Expression Of TH22Cells And Its Cytokine Interleukin-22in Murine Model Of Carbon Tetrachloride-induced Liver Fibrosis

Objective To investigate the expressions and relationships of Th22cellsand its related cytokines interleukin-22(IL-22) in a murine model of carbontetrachloride-induced hepatic fibrosis. To explore whether Th22cells and IL-22are involved in the pathogenesis of liver fibrosis.Methods There were36male BALB/c mice which were randomlydivided into three groups, including normal control group, olive oil controlgroup and liver fibrosis group, with12mice for each group. Mice were inducedliver fibrosis by intraperitoneal injection of carbon tetrachloride (CCl4) mixedwith olive oil twice a week at the concentration of20%,2ml/kg. The olive oilcontrol group which received vehicle only. After eight weeks, mice in eachgroup were sacrificed. HE and Masson staining were used to determine thehepatic pathological changes of these groups. Immunohistochemical stainingwas used to detected the expression of α-smooth muscle actin (α-SMA) andIL-22in the livers of mice. The proportion of IL-22+CD4+T cells,IL-22+IL-17-IFN-γ-T(Th22) cells, IL-17+CD4+T(Th17) cells and IFN-γ+ CD4+T（Th1） cells in murine spleens were examined by flow cytometricanalysis. The level of IL-22in murine peripheral blood was detected by enzymelinked immunosorbent assay(ELISA). Additionally, hepatic IL-22, IL-6, tumornecrosis fator-α(TNF-α), arylhydrocarbon receptor(AhR) mRNA in murinehepatic tissue were measured by real-time PCR. Then the correlation analysisbetween the percentage of Th22cells as well as IL-22and liver fibrosis wereperformed.Results (1) HE staining of liver tissue showed that continuous CCl4injection in model group led to cord-like fibers extended from the portal areainto the hepatic lobule, with a large number of inflammatory cells andfibroblasts infiltration, and hepatic cell cords arranged disorder, fibrosisappeared in centrilobular regions, hepatic lobular structure was damaged.Masson’s staining showed that in model group a lot of collagen deposition wereseen in periportal liver tissue as well as central venous, and irregular fibrosisappeared in hepatic lobule. HE and Masson staining demonstrated that murinehepatic fibrosis model was successfully induced by CCl4injection.(2)Immunohistochemical analysis of α-SMA staining shows that a significantincreased expression of α-SMA was detected in the model group（0.38±0.09）,compared to normal control group(0.07±0.03) and olive oil controlgroup(0.08±0.03); The expression of IL-22(0.21±0.05) was significantly higherthan normal control group (0.06±0.03) and olive oil control group (0.07±0.02),(all P<0.05).(3) The percentage of IL-22+CD4+T cells （8.59±2.00）%inspleens of model group was significantly increased as compared to normalcontrol group（2.40±0.57%）%and olive oil control group（2.27±1.21%）%; Thepercentage of Th22cells （8.69±3.27）%was significantly increased ascompared to normal control group（3.28±0.87）%and olive oil control group （3.12±1.56）%; The percentage of Th17cells （2.02±1.06）%was higher thanthose in normal control group（0.62±0.28）%）%and olive oil control group（0.63±0.25%）%; The percentage of Th1cells （2.35±1.40）%in model groupwas also higher than those in normal control group（1.26±0.46）%and olive oilcontrol group（1.25±0.52）%,(all P<0.05).(4) Compared with normal controlgroup（24.80±3.66）pg/ml and olive oil control group(26.62±8.62) pg/ml,significant elevation of circulating IL-22was demonstrated in modelgroup(65.08±12.29) pg/ml,(P<0.01).(5) The expression of IL-22mRNA(3.10±0.60) in hepatic tissue of model group was significantly increasedas compared with those of normal control group （1.01±0.44）and olive oilcontrol group (0.99±0.24); The expression of IL-6mRNA (1.90±0.47) ofmodel group was significantly increased as compared with those of normalcontrol group （0.57±0.16） and olive oil control group (0.63±0.26); Theexpression of TNF-α mRNA (1.39±0.50) of model group was significantlyincreased as compared with those of normal control group（0.25±0.11） andolive oil control group(0.28±0.09); The expression of AhR mRNA (1.52±0.74)of model group was significantly increased as compared with those of normalcontrol group （0.80±0.28）and olive oil control group(0.79±0.39),（allP<0.05）.(6) The frequency of Th22cells respectively had a positive correlationwith hepatic α-SMA expression (r=0.725, P<0.01) and the levels of IL-22(r=0.660, P <0.05). There was no significant correlation between the frequencyof Th17cells and Th1cells with the levels of IL-22.Conclusion Th22cells are the main cell source of IL-22-secreting CD4+T cells in the spleen of liver fibrosis model. Th22cells may involve in thepathogenesis of liver fibrosis through producing IL-22.