| Objective:Interleukin(IL)-35 is an inhibitory cytokine consisting of p35 and Epstein-Barr virus-induced gene 3(EBI3)and is required by regulatory T-cells(Tregs)for maximal activity.IL-35 can convert human and mouse conventional T-cells(T convs)into a new population of T-cells.These novel inducible Tregs are called iTR35 cells.iTR35 can inhibit the proliferation of T-cells,effectively prevent the occurrence and development of inflammation,which is the key factor to induce infection tolerance.The content of IL-35 in peripheral blood of patients with chronic hepatitis B was significantly increased.It is suggested that IL-35 may play an important role in the pathogenesis of HBV persistent infection,but the regulation of IL-35 against HBV is not clear.In this paper,we discuss the mechanism of IL-35 involved in HBV related cellular immune response and the signaling pathway,and provide new ideas for the new antiviral therapy.Material and Methods:1.MaterialThe whole protocol was performed with 61 treatment-naive chronic active hepatitis B patients,who were recruited from the First Affiliated Hospital of Medical College,Zhejiang University.Age-and sex-matched healthy volunteers(n=42)and resolved HBV individuals(n=21)served as controls.2.Methods(1)Peripheral blood mononuclear cells(PBMC)were isolated by Ficoll lymphocyte separation medium.(2)CD4+ T lymphocytes were isolated by immunomagnetic beads(3)EBI3 and p35 protein levels were detected by flow cytometry.Similarly,IL-35 concentration was detected by enzyme linked immunosorbent assay(ELISA).(4)The proportions of CD25+?CD45RA+ and CD45RO+ cells among CD4+ T-cells were determined by flow cytometry.(5)We used ELISA to detect IL-10 concentrations in the culture supernatant of CD4+T-cells.(6)PBMCs were stimulated with HBV core antigen peptide 18-27 for 11 days at 37 ? in the presence of 100 ng/ml IL-35 or PBS(control).(7)CD4-T-cells from chronically infected patients were stimulated with HBV core antigen for 48 h with or without IL-35.Then,IFN-y producing cells were dectected by ELISPOT.(8)We used ELISA to detect IL-6 concentrations in the culture supernatant of adherent cells(ACs).(9)DC surface markers,including CD11c,CD80,CD86 and HLA-DR were detected by flow cytometry.(10)Immunofluorescence staining was used for localization of STAT1?STAT4 signal pathway after IL-35 stimulation of CD4+ cells.(11)To determine whether the STAT1?STAT4 signal pathway was activated or not after IL-35 stimulation by flow cytometry(12)Serum ALT and AST were detected by automated biochemical techniques.Serum TB was determined by chromatomery.The serum HBeAg was performed by Chemiluminescent Microparticle Immunoassay(CMIA)kit and the serum load of HBV DNA was determined by ABI 7300 fluorescent quantitative PCR,with a detection limit of 300 viral genomes copies/mL.(13)Statistic alanalysis:SPSS 17.0 statistic software and GraphPad Prism 6.0 software.Results(1)After stimulation of CD3/28 in vitro,EBI3 protein level in CD4+T-cells and Tregs in the peripheral blood of CHB patients was significantly increased in HBV carriers and normal people(both P<0.001).Similarly,IL-35 concentration was detected by ELISA,finding a significant increase in CD4+T-cells from CHB patients,compared with healthy controls and resolved individuals(P=0.041,P<0.004).(2)The expression of EBI3 in CD4+T cells was positively correlated with HBV DNA(P=0.036,r=0.525),but not with ALT,AST or TB(P=0.275,r=0.291;P=0.378,r=0.236;P=0.336,r=-0.257).(3)CD4+ T cells were cultured with an antigen-specific(HBV core antigen)with or without different concentrations of IL-35.CD4+ T-cell surface markers CD45RA was analysed.The proportions of CD45RA+ cells among CD4+ T-cells were 36.09±5.93%(Ag only),32.75±6.24%(Ag+10ng/ml IL-35),33.50±5.41%(Ag+40ng/ml IL35),32.23±5.43%(Ag+100ng/ml IL35),(P<0.05).(4)We used ELISA to detect IL-10 levels in the culture supernatant of CD4+T-cells incubated with or without IL-35 in vitro.The IL-10 concentrations in the four groups stimulated by CD3/28 coated beads were as follows:128.3±36.5 ng/1(CD3/28 only),143.1 ±36.08 ng/1(CD3/28+10 ng/ml IL-35),155.5±36.14 ng/1(CD3/28+40 ng/ml IL-35)and 160.8 ±32.75 ng/1(CD3/28+100 ng/ml IL-35),(all P<0.05).(5)The proportion of HBV specific CTLs among CD8+ T-cells was significantly lower in the IL-35 treated group than that in the IL-35-untreated group(1.631 ±0.436 vs 1.019±0.269,P = 0.003).(6)IFN-y producing cells were detected by ELISPOT.The cell counts were as follows:237±28SFU/well[Antigen(Ag)only],216±35SFU/well(Ag+10 ng/ml IL-35),185 ±36 SFU/well(Ag+40 ng/ml IL-35)and 170±36 SFU/well(Ag+100 ng/ml IL-35).The number of IFN-y producing cells was slightly decreased by 10 ng/ml IL-35(P = 0.37).At higher concentrations however,IL-35 dramatically reduced the number of IFN-y producing cells(40 ng/ml IL-35:P = 0.01;100 ng/ml IL-35:P = 0.007).(7)The IL-6 concentrations from ACs were 375.9±101.9 pg/ml(Ag alone)and 313.0 ±99.23 pg/ml(Ag +100 ng/ml IL-35),(P=0.002).(8)The proportion of CD11c+ cells among PBMCs stimulated with HBV core antigen in vitro was significantly decreased by IL-35(100 ng/ml)treatment(IL-35 untreated group:10.10±3.65%;IL-35 treatment group:8.96±3.40%;P?0.02).(9)Immunofluorescence results showed that IL-35 promoted the phosphorylation of STAT1 and STAT4.(10)Flow cytometry showed that IL-35 was significantly increased the antigen-specific expression of STAT1 and STAT4 on CD4+ T-cells(P<0.019,P=0.029).Conclusion(1)IL-35 was highly expressed in peripheral blood of CHB patients and was closely related to the occurrence and development of chronic hepatitis B.(2)IL-35 can not only inhibit the expression of CD4+CD45RA+ T cells and DCs but also can inhibit antigen-specific IFN-? secretion by CTLs in vitro.This is the key factor affecting the occurrence and development of chronic hepatitis B.(3)IL-35 activated the STAT1/STAT4 signaling pathway on CD4+ T cells,indicating that IL-35 is likely to inhibit the proliferation and differentiation of HBV Teff through JAK/STAT pathway. |
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