Liver Immune Mechanisms Of HBV-induced Systemic Immunotolerance

The liver exhitits a distinctive form of immune tolerance, in which orthotopic liver transplantation are predisposed to induce tolerance and foreign antigenic material or other liver targeted pathogens (such as:HBV, HCV and malarial parasite) could not induce an immune response. Futhermore, liver virus persistence results in systemic immune tolerance. For example, HBV carrier could not produce protective levels of anti-HBs when compare with normal person after HBV vaccination. However, the precise mechanism as to how liver participates in induction of systemic immune tolerance without immune resoponse remains poorly understood.In this study, we established adult mouse model for HBV chronic infection by hydrodynamical injection with6μg of pAAV/HBV1.2palsmid to explore the mechanism about the liver immune tolerance and systemic tolerance. Then we evaluated the state of liver tolerance by determining serum levels of HBsAg or anti-HBs using IRMA and serum HBV DNA copies using real-time PCR. Serum transaminase ALT levels and liver pathologic changes were observed to examine the liver injury. These data together helped to determine whether the mouse HBV model was available and the mice whose serum HBsAg surpassed100ng/ml after4weeks HBV plasmid injection were considered to be HBV carrier mice. We found HBV carrier mice induced peripheral tolerance after1μg of CFA/HBsAg immunization and tolerance could be transferred by adptive splenocytes transfer. By cells depletion, effects of the specific lymphocyte interactions in the process of liver tolerance were investigated. We used ELISA to measure cytokine levels after ex vivo activation including IL-10, TGF-P, IFN-y and IL-4. Flow cytometry analysis provided us with the precise information about lymphocyte phenotype and activation. Our major results are shown as followed:1. Establishment of a mouse model for HBV persistenceHuang and colleagues have established a mouse mode of persistent HBV infection by hydrodynamic injection of the plasmid pAAV/HBV1.2into the tail veins of C57BL/6mice. We found that HBV persistence in mice was determined by the doses of the plasmid in the model. C57BL/6mice were injected hydrodynamically with6μg and 20μg of pAAV/HBV1.2plasmid, respectively. In the6μg group, the mice kept high serum levels of HBsAg, and almost all mice were still HBsAg positive at5weeks post injection. Cytoplasmic and nucleic HBcAg were detected in the livers of HBsAg positive mice at5weeks post injection of pAAV/HBV1.2. ALT and Histological analysis of the liver sections from HBsAg positive mice showed that there was no inflammatory response or injury in the liver. On the contrary, the HBsAg level dropped quickly during3to5weeks after pAAV/HBV1.2injection and all mice were negative for HBsAg at5weeks post injection in the20μg group. So, in the subsequent experiments, we chose the6μg of pAAV/HBV1.2to establish the mouse model for HBV persistence.To easily test which organs expressed the HBsAg after hydrodynamic injection of pAAV/HBV1.2, we digested GFP expression cassettes and inserted into the plasmid. By the achievment of GPF and HBV coexpression, we can observe the infected cell directly by fluorscence or immunohistochemistry. The GFP expression was found only in liver, but not in heart, lung or kindny. Therefore, the hepatocytes were targeted by pAAV/HBV1.2and initiated to express HBV related proteins by hydrodynamic injection.2. Induction of peripheral tolerance toward HBsAg in HBV carrier mice.To investigate the immune response toward HBsAg in HBV carrier mice, we carried out the flow cytometry analyses of the NK, T and B cells isolated from liver and spleen. We found the presentage of these cells and surface maker of NK cells (such as:NKG2D, CD69and NKG2A) were unaffected in HBV carrier mice when compared with control mice, indicating the weak immune response in HBV carrier mice and NK cell did not play a key role in the process.To determine whether the sustained HBV expression in liver could induce peripheral tolerance, control or HBV carrier mice were immunized intrasplenically with1μg of CFA/HBsAg. Protective levels of anti-HBs can be detected in control mice after the vaccination, but not in HBV carrier mice. When the mice were immunized at HBV persistence1day, HBsAg immunization could overcome tolerance and all mice produced the high levels of anti-HBs. These data suggested that it was difficult to reverse the tolerance which was induced by liver HBV persistence within about1week by peripheral immunization of HBsAg. 3. Deliver of HBV-immune tolerance to naive miceTo investigate whether immune tolerance of HBV-carrier mice can be transferred to naive mice in vivo, splenocytes from control or HBV carrier mice were transferred into wild type (WT) mice. The serum levels of anti-HBs were determined after being challenged with HBV vaccine. We found the transferred splenocytes from HBV-carrier mice significantly inhibited the production of anti-HBs against HBsAg in WT mice. The degree of inhibition was positively correlated with the amount of transferred splenocytes from HBV carrier mice to WT mice prior to HBV vaccination. Next, using the same experimental procedure, mice transferred with splenocytes were challenged with OVA in order to determine whether the observed suppression was specific to HBsAg. As expectation, mice transferred with splenocytes from HBV carrier mice produced the same serum levels of anti-OVA IgG as that of WT mice, suggesting that the transferred splenocytes from HBV-carrier mice could specifically inhibit anti-HBV immunity in WT mice.4. HBV carrier mice produce regulatory HBsAg-specific CD4+T cellsAfter the adoptive splenocytes transfer, we found Rag1-/-HBV carrier mice, but not WT HBV carrier mice, transferred with anti-HBs (+) splenocytes, but not control splenocytes, could eliminate HBV totally within two weeks. To further observe the direct suppressed effect of regulatory cells from HBV carrier mice, the recipient Rag1-/-AHBV+mice were transferred intravenously with splenocytes isolated from either HBV carrier mice, anti-HBs (+) mice, or mixture from HBV carrier mice and anti-HBs (+) mice (ratio1:1), and then serum HBsAg levels were measured at2weeks after cells transfer. We found HBsAg levels of recipient transferred mixture of splenocytes were obviously higher than recipient transferred anti-HBs (+) splenocytes only. The interpretation of these findings was that HBV carrier mice generated some regulatory cells which were absent in Ragl"7" HBV carrier mice to inhibit anti-HBV immunity.We further confirmed that Ragl^HBV carrier mice were absence of HBsAg-specific regulatory cells via adoptive splenocytes transfer. Suppression of anti-HBs production was lost by transferring splenocytes from Ragl-/-HBV carrier mice instead of HBV carrier mice. Furthermore, anti-HBs production was found to be unaffected after transferring of CD4+T cells with the purity of92%compared with total splenocytes from HBV carrier mice, indicating the regulatory cells in HBV carrier mice were CD4+T cells.5. The regulatory HBsAg-specific CD4+T cells might be Trl-Iike cellsThe immunotolerance induced by HBV was not due to development of CD4+CD25+Foxp3+Treg cells, as we found no significant increase in Treg cells in HBV carrier mice. To explore the possible role Trl like cells play in the induction of tolerance to the HBsAg, at week2post pAAV/HBV1.2injection, liver MNCs and splenocytes were isolated and activated ex vivo to determine the IL-10, IFN-y, TGF-β and IL-4production by ELISA. We found IL-10and IFN-y production by liver MNCs were strongly elevated in HBV carrier mice when compared with control mice, but production of TGF-β and IL-4kept unchanged in two groups, which strongly suggesting the involvement of Trl like cells in HBV-induced immunotolerance.6. Kupffer cells are critical in induction of systemic toleranceTo explore the role of KCs in induction of tolerance, mice were depleted transiently of KCs by a single intravenous administration of clodronate liposome on day-2prior to HBV plasmids injection. Strikingly, this treatment resulted in total elimination of HBV during3to4weeks. However, HBsAg found to be unaffected at8weeks in Rag1-/-HBV carrier mice after depletion of KCs. The performance of adoptive splenocytes transfer indicated depletion of KCs, but not NK/NKT, resulted in functional deficiency of regulatory cells in HBV positive mice. It suggested strongly that KCs contribute to induction of regulatory HBsAg-specific Trl like cells, which was responsible for suppression of HBsAg-activated immune response and maintenance systemic tolerance.7. Inactivation of Tfh cells to HBV vaccination in peripheral lymph nodes in HBV-carrier miceIt was well known there were many definite steps between antigen immunization and antibody production, including DCs activation, percentage of Tfh cells and GC B cells increase, generation of germinal center, and then secretion of antibody. To further explore the mechanism how Trl like cells maintained the tolerance and inhibited the production of anti-HBs after HBV vaccination in HBV carrier mice, we measured the percentage of GC B cells, Tfh cells, and DC cells using flow cytometry. After HBV vaccination, the frequencies of GC B cells and Tfh cells increased significantly in control mice, but not in HBV carrier mice. However, a dramatic relative increase in DC numbers and expression of costimulatory molecular CD86was evident at4days following HBV vaccination both in control and HBV carrier mice. These data suggested that the exact step between DCs and Tfh cells was inhibited in HBV carrier mice, leading to suppression of anti-HBs production after HBsAg immunization.These data suggested that DCs activation and expansion were not influenced but Tfh cells were inhibited in HBV carrier mice, leading to the dysformation of GC and subsequent reduction in anti-HBs synthesis after HBsAg immunization.Conclusion:The present work described an immunotolerance model whereby liver persistent expression of HBV could cause peripheral immune tolerance toward HBsAg immunization. We found KCs in liver played key roles in inducing the generation of regulatory HBsAg-specific Trl like cells which mediated systemic tolerance via migrating to lymph nod and spleen and inhibiting the Tfh and GC B cells activation after HBsAg-vaccination. Our study firstly elaborated a KCs-Trl like cells-follicular Th cells interplay mediated the systemic immunotolerance Induced by liver HBV persistence.