Development Of Primary Biliary Cirrhosis Animal Model And Investigation Of Immune Tolerance Related Mechanisms

Primary biliary cirrhosis is a slowly progressive autoimmune disease of the liver that primarily affects women. The pathogenesis of PBC is unknown and the incidence is growing with the rapid development of diagnostic technologies. PBC is characterised immunologically by the breakdown of immune self-tolerance to highly conserved mitochondrial and nuclear antigens. In over 95% of patients, anti-mitochondrial antibodies are directed at the members of the 2-oxoacid dehydroganese complex family of multi-enzyme complexes. Besides, PDC-E2 specific autoreactive T cells can be detected in most patients with PBC but not in normal ones. However, the reason for the breakdown of immune tolerance and the regulatory mechanisms are still unclear.After T cells are activated by responding to antigenic stimuli, they are generally killed by apoptotic mechanisms. Among the apoptotic mechanisms, activation-induced cell death (AICD) plays a central role, especially in killing autoreactive T cells and in preventing autoimmune responses. AICD in T cells in vivo has been proposed to limit the expansion of an immune response by eliminating effector cells that are no longer needed. Organ-specific autoimmune diseases are characterized by tissue destruction and functional decline due to autoreactive T cells that escape self-tolerance. Primary biliary cirrhosis is characterized immunologically by lymphocytes infiltration in portal areas, destruction of biliary epithelial cells and cholestasis, which lead to progressive liver damage.The portal cellular infiltrate in PBC contained a preponderance of CD4 cells in comparison with CD8 cells, with a CD4/CD8 ratio of 2.45:1, which indicate that CD4+T cells play important roles in the immune inflammation process. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis showed that IFN-γmRNA-positive cells were detected primary around damaged bile ducts. The data indicate that Th1 cells are the more prominent T cell subset in the lymphoid infiltrates in PBC.Polyinosinic polycytidylic acid (polyI:C) is ligand for toll like receptor 3, which can induce the secretion of IFN-α. Prolonged courses of IFN-a have been associated with the onset of autoimmune events. These ranged from the appearance of autoantibodies such as antinuclear, anti-thyroid, and anti-islet-cell antibodies in 80% of patients treated to the exacerbation, or, in some cases, to the development of a variety of autoimmune diseases, including hyper- or hypothyroidism, type I diabetes, psoriasis, and autoimmune hepatitis. In 1995, D'Amico reported for the first time on a case of primary biliary cirrhosis (PBC) induced by IFN-a in a patient with chronic hepatitis C. Alkaline phosphatase levels reached about three times. Further evaluation revealed the constant presence of high titers of serum anti-mitochondrial antibody.The patient underwent a second liver biopsy that showed infiltration of the portal areas with mononuclear cells. Besides, Takii found that the expression levels of type I IFN-αwas significantly higher in portal tract and liver parenchyma as compared to AIH and CHC.In clinical treatment for PBC, some medicines can be used to relieve pruritus, osteoporosis, hyperlipidemia and portal hypertension. However, survival is not adversely affected by the treatment and can not cure PBC fundamentally. According to the reasons underlined above, it is of great importance to investigate an effective method for PBC treatment from immunology aspect.Based on the facts showed above, we plan to develop a kind of PBC animal model by M2 and polyI:C injection and study activation induced cell death of CD4+T cells from spleens and livers to understand peripheral immune tolerance mechanism in PBC model. In order to prevent PBC, we injected spleen cells which were incubated with M2 antigen at the presence of ECDI though caudal vein to reconstruct immune tolerance. All the studies underlined above aim to provide clues for advanced investigation of PBC pathogenesis and clinical treatment.Part I. Fermentation and purification for M2 autoantigen E2 subunit of PDC, OGDC and BCOADC are the predominant self-antigen for patients with PBC. Immunodominant epitopes of PDC, OGDC and BCOADC have been cloned together and expressed in our laboratory. In order to develop PBC animal model and to investigate its immune tolerance, we prepared M2 protein through fermentation. At first, the protein was induced by IPTG to express in tubes and flasks. Second, a lot of M2 was obtained through 3.5L fermentor. At last, the protein was purified by Amersham protein purification system.Results showed that high expression of protein was identified 4h after induction by 1mg/ml IPTG. In the 3.5L fermentor, dissolved oxygen decreased 2.5h later and then glycerol was used as carbon source. We had compared glycerol and glucose as carbon source and found that glycerol can promote M2 expression dramatically. Highest cell density (OD600) was determined 6h after IPTG induction. Dissolved oxygen was controlled up to 30%, the maximum rate of rotation was 500rpm/min and pH was adjusted to 6.8-7.4. At last OD600 was 12.7, bacterial weight was 30g and the concentration of M2 we obtained was 1.5mg/ml.Part II. Development of primary biliary cirrhosis animal modelPBC is a kind of chronic persisting liver disease which is usually accompanied by virus infection and some other pathogens. It is so difficult to obtain enough liver tissues from patients for scientific research that a useful animal model is of great importance.M2 and polyI:C were employed to immunize C57BL/6 female mice at dose of 50μg and 5 mg/kg, 10mg/kg. Mice were sacrificed in different times and serum, liver, kidney, brain, intestine and stomach were separated for further determination. Results are as follows: in M2 group, the positivity of ANA and AMA was 30% and 80%, the AMA titers were all more than 1:100. However, both were negative in control group. In polyI:C group, ANA in control, 5mg/kg and 10mg/kg group were up to 100% positive in the 12th, 8th, and 4th week. The titers of ANA in control and 5mg/kg group were more than 1:100 since the 8th week but in 10mg/kg group it was since the 4th week. Besides, ANA titers more than 1:10000 can be found in each polyI:C group since 8th week. AMA in mice of control group were all negative, however, in 5mg/kg group, the positivity increased with time after polyI:C injection and it was up to 80% in 16th week. Differently, in 10mg/kg group, AMA positivity was 80% in the 4th week but decreased to 20% in the 20th week.Liver tissues were collected and stained with H.E and the number of portal areas with more than 100 mononuclear cells was counted as cell infiltrated portal areas. Data showed that the number of infiltrated portal areas increased until 16th week. The difference between 16th and 20th week had no statistic significance. No infiltrated portal areas were found in the other organisms in mice of each group. Serum AKP in both polyI:C groups increased with time and the average levels were all more than 60U/L, however, it was below 60U/L in control group. No infiltration and AKP change was found in M2 group. There was no significant difference for the ratios of CD4+ and CD8+T between each group. In conclusion, the PBC model developed by polyI:C 5mg/kg injection was more typical than the others and can be used for the following research.Part III Proliferation and activation induced cell death of lymphocytes in primary biliary cirrhosis animal model.Activation induced cell death (AICD) of autoreactive T cells in vivo plays a central role in maintaining peripheral immune tolerance. Defective AICD of effective T cells may result in autoimmune diseases. Studies indicated the predominant of Th1 cells around portal tracts.In this part, we develop PBC model again by injecting 5mg/kg polyI:C. Lymphocytes and CD4+ T cells were separated from spleens and livers after 16th week and stimulated by M2, conA and anti-CD3. Cell proliferation was determined by CCK8 assay and AICD was analyzed by flowcytometry (FCM). We also determined the expression of apoptosis related genes (Fas, FasL and TRAIL) and proteins (FLIPL and caspase-8). Results showed that: 1) No significant difference was found between cell proliferation of M2 group and control mice. However, the ability of cell proliferation of lymphocytes in PBC mice was stronger than that in control mice (P<0.001). 2) Cell apoptosis rates in control groups were lower than that in PBC mice (P<0.001), but no difference was determined in control groups (P>0.05). Furthermore, in PBC mice, the apoptosis rates of T cells from livers were lower than that from spleens significantly (P<0.001). 3) The expression of FasL and TRAIL in spleens and livers of PBC mice was lower than that in controls (P<0.01), but no difference was found with Fas. 4) The expression of caspase-8 in CD4+T cells in PBC mice was not different from that in control ones. However, the expression of FLIPL in PBC mice was 5 times higher than that in controls. Moreover, in PBC mice, the expression of FLIPL in livers was higher than that in spleens (P<0.001).In conclusion, the anti-apoptotic ability of CD4+ T lymphocytes plays an important role in immunological tolerance of PBC mouse model, and the enhancement of the ability was to some degree correlated with the elevated FLIPL expression. Besides, the inhibition of FasL and TRAIL expression may also of help in the enhancement of the anti-apoptotic ability in lymphocytes and in the aggravation of portal area inflammation.Part IV Prevention of primary biliary cirrhosis through immune tolerance reestablishmentThere is no curable method for PBC nowadays and symptomatic treatment methods for pruritus, osteoporosis, hyperlipidemia and portal hypertension are better choices for clinicians. Liver transplantation is the best and only means for patients with PBC in advance stage. Accordingly, it is imminent to find a better way from immunology aspect for PBC treatment. Spleenic cells from na?ve mice were incubated with M2 in the presence of ECDI and the cells were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with BSA were injected as controls. 16 weeks later, AMA, AKP and portal inflammation were assayed for evaluating the prevention effect. Results are as follows: 1) AMA positive rate in tolerance group was lower than that in BSA and PBC groups (P=0.007, P=0.003). The difference between BSA and PBC was not significantly. 2) Serum AKP levels in tolerance, BSA and PBC group were 80.5±9.8U/L, 93.8±15.7U/L and 92.5±17.7U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P=0.0095, P=0.029). 3) The rates of portal areas with cell infiltration were 42.67±12.3%, 57.07±11.35% and 51.53±9.96%, separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P=0.039) and BSA group (P=0.0024).In conclusion, primary biliary cirrhosis in mouse model was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.