The Immunologic Mechanisms Of Liver Fibrosis In HBV Transgenic Mice

Liver fibrosis is huge threaten to human health. Long term alcohol drinking, fatty liver and chronic virus hepatitis infection are the three major causes of liver fibrosis. Liver fibrosis is a common end for many chronic liver diseases. Serious liver fibrosis could lead to cirrhosis or even hepatocellular carcinoma (HCC). There are many kinds of cell participate in liver fibrosis, including parenchyma and non-parenchyma cells. Among which the most significant cell is hepatic stellate cell (HSC). Upon activation, HSC secrete a lot of collagen, inducing extracellular matrix (ECM) deposition. Recent studies suggested that the hepatocyte also has a role in liver fibrosis. At the same time, liver is predominant for the innate immunity, the innate cells in the liver could affect the progress of liver fibrosis.These are many Chinese hepatitis B virus (HBV) carriers, which is the main causes for later liver fibrosis. How HBV infection accelerates liver fibrosis and the immune cells function are not completed studied. One of the most important reasons is the lack of suitable mice model. In this study, we compared liver fibrosis after the long term hepatotoxin carbon tetrachloride (CCl₄) administration in C57BL/6 mice and HBV-tg mice. We tested the liver injury, pathological changes and fibrosis related molecules. To find the mechanism, we used cell counting and ratio analysis, cell depletion, purification, adoptive transfer, co-culture of HSC and NKT cells, isolate and culture primary hepatocyte etc. Through the above methods, we have these results:1. Spontaneous liver fibrosis in HBV-tg miceWe found the six month old HBV-tg mice had spontaneous liver injury compared with C57BL/6 mice by detecting alanine aminotransferase(ALT)and pathology. The six month old HBV-tg mice also had much more liver fibrosis than C57BL/6 mice by liver Sirius red staining and qPCR of fibrosis related genes.2. HBV-tg mice are oversensitive to CCl₄-induced liver fibrosisC57BL/6 and HBV-tg mice were injected CCl₄ twice a week and lasted from 2 to 14 weeks. We found much more liver fibrosis in HBV-tg mice than C57BL/6 mice. For the first time, we suggest HBV-tg mice for a good mice model to study HBV related liver fibrosis.3. NKT cell activate HSC and aggravate liver fibrosis in HBV-tg miceExperiments showed that Rag1-/- mice adoptively transferred lymphocytes from HBV-tg mice could induce liver fibrosis but not the cells from C57BL/6 mice. Further studies showed the percentage and number of liver NK and NKT cells are much more in HBV-tg mice than C57BL/6 mice after CCl₄ injection. We found in HBV-tg mice NKT cells could activate HSC by using cell depletion and adoptive transfer experiments. And NKT cell could also up-regulated fibrosis related genes, thus increasing liver fibrosis in HBV-tg mice.4. NKT cells activate HSC through secreting of cytokinesHow NKT cells activate HSC? We found NKT cell number increased in HBV-tg mice than C57BL/6 mice after CCl₄ injection. The number of NKT cell secreting IFN-γ, IL-4 and IL-13 were also more in HBV-tg mice. NKT cell could activate HSC in an in vitro co-culture of sorted liver NKT cells and perfused liver HSC. If we added neutralizing cytokine antibodies against IL-4 or IL-13, the activation was disappeared but not the antibody against IFN-γ. These results suggested NKT cell activate HSC by the Th2 cytokines (eg, IL-4, IL-13).5. Hepatocyte from HBV-tg mice has spontaneous epithelial-mesenchyma-transition (EMT).Besides NKT cell accelerating liver fibrosis, we also found the hepatocyte promoted liver fibrosis in HBV-tg mice. When cultured and compared the hepatocyte from six months of age C57BL/6 mice and HBV-tg mice, we found more mesenchyma cell phenotype from HBV-tg mice than C57BL/6 mice. The EMT marker vimentin also increased in the hepatocyte from HBV-tg mice than C57BL/6 mice by qPCR, which is a solid evidence for the EMT in HBV-tg mice. We also found the fibrosis related genes up regulated in hepatocyte from six month old HBV-tg mice than that of C57BL/6 mice, which demonstrated that the EMT from HBV-tg mice hepatocyte could contribute to liver fibrosis.6. The hepatocyte from CCl₄ induced liver fibrosis has EMT and contributes to liver fibrosis The hepatocyte was perfused and cultured from CCl₄ induced liver fibrosis of C57BL/6 mice and HBV-tg mice, and was visualized at different time points. The hepatocyte from HBV-tg mice showed more fibroblast like phenotype. The EMT marker vimentin was increased suggested the more EMT in hepatocyte of HBV-tg mice during CCl₄ induced liver fibrosis. The fibrosis related genes were also increased more in HBV-tg mice than C57BL/6 mice at some of the time points. These results showed the hepatocyte also contribute to CCl₄ induce liver fibrosis.7. NK cells inhibit EMT in HBV-tg miceWe found EMT marker vimentin increased in HBV-tg mice after CCl₄ injection for 24 hours. NK cells could inhibit EMT by cell depletion experiment. We hypothesis that NK cells could inhibit hepatocyte EMT through the DR5 up-regulated in CCl₄ injected hepatocyte.8. EMT in HBV-tg mice contribute to HBV induced HCCThe above results suggested there is more EMT in HBV-tg mice. Recent studies showed EMT could accelerate the carcinoma. We found the old HBV-tg mice could spontaneously develop liver tumor and two times a week CCl₄ injection for 14 weeks could induce liver tumor from all of the HBV-tg mice. So we suggested HBV-tg mice as a good animal model for study HBV induced carcinoma.Conclusion: HBV-tg mice is an important mice model for study HBV related liver fibrosis. The immune cells could accelerate liver fibrosis, especially liver NKT cells activate HSC through secreting of cytokines. On the other hand, the hepatocyte from HBV-tg mice could undergo EMT and participate in liver fibrosis, which is a contribution for HCC.