| Research background and objectiveHepatitis B induced by HBV is one of the most grievous and prevalent health problems, affecting 93 million people in China and 350 to 400 million people worldwide. HBV infection-associated diseases annually result in approximately 1 million deaths worldwide from hepatic decompensation, liver failure, and HCC. HBV is a noncytopathic DNA virus that can cause acute and chronic hepatitis. Recent research suggested that HBV-specific CD8+T cells have been shown to inhibit HBV replication by cytopathic and noncytolytic mechanism and noncytolytic downregulation of viral replication seems to play a critical role in HBV infection. Recently a study provided direct evidence that virus-specific CD8+ T cells efficiently control HBV replication by noncytolytic mechanisms mediated by IFN-γ and TNF-α. CD8+T cells not only kill infected cells, but also induce a noncytopathic effect that blocks conversion of pg RNA to DNA-containing capsids and removes ccc DNA. Moreover the noncytolytic regulation of HBV replication and inhibition-of ccc DNA by antiviral cytokines would be a major forward step in finding antiviral therapies for chronic hepatitis B. In general, T cell-derived antiviral cytokines are crucial factors in noncytolytic inhibition of HBV replication.The immunostimulatory cytokine IL-21 is an IL-2 family cytokine produced by multiple CD4+ T cell subsets, prominently T helper 17(TH17) cells, follicular helper T cells, and natural killer T cells. IL-21 is pivotal to drive the expansion and promote the survival of both CD4+ and CD8+ T cells, and IL-21 also can selectively enhance the production of IFN-γ and TNF-α to combat with chronic viral infection. The observation suggested an age-dependent outcome of HBV infection in humans, in which decreased IL-21 production in younger patients may hinder the generation of crucial CD8+ T and B cell responses. The relevant studies have found that IL-21 may play an important role in controlling HBV replication. Up till the present moment, IL-21-mediated HBV clearance from infected hepatocytes has not been fully elucidated in vitro because of the lack of a relevant cell culture system. In the present study, we adopted a co-culture system of peripheral blood mononuclear cells with Huh7.93 cells to investigate the mechanism on HBV replication of IL-21.MethodPart one: The establishment of PBMCs and Huh7.93 cell co-culture system.Normal human volunteers PBMCs with Huh7.93 cells as well as chronic HBV infected patients PBMCs with Huh7.93 cells were co-cultured for 7d. The changes of cytokines of IFN-γ, TNF-α, IL-21, and IL-10 level in cell culture supernatant and HBV replication were detected, the relationship between cytokines and HBV replication were analyzed.Part two: Interleukin-21 inhibits the replication of HBV in vitro.We studyed at different concentrations of IL-21, IL-10 stimulates Huh7.93 cells, and to investigate the relationalship between HBV replication and concentrations of IL-21, IL-10. We observed the change of HBV replication and cytokines level in co-culture system.Part three: The relationship of interleukin-21 with chronic hepatitis B patients.This part content was to detection the cytokine levels of IL-21, IL-10, IFN-gamma, and TNF- ɑin the peripheral blood of patients with chronic hepatitis B. We also study the HBV DNA, HBe Ag and HBs Ab level in peripheral blood of patients. Explore the cytokines level of IFN-gamma, TNF-ɑ, IL-21, and IL- 10 in chronic HBV infection.ResultsPart one: The establishment of PBMCs and Huh7.93 cell co-culture system.The levels of HBV DNA were obviously increased in the co-cultured supernatant of Huh7.93 with n PBMCs or c PBMCs than Huh7.93 monoculture. As shown by Southern blotting, all produced the expected pattern of relaxed circular(RC) DNA, doublestranded linear(ds L) DNA and single-stranded(ss) DNA of HBV in co-cultured cell lysates were significantly higher than Huh7.93 monocultures. The results implicated that Huh7.93 cells co-cultured with PBMCs could lead to significant enhancement of HBV expression.Part two: Interleukin-21 inhibits the replication of HBV in vitro.HBV DNA concentration in supernatant from co-cultured cells stimulated by 200ng/ml recombinant human IL-21 were significantly lower than supernatant without IL-21. Southern blot analysis of HBV replication intermediates further disclosed that 200ng/ml IL-21 could significantly inhibit HBV replication in the cell lysate of Huh7.93 cells co-cultured with c PBMCs. The concentration of IL-10 in co-cultured supernatant was down-regulated when compared with control supernatant without IL-21 stimulation.Part three: The relationship of interleukin-21 with chronic hepatitis B patients.Our study shows that IL-10, ALT, and AST levels in severe chronic hepatitis B patients serum compared with mild-to-moderate chronic hepatitis B patients increased obviously. Correlation analysis showed that the level of HBV DNA was negatively related to the level of IFN-gamma and IL-21. HBe Ag quantitative level and also negatively related to the level of IL-21.Conclusion(1) In the present study, we adopted a co-culture system of peripheral blood mononuclear cells with Huh7.93 cells to simulate the state of the body’s immune function, and to investigate the immunologic mechanism of HBV replication. We found that HBV replication was increased in Huh7.93 cells co-cultured with PBMCs. For the first time successfully made a simple and reliable cell co-culture system, and provides a new method of immune clearance mechanism for the study of HBV infection. Our study has a important practical value.(2) Here we showed that IL-21 results in directly inhibit HBV replication in Huh7.93 cell monoculture in vitro culture conditions, and also lead to a marked inhibition of HBV replication in co-culture cells. Our data also indicated that IL-21 could promote the excretion of IFN-γ and reduced the production of IL-10. Collectively, our data implicated that hyposecretion of IFN-γ and IL-21, and the increase production of IL-10 were highly associated with HBV persistent infection, and IL-10 could potentially promote HBV replication. These observations indicated that cytokine network system has important regulation effects on HBV replication.(3) To confirm the effect of IL-21 on HBV replication in chronic hepatitis B patients, we observed the level of IL-21 in serum of patients with chronic hepatitis B and the relationship between HBV replication and IL-21. Our research indicated that HBV DNA level and HBe Ag level in serum of chronic hepatitis B were negative correlation to IL-21. Reveals inadequate secretion of IL- 21 in patients with chronic hepatitis b is an important factor of HBV persistent infection. |
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