stimulus [tumour necrosis factor (TNF)-alpha] and their subsequent functional capabilities. Expression of maturation-associated phenotypic markers [human leucocyte antigen (HLA)-DR, CD83, CD86, CD40], allostimulatory capacity in mixed lymphocyte reactions (MLRs) and CD40-ligand-induced cytokine and chemokine generation were compared in CHC- versus NHD-MoDCs. TNF-alpha-stimulated CHC-MoDCs up-regulated phenotypic markers, but to significantly lower levels than NHD-MoDCs. At physiological ratios of DCs to T cells, CHC-MoDCs were less allostimulatory than NHD-MoDCs, but not when DC numbers were substantially increased. CHC- and NHD-MoDCs generated equivalent amounts of cytokines [TNF-alpha, interleukin (IL)-1beta, IL-6, IL-12p70, IL-15, IL-10] and chemokines [interferon-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1alpha, regulated upon activation, normal T expressed and secreted (RANTES)] after CD40 ligation. Because the functional defect was not apparent at high MoDC : T cell ratios, autologous MoDC therapy with sufficiently high numbers of DCs could, in theory, overcome any impairment of MoDC function in CHC.     

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-alpha, polyinosine-polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG-I, but TNF-alpha, 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-beta, TNF-alpha 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.     

CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production

It is becoming increasingly clear that the T-cell-mediated immune response is important in many diseases. In this study, we used concanavalin A (Con A) -induced hepatitis to investigate the role of CD49a in the molecular and cellular mechanism of the T-cell-mediated immune response. We found that CD49a^−/− mice had significantly reduced levels of serum alanine aminotransferase and were protected from Con A-induced hepatitis. CD49a deficiency led to decreased production of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) after Con A injection. Furthermore, we found that hepatic CD4⁺ T cells and invariant natural killer T cells up-regulated CD49a expression, along with enhanced activation after Con A injection, leading to production of inflammatory cytokines by these T cells. Blockade of CD49a in vivo ameliorated Con A-induced hepatitis with reduced production of IFN-γ and IL-17A. Hence, CD49a promoted Con A-induced hepatitis through enhancing inflammatory cytokine production (IFN-γ and IL-17A) by CD4⁺ T and invariant natural killer T cells. The protective effect of CD49a blockade antibody suggested a new target therapeutic molecule for intervention of T-cell-mediated liver injury.