Hai-Tao Yu

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.

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 3 mo and 6 mo 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.

Elevated vascular endothelial growth factor levels induce hyperpermeability of endothelial cells in hantavirus infection.

Objective: To investigate the role of vascular endothelial growth factor (VEGF) in haemorrhagic fever with renal syndrome (HFRS). Methods: VEGF, soluble VEGF receptor (sVEGFR)-2, angiopoietin (Ang)-1, tumour necrosis factor (TNF)-α and interferon (IFN)-γ levels were measured in serum samples from 68 patients with HFRS. Cultured human umbilical vein endothelial cells (HUEVCs) were infected by Hantaan virus (HTNV) and/or stimulated with recombinant VEGF; dextran permeability of the cells was determined. Claudin-1 and vascular endothelial (VE)-cadherin levels were determined by real-time reverse transcription-polymerase chain reaction and Western blot analyses. Results: Serum VEGF, TNF-α and IFN-γ levels were significantly elevated, whereas sVEGFR2 and Ang-1 levels were reduced, during the acute phase of HFRS. In vitro cell permeability was unaffected by HTNV infection or VEGF stimulation alone, but the combination of HTNV infection and VEGF treatment significantly increased the permeability of endothelial cell monolayers in a time-dependent manner. Claudin-1 and VE-cadherin were downregulated at both the mRNA and protein level by combined HTNV infection and VEGF stimulation. Conclusions: Elevated VEGF induced by HTNV infection may play an important role in the vascular hyperpermeability that is characteristic of HFRS.

Hantaan Virus Triggers TLR4-Dependent Innate Immune Responses

The innate immune response induced by Hantavirus is responsible for endothelial cell dysfunction and viral pathogenicity. Recent studies demonstrate that TLR4 expression is upregulated and mediates the secretion of several cytokines in Hantaan virus (HTNV)-infected endothelial cells. To examine viral interactions with host endothelial cells and characterize the innate antiviral responses associated with Toll-like receptors, we selected TLR4 as the target molecule to investigate anti-hantavirus immunity. TLR4 mRNA-silenced EVC-304 (EVC-304 TLR4-) cells and EVC-304 cells were used to investigate signaling molecules downstream of TLR4. The expression of the adaptor protein TRIF was higher in HTNV-infected EVC-304 cells than in EVC-304 TLR4- cells. However, there was no apparent difference in the expression of MyD88 in either cell line. The transcription factors for NF-κB and IRF-3 were translocated from the cytoplasm into the nucleus in HTNV-infected EVC-304 cells, but not in HTNV-infected EVC-304 TLR4- cells. Our results demonstrate that TLR4 may play an important role in the antiviral immunity of the host against HTNV infection through an MyD88-independent signaling pathway.

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.

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.

Involvement of the Akt/NF-κB pathways in the HTNV-mediated increase of IL-6, CCL5, ICAM-1, and VCAM-1 in HUVECs.

Background Hantaan virus (HTNV) infection causes a severe form of HFRS(hemorrhagic fever with renal syndrome)in Asia. Although HTNV has been isolated for nearly forty years, the pathogenesis of HFRS is still unknown, and little is known regarding the signaling pathway that is activated by the virus. Methodology/Principal Findings Cardamonin was selected as a NF-κB inhibitor, and indirect immunofluorescence assays were used to detect the effect of cardamonin on HTNV-infected HUVECs. The effect of cardamonin on the HTNV-induced phosphorylation of Akt and DNA-binding activity of NF-κB were determined using Western blot analysis and electrophoretic mobility shift assays (EMSAs), respectively. Then, flow cytometric and quantitative real-time PCR analyses were performed to quantify the expression levels of the adhesion molecules ICAM-1 and VCAM-1, and the concentrations of IL-6, IL-8, and CCL5 in HUVEC supernatants were examined using ELISA. The results showed that cardamonin did not effect the proliferation of HUVECs or the replication of HTNV in HUVECs. Instead, cardamonin inhibited the phosphorylation of Akt and nuclear transduction of NF-κB and further reduced the expression of the adhesion molecules ICAM-1 and VCAM-1 in HTNV-infected HUVECs. Cardamonin also inhibited the secretion of IL-6 and CCL5, but not IL-8. Conclusion/Significance HTNV replication may not be dependent upon the ability of the virus to activate NF-κB in HUVECs. The Akt/NF-κB pathways may be involved in the pathogenesis of HFRS; therefore, cardamonin may serve as a potential beneficial agent for HFRS therapy.

Elevated Serum Concentrations of Inflammatory Cytokines and Chemokines in Patients with Haemorrhagic Fever with Renal Syndrome

OBJECTIVE: Prospective case—control study, undertaken to investigate serum cytokine and chemokine concentrations during all clinical phases and in different clinical types of haemorrhagic fever with renal syndrome (HFRS). METHODS: Serum was collected at various disease phases from patients with HFRS (n = 35) and healthy control subjects (n = 10). Tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-4, interferon (IFN)-γ, IL-8, interferon inducible protein-10 (IP-10) and chemokine (C—C motif) ligand 5 (also known as ‘regulated upon activation, normal T-cell expressed and secreted’ [RANTES]) were quantified using commercial enzyme-linked immunosorbent assay kits. RESULTS: Serum concentrations of TNF-α, IL-6, IFN-γ, IL-8, IP-10 and RANTES (but not IL-4) were significantly higher in patients compared with controls. Highest concentrations were generally found during the febrile, hypotensive and oliguric disease phases, as well as in clinically severe and critical cases. CONCLUSION: Serum concentrations of proinflammatory cytokines and chemokines increased in line with disease severity in HFRS patients.