Jiu-Ping Wang

Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4(+)CD25(+) regulatory T cells in chronic hepatitis B virus infection.

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4(+)CD25(+)CD127(low/-) Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14(+) monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4(+)CD25(+) Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection.

Circulating Toll-like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C.

Wang J‐P, Zhang Y, Wei X, Li J, Nan X‐P, Yu H‐T, Li Y, Wang P‐Z, Bai X‐F. Circulating Toll‐like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C. APMIS 2010; 118: 261–70.

Circulating CD4+CD25high regulatory T cells and expression of PD-1 and BTLA on CD4+ T cells in patients with chronic hepatitis B virus infection.

The roles of regulatory T cells (Tregs) and PD-1 in hepatitis B have not been clearly described. Also, the role of B and T lymphocyte attenuator (BTLA), which serves as a negative regulator of T-cell activation, is still unknown in hepatitis B. In this study, we analyzed the frequency of circulating CD4(+)CD25(high) Tregs in patients with chronic hepatitis B (CHB), and subsequently investigated expression of PD-1 and BTLA on CD4(+) T cells, as well as their relationships with the clinical index of CHB patients. A total of 39 CHB patients and 19 healthy persons as controls were enrolled in the study. We found that the frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T cells was significantly increased in CHB patients compared with normal controls. However, BTLA expression on CD4(+) T cells showed no significant difference between the two groups. The frequency of Tregs was significantly higher in patients with HBV DNA titers >or=10(8) than in those with HBV DNA titers <10(8). Circulating CD4(+)CD25(high) Treg frequency and PD-1 expression on CD4(+) T cells correlated positively with serum HBV DNA load in CHB patients. Our findings suggest that the increased frequency of CD4(+)CD25(high) Tregs and PD-1 expression on CD4(+) T lymphocytes may inhibit the cellular immune response against HBV and affect viral clearance, leading to the persistence of chronic HBV infection.

Contributions of PD-1/PD-L1 pathway to interactions of myeloid DCs with T cells in atherosclerosis

Although inflammatory cells contribute to immunopathogenesis of atherosclerosis, underlying molecular mechanisms remain largely undefined. Recently, it has been demonstrated in mouse model that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway plays a critical role in proatherogenic immune responses. Here we examined the expression of PD-1 and PD-L1 on peripheral blood mononuclear cells by flow cytometry in 76 patients with coronary artery disease (CAD), and 25 healthy volunteers. The expression of PD-1 and PD-L1 is significantly down-regulated on T cells and myeloid dendritic cells (mDCs) in CAD patients than in healthy individuals, respectively. More importantly, we found that decreased PD-L1 expression on mDCs is related with the increased T cell immune responses in CAD patients. In addition, stimulation of PD-L1 expression in vitro could attenuate the stimulatory ability on allogeneic T cell proliferation and its cytokine production, including IFN-gamma and IL-2, and also influence the production of IL-10 and IL-12 by mDCs. Taken together, we can draw a conclusion that PD-1/PD-L1 pathway plays a key role in the regulation of proatherogenic T cell immunity by intervening antigen presenting cell (APC)-dependent T cell activation, which associates with pro-inflammatory or anti-inflammatory cytokine production, and further studies need gain insight into that this pathway represents a strategy of immunotherapy for atherosclerosis.

Inhibition of Viral Replication Downregulates CD4+CD25high Regulatory T Cells and Programmed Death-Ligand 1 in Chronic Hepatitis B

Chronic hepatitis B is characterized by an impaired immune response to hepatitis B virus (HBV). Telbivudine treatment has significantly improved the clinical outcome of chronic HBV infection. However, the underlying mechanism behind the antiviral response of patients treated with nucleoside analogs remains unclear. To gather more evidence about the mechanism responsible for the weak immune response, in this study we analyzed the effects on HBV viral load of treatment with the nucleoside analogue telbivudine and the percentage of Tregs, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression, and related cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum of 28 patients with chronic hepatitis B were collected at baseline, and 3u2009mo and 6u2009mo after therapy was begun. In parallel with the decline in viral load and serum ALT normalization, we found a decline in circulating CD4(+)CD25(high) Tregs, PD-L1 on CD4(+) T cells, and IL-9 production. The expression of PD-1 on CD4(+) T cells and the production of IFN-γ did not increase during therapy. Our findings suggest that the antiviral effect of the nucleoside analogs may be attributable not only to their direct effect on virus suppression, but also to their immunoregulatory capabilities.

Analysis of the immune response to Hantaan virus nucleocapsid protein C-terminal-specific CD8+ T cells in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

Inhibition of Hepatitis C Virus Infection by Interferon-γ Through Downregulating Claudin-1

Hepatitis C virus (HCV) is a serious global health threat and current medical treatment options are limited. Interferon (IFN)-gamma is an important proinflammatory cytokine with antiviral activity. However, the mechanism of IFN-gamma in anti-HCV infection remains unclear. In this study, we investigated the role of IFN-gamma on HCV infection of polarized Caco-2 cells using cell culture-derived HCV (HCVcc). We found that downregulation of claudin-1 (CLDN1) induced by IFN-gamma resulted in disruption of barrier function as demonstrated by measurement of transepithelial electrical resistance and dextran permeability. Further, results from confocal microscopy and Western blot analysis showed that in addition to the reduction of CLDN1 expression, IFN-gamma treatment also led to significant changes in the distribution of CLDN1, CD81, and scavenger receptor class B type I. Moreover, infection assays revealed that IFN-gamma-treated cells showed decreased susceptibility to HCVcc infection. These results suggest a novel mechanism that IFN-gamma may inhibit HCV infection by regulating CLDN1 expression and distribution of HCV receptors.

Dysregulation of the β3 integrin-VEGFR2 complex in Hantaan virus–directed hyperpermeability upon treatment with VEGF

Hantaviruses infect human endothelial cells (ECs) and are known to cause vascular-permeability-based diseases, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The αvβ3 integrins, which are highly expressed on the surface of ECs, serve as hantavirus receptors. Specifically, the β3 integrin and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) form a functional complex and interact with each other. Signaling through this complex causes cytoskeletal reorganization, which is one of the most important mechanisms underlying hyperpermeability. In this study, we show that VEGF dramatically enhances Hantaan virus (HTNV)-directed permeability and increases the reorganization of the cytoskeleton and the disruption of junctional organizations in an EC monolayer at 3 days postinfection. HTNV infection reduced the effect of VEGF on adhesion, migration, and the upregulation of β3 expression, but the infection alone upregulated the expression of β3 and VEGFR2. These results indicate that in addition to its role in blocking β3 integrin activation as reported previously, HTNV blocks the function of the complex of VEGFR2 and β3 integrin, and the dysfunction of the complex may contribute to cytoskeletal reorganization in an HTNV-directed hyperpermeability response to VEGF.

Down-regulation of CXCR4 expression by SDF-KDEL in CD34+ hematopoietic stem cells: an anti-human immunodeficiency virus strategy.

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34(+) hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34(+) hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10(3) TCID50 HIV-1 infected CD34(+) hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P<0.05).

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.