Involvment Of Kupffer Cells And Interleukin-15in HBV-induced Immuntolerance

The liver is the largest internal organ in body and plays an important role in maintaining normal metabolism and detoxifying the body. Also, the liver has been recognized as a unique immune organ which favours the induction of immune tolerance rather than immunity, such as oral tolerance, liver transplant tolerance. Some liver pathogens (HBV, HCV, and malaria) can establish persistent/chronic infections in the liver that lead to inflammatory liver disease.HBV infects the liver as its primary target. The immune system of HBV infected patient has become tolerant to HBV and shows no response to periphery HBV vaccine. Up to now, most in vivo studies on the mechanisms of HBV tolerance have been performed by using HBV transgenic mice; however, HBV transgenic mice are inherently tolerant to HBV virus and are not suitable for studying the mechanisms of HBV tolerance in HBV infected patient who is infected after birth and have limitations in addressing the factors at the onset of infection that influence the final outcomes of chronic HBV infection. Hydrodynamic injection of pAAV/HBV1.2leads to a persistent HBV gene expression in mice, which mimics human chronic HBV infections in the immune tolerant stage. So in the current study, we used this model to study the mechanism of HBV induced immune tolerance and examine the effects of IL-15on immune response to HBV.In this study, a mouse model for HBV tolerance was established by hydrodynamical injection of pAAV/HBV1.2plasmid into C57BL/6mice. The serum levels of HBsAg、HBeAg and Anti-HBs were measured by radioimmunoassay. Intrahepatic HBcAg expression was determined by immunohistochemistry. Serum HBV DNA was measured by quantitative PCR. In experiments of cell depletion, cell purification and cell transfer, we examined the exact role of specific cell population in HBV immune tolerance. With the aim to investigate the immunomodulatory effects of IL-15in vivo, we constructed a gene transfer vector to endogenously overexpress IL-15in mice. The status of lymphocyte subsets was analyzed with flow cytometer. Serum levels of cytokines were measured by ELISA. Our major findings are shown as follows: Involvement of Kupffer cells in HBV-induced immuntoleranceHydrodynamic delivery of the plasmid pAAV/HBV1.2into mouse liver allowed persistent HBV viremia and antigenemia in C57BL/6mice. Secretion of HBsAg and HBeAg into the serum remained high6weeks post pAAV/HBV1.2administration. Both cytoplasmic and nucleic HBcAg were detected in the livers of HBV-carrier mice at6weeks after injection. There were no detectable anti-HBs antibodies in the sera of these animals. The animal model mimics human chronic HBV infections in the immune tolerant stage. If mice were immunized with HBsAg vaccine ahead of tail vein hydrodynamic injection, all HBsAg-vaccinated mice could eliminated HBV virus by decreasing HBsAg and HBeAg in their sera and HBcAg in liver tissues. However, if HBV-carrier mice were immunized with HBsAg vaccine, serum anti-HBs can not be detected in HBV carrier mice and HBsAg vaccine can not cure HBV persistence in HBV carrier mice. Thus, HBV persistence in liver could induce peripheral immune tolerance. Meanwhile, the HBV-carrier mice preserved a strong immune response to other antigen such as OVA, so that peripheral tolerance toward HBsAg was antigen-specifically induced in HBV-carrier mice.Splenocytes from naive or HBV carrier mice were transferred to ragl-/-mice. After being challenged with HBV vaccine, ragl-/-mice with the immune system of naive mice developed protective levels of anti-HBs, whereas anti-HBs levels were nearly undetectable in ragl-/-mice with the immune system of HBV carrier mice. If we replaced CD4+T cells of splenocytes from HBV carrier mice with CD4+T cells from naive mice and transferred the reconstructive splenocytes to ragl-/-mice and vaccined with HBsAg, we found the reconstructive immune system could response to HBsAg, indicating that the immune tolerant status of HBV carrier mice was associated with the dysfunction of CD4+T cells.The inherent tolerogenicity of the liver are mediated by tolerogenic antigen-presenting cells. Although phenotype of kupffer cells has no obvious change after pAAV/HBV1.2injection, the mice received NPCs or only KCs from HBV carrier mice secreted obvious low anti-HBs levels after HBsAg immunization, suggesting that kupffer cells are critical in induction of immune tolerance.Prevention of hepatitis B virus-induced immunotolerance by liver over-expression of interleukin-15via promoting IFN-beta productionWith the aim to investigate the immunomodulatory effects of IL-15in vivo, we constructed a gene transfer vector to endogenously overexpress IL-15in mice. We found that hydrodynamic injection of the plasmid pLIVE-IL-15resulted in a sustained concentration of IL-15in serum. Liver IL-15over-expression resulted in great suppression of HBsAg and HBeAg expression early after pAAV/HBV1.2injection and even eliminated HBV virus at five weeks post injection. So, IL-15liver gene transfer induced enhanced immune responses to HBV.IL-15is a pleiotropic cytokine known to modulate both innate and adaptive immune cells. Over-expression of IL-15similarly decreased HBsAg and HBeAg levels in Ragl-/-mice, suggesting that IL-15exerted anti-HBV activity in a T-cell and B-cell independent manner. Interestingly, despite an increase in NK cell numbers in both spleen and liver of IL-15-expression mice, the anti-HBV effect of IL-15was neither dependent on presence of NK cells in mAb depletion experiment and by using NK-deficient mice (IL-2Rγc-/-).Type Ⅰ IFNs (IFN-α/β) and IFN-y are known to inhibit HBV gene expression noncytopathically. IL-15inhibits HBV gene expression in IFN-γ-/-mice, suggesting that IFN-y did not play a critical role in IL-15-mediated anti-HBV activity. IL-15treatment increased intrahepatic IFN-β mRNA expression as well as circulating serum IFN-β levels, whereas IFN-a mRNA and protein levels were unchanged. Blockade of IFN-β function led to a significant increase in expression of HBsAg and HBeAg in IL-15treated mice, indicating the anti-HBV activity of IL-15was probably mediated by IFN-β. Depletion of macrophages with clodronate lyposomes did not block the ability of IL-15to inhibit HBV expression in our study, suggesting that IFN-β was not produced by macrophages.Conclusion:The characteristics of the mouse model for HBV-induced immuntolerance are analogous to those of human chronic HBV infections. The status of HBV induced immuntolerance is associated with dysfunction of CD4+T cells. Kupffer cells play an important role in inducing periphery immune tolerance. Liver over-expression of IL-15prevents HBV-induced immune tolerance in an IFN-β-dependent manner. So, the study on the mechanism of HBV-induced immuntolerance and a novel function of IL-15in anti-HBV immunity might provide an implication in clinical practice.