The Regulatory Mechanism Of Liver Antiviral Immune Response

Liver is a unique immune organ with many resident innate and adaptive immune cells, which induce various complex and sophisticated immune responses. Many kinds of virus target the liver, while different and even opposite outcomes generated in the interplay between liver immune system and hepadnavirus, such as immune clearance and immune tolerance. It has important significance to reseach the regulatory mechanism of liver antiviral immune response, which can expand the understanding of liver immunology and provide the basis for the clinical prevention and treatment of virus associated hepatic disease.The difference in the liver antiviral immune responses is resulted from various factors including microbiota, virus characterization, peripheral immunity, etc.The metabolic products of microbiota coming from the portal vein constitutively stimulate the liver immune system, thus the liver immune tolerance is induced to protect from overactive immune response induced tissue damage. The classical view believed that microbiota maintains the unique immune microenvironment thus induce almost all of the liver resident immune cells acquire the immune regulatory function. However increased evidences indicated that microbiota also played an important role in promoting various immune responses, such as inducing lymphoid tissue formation, maintaining the mucosal immune homeostasis, inducing lymphocyte development and differentiation. Nevertheless, the exact role of microbiota in liver antiviral immune response is still unknown.Different virus will induce different characteristic liver immune responses. For example, HAV and adenovirus often induce acute immune clearance; while HBV and HCV often formatted chronic persistent infection by inducing immune tolerant. It indicated that the virus itself had the ability to regulate the liver immune response. For example, NK cells activation and cytotoxicity decreased during chronic HBV infection, but the precise mechanism is not clear.The hepadnavirus will be cleared when virus specific immune memory cells exist in peripheral immune system. Several hepadnavirus vaccine achieved success by this feature, but there still are parts of the vaccinated people who cannot develop the protective memory immune response. There is an urged need for the development of new vaccine adjuvant and immune project. In this work, we studied the anti-viral immune responses and their respective mechanisms in the liver from the three points discussed above.Ⅰ, Microbiota maintain hepatic γδT-17cell to enhance liver antiviral immune responseIn this study, the microbiota of adult C57BL/6mice was depleted by feeding the mice with antibiotics added water (refered as ’Atb mice’ below). Normal mice and Atb mice were challenged with adenovirus to evaluate their antiviral ability. FACS was used to detect the frequency, activation and cytokine secretion by the lymphocytes. Real-time PCR was used to detect the transcripts of virus and immune molecules. Cytokines in cell culture supernatant were detected by ELISA. Virus fluorescent protein expression was detected by liver frozen section. Cell depletion was performed to study the function of specific lymphocyte. Cytokines and stimulus were used to explore their roles in promoting hepatic γδT-17cells. Major findings of this study are shown as followed:1. Enhanced susceptibility to adenovirus infection of Atb mice.The peripheral immune response to HBsAg vaccine was normal in Atb mice; after challenge with adenovirus-eGFP, higher expressions of virus associated eGFP protein and mRNA were observed in Atb mice, which indicated that microbiota depletion led the enhanced susceptibility to adenovirus of the liver.2. Reduced IL-17production of hepatic γδT cells was responsible for the impaired antiviral ability of Atb mice.Enhanced susceptibility to adenovirus disappeared after depletion of immune system in Atb mice, which indicated that the susceptibility was associated with the immune system. NK cells’ frequencies and activation were normal in Atb mice, depletion of NK/NKT cells by PK136antibody did not influence the liver inflammatory damage, and these together indicated that NK and NKT cells were not responsible for the reduced IL-17expression. IL-17production by hepatic γδT cells decreased in Atb mice, meanwhile IL-17mRNA expression in liver and IL-17protein in the culture supernatant of liver lymphocytes also decreased in Atb mice. Moreover, administration of IL-17to Atb mice could recover their antiviral ability. 3. γδT-17cells accumulated in the liver of adult mice.IL-17production ability of γδT cells from different tissues ranked as followed: liver> peritoneal cavity> lung> spleen> thymus> IEL, it suggested γδT-17cells preferred resident in mucosal related tissues. Unlike adult mice, neonatal mice’s hepatic γδT cells produced low levels of IL-17and γδT-17associated molecules. These together indicated that γδT cells accumulated in the liver after birth might be driven by microbiota.4. IL-7and IL-1signalings were not responsible for the reduction of γδT-17cells in Atb mice.Administration IL-7to Atb mice could not recover the impaired IL-17secretion by γδT cells. Though Kupffer cells derived IL-1(3decreased in Atb mice, IL-17expression by hepatic γδT cells did not reduce but increased when Kupffer cells were depleted. These results indicated that microbiota maintained hepatic γδT-17in an IL-1and IL-7independent manner.5. Microbiota derived multiple TLR ligands promoted hepatic γδT cells producing IL-17.Single kind of stimulus of TLR ligands or curdlan could not recover the hepatic γδT-17cells in Atb mice, but ligands of TLR2, TLR4and TLR9administrated together significantly improved γδT-17cells in Atb mice. Moreover, though there was no difference of hepatic γδT-17expression between TLR2TLR4TLR9triple knockout mice and wild-type mice, microbiota depletion could not influence the hepatic γδT-17cells of TLR2/4/9triple knockout mice. Correspondingly, single kind of antibiotic treated mice did not reduce the hepatic γδT-17cells, but any two kinds of antibiotic together treated mice reduced their γδT-17cells. These results together indicated microbiota maintain hepatic γδT-17in a multiple TLR ligands dependent manner.In conclusion, our research indicated that microbiota promoted liver antiviral immune response by maintaining hepatic γδT-17cells; additionally microbiota maintained hepatic γδT-17was independent of IL-1and IL-7signalings and dependent on multiple TLR signalings. These findings revealed the function and mechanism of microbiota in promoting the liver antiviral immune responses. Ⅱ、Mechanism of the up-regulated expression of NKG2A on NK cells in HBV carrier mice.HBV carrier mice were established by hydrodynamic injecting C57BL/6mice with pAAV/HBV1.2plasmid, which imitate human HBV carrier, the change of NK cells’phenotype and function and its mechanism were studied. FACS was used to analyze the phenotype and cytokine secretion of lymphocytes. Cell transfer were performed to research the function of specific immune cells. The blocking antibodies were applied to detect the specific signaling which influence NK cells’antiviral function. In vivo lymphocyte proliferation was detected by BrdU incorporation. In vitro co-culture was used to observe the factors that influence NK cells’phenotype and function. Major findings of this study are shown as followed:1. NKG2A expression on NK cells up-regulated in HBV carrier mice.NKG2A expression on NK cells, especially hepatic NK cells, increased in HBV carrier mice, and the high frequency of CXCR6expression on NKG2A+NK cells might induced its liver tropism. NKG2A expression decreased in HBV carrier mice who spontaneously cleared HBV. These findings indicated that NKG2A expression positively correlated with HBV virus load.2. NKG2A suppressed the antiviral ability of NK cells in HBV carrier mice.Serum HBsAg concentration of HBV carrier Ragl-/-mice continued drop after injection of NKG2A blocking antibody, while HBsAg did not change in isotype antibody injected mice. It indicated NKG2A blockade could recover NK cells’ antiviral ability in HBV carrier mice.3. Hepatic CD4+regulatory T cells derived IL-10up-regulated NKG2A expression on NK cells in HBV carrier mice.Hepatic NKG2A+NK cells but not NKG2A-NK cells absorbed more BrdU in HBV carrier mice, which indicated that NKG2A+NK cells were induced proliferation in HBV carrier mice. In vitro co-culture and in vivo cell transfer experiment indicated that hepatic CD4+regulatory T cells from HBV carrier mice could induce NKG2A up-regulation on NK cells; additionally this process was dependent on IL-10and IL-10R signal. Moreover, recombinant human IL-10could also directly up-regulate NKG2A expression on NK cells in vitro. In conclusion, our research indicated that hepatic CD4+regulatory T cells derived IL-10increased in HBV carrier mice, and IL-10could suppress NK cells’ antiviral ability by up-regulating NKG2A expression; nevertheless, blocking NKG2A signal could significantly enhance NK cell mediated HBV clearance. These findings revealed the mechanism of HBV associated NK cell tolerance and provided new target for the therapy of chronic HBV patients.Ⅲ、IL-12promotes immune responses to HBsAg vaccineIL-12was used as co-adjuvant of alum based HBsAg vaccine and different vaccine protocols were administrated on adult C57BL/6and BALB/c mice to evaluate the effects of IL-12in promoting HBsAg vaccine. Radioimmunoassay was used to detect HBV related protein in serum. HBsAg specific antibody subtype and IFN-y secreted lymphocytes were detected by ELISA and ELISPOT. Mice were hydrodynamic injected with pAAV/HBV1.2plasmid to imitate human HBV infection. Major findings of this study are shown as followed:1. IL-12and alum worked as the co-adjuvant of HBsAg vaccine.Traditional Alum adjuvant induced higher titers of total anti-HBs than IL-12, while IL-12adjuvant induced higher titers of HBsAg specific IgG2a subtype antibody; nevertheless, using IL-12and Alum as co-adjuvant could obtain both advantages.2. Optimized the project of IL-12co-adjuvant based HBsAg vaccine.IL-12mixed together with HBsAg vaccine promoted stronger Th1polarization response than that used apart from vaccine; administration IL-12in the first vaccination was the necessary and sufficient condition for Thl polarization; IL-12promoted Th1polarization of HBsAg vaccine in a dose dependent way.3. IL-12co-adjuvant enhanced the antiviral ability of HBsAg vaccinated mice.Compared with HBsAg vaccine (Alum alone) immunized mice, IL-12co-adjuvant immunized mice produced much more anti-HBs and declined HBsAg and HBeAg expression more quickly after hydrodynamic injection with pAAV/HBV1.2plasmid.In conclusion, IL-12used as the adjuvant of HBsAg vaccine could enhance the antiviral ability through inducing Thl type immune response; and the best IL-12delivery mode was mixing IL-12in vaccine at the first immunization. These findings were helpful for the development and utilization of a new clinical immune adjuvant.