The Role And Mechanism Of CD4~+CD25~+FOXP3~+Treg And Its Effector Molecule Fibrinogen-like Protein2in Autoimmune Hepatitis

[B ACKGROUND&OBJECTIVE]Autoimmune hepatitis (autoimmune hepatitis, AIH) is characterized by elevated serum transaminases, a variety of autoantibodies appeared in circulatory system, hypergamma-globulinemia、liver tissue interface hepatitis、lymphatic cell infiltration and fibrosis and occurred more frequently in women, whose cause and pathogenesis has not been fully elucidated.The course of the disease may involve complex interactions between a variety of factors such as genetic susceptibility, environmental inducing factors, autoantigen, immune disorders, ultimately the immune tolerance is broken up, causing the immune response against liver autoantigen, which leads to liver cells destruction resulting in inflammation and necrosis of the liver and may progress to liver fibrosis and cirrhosis. Current domestic and international research on the pathogenesis of autoimmune hepatitis is mainly concentrated on the genetic susceptibility factors (AIH susceptibility allele in HLA; The effect of TCR in genetics), liver cell injury mechanism (CD4+T and CD8+T are involved), immunoregulatory network (the autoantigen proposed, immune cell activation, cytokine network (TH1/TH2cell imbalance)), as well as cross-reactivity (Molecular Simulation between foreign antigens and autoantigens autoimmune is one of the causes of autoimmune disease, such as viral infections, drugs stimulation, destruction of self-tolerance, which ultimately lead to the occurrence of the disease), immunomodulatory and liver cell injury are most cited as a concern.Immune-mediated inflammatory damage in the disease is in an important position. Both Thl cell-mediated cellular immune response and TH2cell-mediated humoral immunity are involved in the development of autoimmune hepatitis. Recent studies found that TH17cells are also important cells inducing autoimmune disease tissue damage. IL-17A is a characteristic cytokines and important inflammatory mediators secreted by TH17cells, which can activate the inflammatory cascade effect leading to tissue damage through binding the receptor; inflammation is also important factor for initiation and promotion of the liver fibrosis progress. The pathological features of autoimmune hepatitis is inflammatory cell infiltration in the portal area and invading to the surrounding liver parenchyma. The immunohistochemical study confirmed the infiltration of inflammatory cells mainly include CD4+T cells, CD8+T cells, and monocytes, macrophages, B lymphocytes, natural killer cells, etc also included. Therefore, the study of the effect of immune network consisted of a variety of immune cells and cytokines secreted by them on the immune liver injury is conducive to the understanding of the pathogenesis of autoimmune hepatitis.CD4+CD25+FOXP3+regulatory T cells (Treg) are a group of full-time cell for the rejection of auto or foreign antigen immune response, play an important role in the maintenance of immune balance. Previous studies have generally agreed that Treg number and function may be defective in patients with autoimmune liver, which is more obvious in the active phase of the disease. The peripheral blood Treg proportion decreased in patients with biliary cirrhosis, intrahepatic CD8+T lymphocytes/FOXP3+Treg ratio is higher than that in chronic HCV infection and patients with autoimmune hepatitis in early phase, meanwhile the Treg function defects. These findings suggest that Treg dysfunction may contribute to the occurrence of autoimmune liver disease, and play an role in aggravating the disease. Recently, some scholars believe that the number and function of Treg in patients with autoimmune hepatitis has not declined. The difference of specific staining mark of Treg and functional experimental methodology, and the different detection site and evaluation criteria may lead to different results and the existence of the dispute. Therefore, for different sites of the peripheral blood and liver, re-evaluate the number and function of Treg in patients with autoimmune hepatitis contribute to a deep understanding and exploring the pathogenesis of AIH.Conventional idea is that Treg cells play a role by the main inhibitory cytokines IL-10, TGF-β, but in recent years some study confirmed FGL2can exhibit immunosuppressive effects as Treg new effector molecule, in autoimmune kidney glomerulonephritis and MHV-3induced rat fulminant hepatitis, the study showed that, fiber mediated prime protein2(Fibrinogen Like Protein2) is one of the the mechanisms CD4+CD25+FOXP3+regulating T cells play an inhibitory effect, recently some study also found the FGL2was closely related to the severity of with chronic C hepatitis, however, about the role of FGL2in autoimmune hepatitis has been rarely reported.Therefore, we collected the clinical data and specimens of patients with autoimmune hepatitis, and the established experimental autoimmune hepatitis mouse model to assess the number and function of Treg cells and explore the the role of FGL2expressed in Treg and action mechanism of FGL2. Meanwhile, the relationship between the expression of the plasma FGL2and inflammation and pathological changes in autoimmune hepatitis was preliminarily studied and explored in order to provide a new theoretical basis and strategies for the diagnosis and treatment of autoimmune diseases.1%Autoimmune hepatitis patients were selected as study objects(1) Peripheral blood and liver biopsy specimens were obteined from AIH patients and healthy controls to observe Treg cells proportion in lymphocytes and FGL2expression in liver tissue.(2) The relative between clinical parameters and cytokines of TH1/TH2/TH17as well as FGL2expression were analysed based on the detection of cytokines of TH1/TH2/TH17and FGL2expression in peripheral blood and then FGL2expression levels were assessed according to liver inflammation and fibrosis staging.2、Establish model of C57BL/6mice with experimental autoimmune hepatitis (EAH)(1) Observe Treg and FGL2expression in blood, liver and spleen Treg and FGL2expression in EAH model.(2) Cytokines of TH1/TH2/TH17and FGL2expression in peripheral blood were detected and the correlation of cytokines of TH1/TH2/TH17and FGL2were analysed. (3) To explore the influencing mechanism of Treg and FGL2on effector cells cocultured in vitro.[METHODS]1、CD4+CD25+FOXP3+Treg cells from AIH patients and healthy controls were analysed by flow cytometry; FOXP3and FGL2expression in liver tissue from AIH patients and healthy controls were detected by immunohistochemical technology; Immuno-fluorescence double dye staining was used to identify whether FOXP3and FGL2expressed in the same cell.2、After the model of C57BL/6mice with experimental autoimmune hepatitis (EAH) were established successfully, CD4+CD25+FOXP3+Treg cells in blood, liver and spleen from EAH and control were analysed by flow cytometry; FOXP3and FGL2expression in liver tissue from EAH and control were detected by immunohistochemical technology and Realtime-PCR; Immunofluorescence double dye staining was used to identify whether both of FOXP3and FGL2expressed on the same CD4+CD25+T cells and liver tissue.3、In vitro experiment:CD4+CD25+Treg, CD4+CD25-T cells and CD8+T cells were sorted by magnetic bead from spleen of immunised2-4w EAH mice model. Elisa was used to detect FGL2expression in cultured CD4+CD25+Treg cell supernatant. FGL2mRNA of the three above sorted cells and FOXP3mRNA, IL-10mRNA, TGF-β1mRNA of CD4+CD25+Treg cell were analysed by real-time PCR. IFN-y and IL-17A expression on CD8+T cell were detected by flow cytometry. Co-culture CD4+CD25+Treg cells and CD8+T lymphocytes, flow cytometry was used to determine Treg secreted FGL2and proliferation, IFN-y and IL-17A expression on CD8+T cell after FGL2blocked by antibodies. Exogenous FGL2was added into CD8+T cultured cells with different concentration, and then detected IFN-y and IL-17A in supernate and CD8+T cytoplasm.4. Flow cytometry ball microarray technology (cytometric bead array, CBA) was used to detect TH1/TH2/TH17cytokines of IL-2, IFN-y, TNF-a, IL-4, IL-6, IL-10and IL-17on0w,1w,2w,3w,4w,5w,6w of twice immunised mice, meanwhile, plasma FGL2were analysed by elisa. Then the correlation of FGL2and TH1/TH2/TH17relative cytokines with ALT and AST were statistical analysed.5、Plasma TH1/TH2/TH17related cytokines were detected by CBA and plasma FGL2 expression was tested by Elisa in patients with AIH. The correlation of FGL2, TH1/TH2/TH17relative cytokines in plasma and clinical features were analysed by statistical software and plasma FGL2expression levels were assessed according to liver inflammation and fibrosis staging of AIH.[RESULTS]1、Compared to controls, the ratio of CD4+CD25+FOXP3+Treg cells from peripheral blood declined in AIH patients while FOXP3and FGL2expression in liver enhanced. It was found that both FGL2and FOXP3express in the same cell.2. It was found that the percentage of CD4+CD25+FOXP3+Treg cells of EAH mice declined in peripheral blood and spleen while increased in liver when compared with control. More FGL2and FOXP3expressions in mRNA and protein levels were found in EAH than in control, Which showed the same trends with AIH patients. Both FGL2and FOXP3can be found in the same cell.3. In vitro experiment showed that CD4+CD25+Treg FGL2mRNA expression level was significantly higher than that of CD4+CD25+T cells and CD8+T cells. The mRNA expression of FOXP3, TGF-β1and IL-10in EAH mice were lower than control while FGL2mRNA as well as FGL2in CD4+CD25+Treg cell culture supernatant were higher than control. IFN-γ and IL-17A secreting CD8+T cells were increased in EAH. Cell hybrid culture experiments show the inhibitory effect of CD4+CD25+Treg to CD8+T cell with cell less proliferation, less IFN-γ and IL-17A secreting in EAH mice than control. The inhibitory effect weakend when neutralized antibody to FGL2was added. Moreover, exogenous FGL2also performed inhibitory effect to CD8+T cells with declined IFN-γ and IL-17A both in cultured supernatant and intracytoplasmic.4. Plasma IL-2, IFN-γ, TNF-a, IL-4, IL-6, IL-10and IL-17increased in EAH mice and reached a peak at the second week, then decreased with time while TNF-a, IL-6and IL-10increased from the third week and reached another peak on the fifth week and declined since then. No obvious relevance was found with ALT and AST.5. Plasma IL-10and IL-17in AIH patients were obviously higher than control while IFN-γ and IL-4decreased, and IL-2, TNF-a, and IL-6showed no significant difference between them. Plasma FGL2expression increased obviously in patients with AIH than controls and showed positive correlation with TBil and IL-17, but no obvious correlation existed between plasma FGL2and ALT, AST, DBil, ALP, r-GT, LDH, ALB, INR, PTA and fibrinogen. In addition, plasma FGL2showed a rising trend as deteriorated liver inflammation (G1-G3) and fibrosis (S0-S4)[CONCLUSION]1、This study found the percentage of CD4+CD25+FOXP3+Treg fall in peripheral blood and raise in the liver partialy compensatory, from two aspects of peripheral and intrahepatic by assessing patients with autoimmune hepatitis and animal model of autoimmune hepatitis. The expression of TGF-β1and IL-10mRNA levels of Treg decreased in EAH. Reduced Treg numbers and decreased inhibitory cytokine expression may contribute to the defect of negative immune regulation of Treg and disease development.2、FGL2expression levels in EAH was higher than normal controls and it highly expressed on Treg. The changes of plasma FGL2level consistented to peripheral blood Treg proportion. Treg inhibiting proliferation effect is abated after FGL2neutralizated with antibody, showed that while the transcription factor of FOXP3and cytokines TGF-β1and IL-10expression levels decreased, FGL2served as another Treg secreted cytokines and played an important immune inhibitory effection, namely the inhibition of CD8+T cell proliferation and cytokine IFN-γ, IL-17A production.3、FGL2and clinical parameter correlation analysis showed that the plasma FGL2expression level are positively related with TBiL. TH1/TH2/TH17cytokines correlation analysis showed that the plasma FGL2and IL-17A is closely related. It illustrated that FGL2participated in the development of autoimmune hepatitis and closely related with liver damage. In EAH, the plasma IL-17A cytokine expression peak increased before FGL2, explaining that FGL2might be compensatory increased to suppress pathogenic effect of IL-17A. These findings help to understand the pathogenesis of autoimmune hepatitis and may provide a new reference for clinical diagnosis and treatment and strategy.