Preliminary Study Relationship Of Th22 Cells And IL-22 With Hepatic Fibrosis

Liver fibrosis disease process with abnormal proliferation and excessive deposition of extracellular matrix characteristics, the body’s immune system also plays an important role in it. Th22 cells, a distinct new CD4+ T helper lineage, are characteristic of producing IL-22. This new CD4+ subset has been shown to be involved in many kinds of autoimmune diseases in animal models and human. It has been shown that Th22 cells and its crucial effector cytokine, IL-17, may participate in the pathogenesis of hepatic fibrosis process. But how the Th22 cells and Th17-related cytokines change during the development of hepatic fibrosis? What’s the relationship of those cytokines? No reports have been presented until now. In order to elucidate the role of Th22 cells in hepatic fibrosis, and explore the new therapeutical target, hepatic fibrosis was induced in male BALB/c mice by CCL4 peritoneal injection, and the hepatic stellate cells were intervented by IL-22, then the following 3 parts researches were carried.Part 1:The dynamic changes of Th22 and its related cytokines in murine hepatic fibrosisObjective:To investigate the alteration of Th22 cells and its related cytokines such as (IL-22, IL-17A, IFN-y, IL-6, TNF-a) in murine liver fibrosis, and to investigate their role in liver fibrosis.Methods:A total of 72 mice were randomly divided into murine hepatic fibrosis model groups (n= 40,10 mice/subgroup) and control groups (n= 40,10 mice/subgroup). Each group was divided into 4 subgroups (WeekO, Week4, Week8, Week 12). The hepatic fibrosis model groups were induced liver fibrosis by intraperitoneal injection of carbon tetrachloride (CC14) mixed with olive oil twice a week at the concentration of 20%,2 ml/kg. The control group which received vehicle only.At 0 (berore injection),4,6 and 8 weeks, seventy-two hours after the last injection, mice were sacrificed. HE and Masson staining were used to determine the hepatic pathological changes of these groups and degree of liver fibrosis was assessed based on Ishak scoring system. Immunohistochemical staining was used to detecte the expression of a-smooth muscle actin (a-SMA) and IL-22 in the livers of mice. Flow cytometric analysis was used to evaluate the frequencies of Th22 subsets in spleen. IL-22, IL-17A, IFN-y, IL-6, TNF-a mRNA in murine hepatic tissue were measured by real-time PCR. The level of IL-22 in murine peripheral blood was detected by enzyme linked immunosorbent assay(ELISA).Results:(1) HE staining of liver tissue showed that continuous CC14 injection in model group led to degeneration, necrosis appeared and then fiber septa were formed gradually. The structure of the hepatic lobules was in disorder and finally pseudolobules were formed. Masson’s staining showed that in model group a lot of collagen deposition were seen in periportal liver tissue as well as central venous, and irregular fibrosis appeared in hepatic lobule. (2) Immunohistochemical staining for a-SMA expression. After CC14 administration, increased a-SMA staining was observed in livers of mice from weekO to week12. In addition to the week 0 point, model group, all other point a-SMA protein expression was higher than control group (P< 0.01). (3) Frequencies of splenic Th22 cells in the model group were significant higher than control group from week4 to week 12(P< 0.01), and continuing to 12 weeks.In addition to the week 0 point, Th22 cell proportion in model group were higher than in the control group of corresponding point (P< 0.01). (4) The expression of Hepatic IL-22,IL-17A,IFN-γ,IL-6,TNF-amRNA in the model group were significant higher than control group from week4 to week12(P< 0.01), and continuing to 12 weeks. In addition to the week 0 point, IL-22,IL-17A,IFN-γ,IL-6,TNF-amRNA in model group were higher than in the control group of corresponding point (P< 0.01). (5)Peripheral blood levels of IL-22,IL-17A,IFN-y,IL-6,TNF-a in the model group were significant higher than control group from week4 to weekl2(P< 0.01), and continuing to 12 weeks.In addition to the week 0 point, IL-22, IL-17A, IFN-y,IL-6,TNF-ain model group were higher than in the control group of corresponding point (P< 0.01). (6) The expression of Hepatic IL-22 protein in the model group were significant higher than control group from week4 to week12 (P< 0.01), and continuing to 12 weeks. In addition to the week 0 point, IL-22 protein in model group were higher than in the control group of corresponding point (P<0.01).Conclusion:The expression of Th22 cells and its related cytokines increase in mice of liver fibrosis.Part 2:Inhibiting liver fibrosis in mice with recombinant IL-22 in vivoObjective:To investigate the effect of rmIL-22 in vivo in liver fibrosis mice so that to establish the pathogenesis role of Th22 in liver fibrosis.Methods:A total of 16 mice were treated with 2 mL/kg body weight of 20% CCl4 intraperitoneally for 8 wk, and then randomly divided into two groups. The mice were given 300 μg/kg of rmIL-22 (0.3 μg/g) daily by intraperitoneal injection for 14 d before being sacrificed (n= 8, rmIL-22 group). The control groups were injected with a carrier solution of 0.5% BSA (in PBS, pH 7.0) (n=8, carrier group). All surviving animals were sacrificed on week 10 after CCl4 injection. The livers, spleens and plasma were collected. HE and Masson staining were used to determine the hepatic pathological changes of two groups and degree of liver fibrosis was assessed based on Ishak scoring system. Immunohistochemical staining was used to detecte the expression of a-smooth muscle actin (a-SMA) and IL-22 in the livers of mice. Flow cytometric analysis was used to evaluate the frequencies of Th22 subsets in spleen. IL-22, IL-22R1, IL-10R2, IL-17A, IFN-y,IL-6,TNF-amRNA in murine hepatic tissue were measured by real-time PCR. The level of IL-22, IL-17A, IFN-y, IL-6, TNF-a protein in murine peripheral blood was detected by enzyme linked immunosorbent assay (ELISA).Results:(1) Compared with the control group, rmIL-22 alleviated the severity of hepatic fibrosis, reduced the fibrosis scores and the number of a-SMA positive cells in the rmIL-22 group (P<0.05). (2) Compared with the control group, rmIL-22 decreased the frequencies of Th22 cells and the levels of IL-22 protein in liver tissue and IL-22 protein in plasma, and decreased the expressions of hepatic IL-22, IL-22R1,IL-10R2 mRNA (P<0.05).(3) Compared with the control group, rmIL-22 decreased the levels of IL-17A, IFN-γ,TNF-α,IL-6 protein in plasma,and decreased the expressions of hepatic IL-17A, IFN-γ,TNF-α,IL-6 mRNA(P<0.05).Conclusion:IL-22 inhibite HSC activation and down-regulate the levels of inflammatory cytokines, thereby ameliorating liver fibrogenesis.Part 3:Inhibiting hepatic stellate cell proliferation with recombinant IL-22 in vitroObjective:To investigate the effects of IL-22 on the proliferation and activation of hepatic stellate cells (HSCs) and the expression of fibrosis-related proteins in vitro.Methods:HSCs were divided into the following groups:① the control group:HSCs were cultured alone; ② the experimental group a:HSCs were cultured with rmIL-22 (10 ng/ml)③ the experimental group b:HSCs were co-cultured with rmIL-22 (20ng/ml). Groups respectively to cultivate 24 h,48 h and 72 h, the dynamic observation of cell morphology change under inverted phase contrast microscope; the activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. The best intervention concentration of IL-22 was detected by MTT assay; The expression of α-SMA,TGF-β1,TIMP-1mRNA mRNA were evaluated by PCR.Results:(1)Under Inverted phase contrast microscope HSCs were observed at the good condition with membrane growth, typical star or polygon, intracellular more grain. At cultured for 48h, immunohisto- chemical staining results showed that brown granules in the cytoplasm within HSCs and light blue nuclear were viewed.The results showed α-SMA(+) and more than 95% of activated HSCs positive. (2) Compared with the control group, different concentration of the IL-22 could inhibit the proliferation of the HSC after 24hours,48hours,72hours; RmIL-22 at 20 ng/ml caused obviously HSCs inhibition (P<0.05) compared with other concentration groups. (3)The expression of a-SMA,TGF-β1,TIMP-1mRNA in experimental group decreased over time and caused the lowest levels compared with other groups (P< 0.01).Conclusion:IL-22 inhibite the proliferation and activation of hepatic stellate cells, thereby reducing the level of fibrosis factors.