Liver Induced Systemic Immune Tolerance And Its Reversal

The liver possesses unique feature in inducing immune tolerance. Persistent exposure to liver-derived antigens usually results in systemic unresponsiveness rather than immunity against these antigens, such as liver transplantation tolerance, chronic hepadnavirus infection and oral tolerance. This feature of liver is closely related to its physiological structure and cell composition, for example, the blood supply from portal vein exposes liver to constant massive stimulation by intestinal microbiota and dietary derived antigens; as the biggest blood reservoir of the body, the blood flow rate is extremely low in liver; the innate immune cells are dominant in liver immunity and a lot of tolerogenic antigen presenting cells reside in liver. All of these characteristic compose the essential basis for liver to induce systemic antigen-specific tolerance through induction of inhibitory immune cells, immune deviation, T cell anergy and clonal deletion.Manipulating liver tolerance has huge clinical potential for diseases intervention, for example, orthotopic liver transplantation could significantly ameliorate the immune rejection of following skin or kidney grafts from the same donor; over-expression of auto-antigens in liver efficiently reduce the incidence of autoimmune diseases; liver-targeted gene expression greatly improve the therapeutic effects of gene therapy by avoiding host immune responses against the transgene. Most importantly, during hepatotropic pathogen infection, such as HBV and HCV infection, persistent virus existence usually leads to systemic immune tolerance toward viral antigens, which is the major reason for failure in mounting effective anti-viral immunity and for development of chronic infection. Hence, investigating the mechanism of liver tolerance, especially seeking for the molecules that can maintain or reverse liver tolerance will be greatly meaningful for treatment of many types of human diseases, such as autoimmune and infectious diseases.In this regard, our study is trying to elucidate this topic by using a liver targeted HBV persistent mouse model, and can be divided into two major parts:I. Interleukin-12-based vaccination therapy efficiently reverses HBV persistence induced systemic tolerance.1. HBV-persistent mouse mimics liver-viral-persistence induced systemic immune tolerance state.Hydrodynamical injection of pAAV-HBV1.2plasmid into C57BL/6mice permits sustained HBV replication and expression in adult mice, among which thirty percentage of injected mice develop long-term HBV carrier state without liver injury, and become unresponsiveness toward peripheral HBV vaccination. Thus these HBV-carrier mice provide us a faithful model to mimic the asymptomatic chronic HBV carriers.2. Interleukin-12-based vaccination therapy efficiently clears HBV from HBV-carrier mice.We found that a combination of IL-12pretreatment and co-injection with HBsAg vaccine, termed as IL-12-based vaccination therapy, can efficiently clear HBV from HBV-carrier mice, leading to significantly reduction or elimination of HBsAg, HBeAg, HBV DNA and HBcAg. This therapeutic approach dose not induce liver injury during treatment, thus could be a safe, efficient and viable strategy for CHB treatment.3. Interleukin-12-based vaccination therapy efficiently reverses HBV induced systemic tolerance.We observed that IL-12-based vaccination therapy can elicit HBV specific recall responses, induce dramatically amplification of follicular helper T cells and germinal center B cells, and promote anti-HBs production and HBsAg-specific T cell proliferation, which together indicate that HBV induced systemic tolerance was reversed by our approach.4. Interleukin-12-based vaccination therapy synergistically reverses HBV induced systemic tolerance.Mechanistically, we found that the reversal of tolerance by IL-12-based vaccination therapy was relied on stimulation of adaptive immunity, especially T cell system. IL-12and HBsAg vaccine have synergistic effects in promoting HBV-priming CD4+T and CD8+T cells amplification, activation and function. Moreover, IL-12also inhibited HBV induced generation of regulatory T cells.Conclusion Ⅰ:We identified a viable therapeutic approach against CHB, termed as interleukin-12-based vaccination therapy, which can efficiently reverse HBV induced systemic tolerance by reinvigorating multiple-specific anti-HBV T cell responses and inhibiting HBV-primed regulatory T cells generation, thus efficiently clears HBV from HBV-carrier mice.II. Interferon-y facilitates hepatic T cell retention for maintaining liver induced systemic tolerance1. Deficiency in IFN-y abolishes HBV-persistence induced systemic tolerance.HBV-carrier WT mice developed systemic HBsAg-specific tolerance, however, although IFN-y deficient led to strengthened HBV persistent state, it also abrogated liver induced systemic tolerance against HBV, leading to robust responses toward peripheral HBsAg vaccination, which were characterized by the reduction of HBsAg and HBeAg, appearance of anti-HBs, amplification of germinal center B cells and recovery of HBsAg-specific proliferation.2. CD4+T cells derived IFN-y contributes to maintenance of HBV induced systemic tolerance.We observed significantly enhanced IFN-y production by hepatic CD4+T cells from HBV-persistent mice upon anti-CD3plus anti-CD28re-stimulation when compared to that from control mice. Moreover, by using transfer and immune-reconstitution experiments, we found that CD4-/-mice reconstituted with IFN-γ deficient CD4+T cells lost their ability to induce systemic HBV tolerance, which was as similar as found in IFN-y deficient mice.3. IFN-y maintains HBV induced systemic tolerance through hepatic retention of HBV-primed CD4+T cells.We found dramatically decreased expression of a T cell chemokine, CXCL9in livers of HBV persistent IFN-y receptor deficient (GRKO) mice than WT mice. Consistently, HBV persistent GRKO mice showed impaired hepatic attraction of HBV-primed CD4+T cell than WT mice. Ectopic expression of CXCL9in livers of GRKO mice restored the HBV induced hepatic T cell retention and systemic tolerance. Moreover, blockade of CXCR3, the receptor of CXCL9, in WT mice partially reversed HBV induced systemic tolerance.4. Liver resident macrophages participates in liver induced systemic tolerance through retention of T cells. We found that among liver cells, the liver resident macrophages (Kupffer cells) were the main cell source of hepatic CXCL9during HBV persistence. Depletion of Kupffer cells by liposome significantly decreased hepatic CXCL9expression, impeded hepatic attraction of HBV-primed CD4+T cells and broke HBV induced systemic tolerance. Furthermore, ectopic expression of CXCL9in livers of Kupffer cells depleted HBV mice could partially restore the tolerance.Conclusion Ⅱ:Interferon-y is critical for maintaining liver induced systemic tolerance. Mechanistically, HBV persistence induces sustained hepatic CD4+T cell-derived interferon-y production, which then promotes CXCL9secretion from liver-resident macrophages, facilitating retention of anti-viral CXCR3+CD4+T cells in the liver to undergo subsequent tolerization.