The Role Of Immunoregulation By TH17 And Treg Cells In The Pathogenesis Of Chronic HBV Infection

Objective: Chronic hepatitis B (CHB) is one of the common chronic infectious diseases in China, which seriously endanger people's health. The immune responses in the host play important roles in the clearance of hepatitis B virus (HBV). Yet up to now, there have been few studies concerning the relations between the Th17 cells and chronic HBV infection. In this study, we investigated the role of immunoregulation by Th17 and Treg cells in the pathogenesis of chronic HBV infection, including asymptomatic carriers (AsC), chronic hepatitis B in low or moderate grade (CHB-LM), chronic severe hepatitis B (CSHB). And as controls, 10 healthy donors were included in this study..Methods:1. Relationship between peripheral Th17 cells, Treg cells and the immune or clinical state. Asymptomatic HBV carriers (AsC), chronic hepatitis B in low or moderate grade (CHB-LM), chronic severe hepatitis B (CSHB) were selected, and healthy donors were enrolled as controls. Heparinized peripheral venous blood were taken from all participants after informed consent. The cell membrane and intracellular were stained by streaming antibody. Flow cytometry were used to detecte the ratio of Th17 cells, Treg cells, Th1 cells in peripheral blood. Analysis whether the different proportion of cells is significant between the healthy control group and the different degrees of chronic HBV infection groups. Analysis whether the ratio is relevant between Th17 cells, Treg cells and immune cells ,such as Th1 cells, CD4 + T cells, CD8 + T cells , NK cells, DC cells of various samples, and whether are relevant with HBV-DNA load and liver function (ALT, AST, TB, DB).2. Correlations between the specific transcription factor of Th17 cells, Treg cells and Th1 cell, such as RORc, Foxp3, T-bet and immune response or clinical state. Real-time PCR were used to detected the mRNA of transcription factors RORc, Foxp3 and T-bet. Analyze whether the gene expression is significantly difference between HC group and varying degrees of chronic HBV infection groups. Analyze the relevance of RORc, Foxp3, T-bet in each sample, and whether is relevant with HBV-DNA load and liver function (ALT, AST, TB, DB) .3. Correlations between related cytokines in plasma and the immune response or clinical state. ELISA was used to detect the concentration of IL-17, TGF-β, IFN-γ, IL-1β, IL-6 and IL-21 in peripheral plasma. Analyze whether the difference of cytokine concentration is significantly among the healthy control group and the different immune state of chronic HBV infection groups, and whether is relevant with HBV-DNA quantification and liver function (ALT, AST, TB, DB) .Results:1. Research on the relationship between Th17 cells, Treg cells in peripheral blood and the immune status or clinical status.Th17/CD4+T% of CSHB patients was significantly higher than another groups,There were no significant among HC group, AsC group and CHB-LM group. When detected Treg/CD4+T%, we found the frequency of Treg cells of AsC group was significantly higher, compared with CSHB group(P<0.05), but there was no significant difference with other groups. Chronic HBV infection may not impact the proportion of CD4+T cells and CD8+T cells in peripheral blood, but can affect the proportion of NK/lym and DCs/lym. NK cells of peripheral blood in CSHB group was decreased, and with statistically significant difference, as compared to the HC group, AsC group and CHB-LM group (P<0.01). There were significant differences between the CHB-LM group and the HC group , between CHB-LM group and AsC group(all P<0.05). DC/lym% of peripheral blood in CHB-LM group was the highest, and with statistically significant difference, as compared with HC group(P<0.05) and CSHB group (P<0.01). Comparison of the various cells in peripheral blood: the proportion of Th17 cells and Treg cell ratio was negatively correlated (P<0.05); Th1 cells and Treg cells was positively correlated (P<0.05). Th17/CD4+T% was significantly and positively correlated with TB (P<0.01), while the Treg/CD4+T% was positive correlated with HBV-DNA quantification (P<0.01); both of them were not significantly correlated with ALT, AST (P>0.05).2. Study the relevance between the specific transcription factor of Th17 cells, Treg cells and Th1 cells, such as RORc, Foxp3, T-bet and immune response or clinical status. Compared to AsC group and CHB-LM group, the expression of RORc in CSHB group was significantly higher, with statistically differences (P<0.05), but without significant difference, compared to those in HC group. There were no significant differences for Foxp3 and T-bet among all groups, but there were positive correlation (r=0.408, P<0.05). Foxp3 and ALT were negatively correlated (r =- 0.419, P<0.05).3. Study on related cytokines in plasma and immune response or clinical status. There were no significant difference in plasma TGF-βamong the groups. To contrast the IL-17, the outcome showed that there was significant difference (P<0.05) between AsC group and HC group, AsC group and CHB-LM group. When compared the IFN-γlevels of plasma in each group found, there was significant difference between CSHB group and HC group, AsC group (all P<0.05). IL-1βand IL-6 of peripheral blood in chronic HBV infection were higher than that of in control group. There were statistically significant differences between HC group and CHB-LM, CSHB group (P<0.05), but compared with the other groups, there was no differences in AsC group (P>0.05). Compared IL-21 of plasma, there were significantly different between CSHB group and HC group, CSHB group and CHB-LM group (P<0.05). IL-17 and IL-21 was positively correlated, IL-1βwas positively correlated with the IL-6 (r=0.262, r=0.253, respectively). IFN-γwere positively correlated with liver function (ALT, AST, TB, DB), without correlation with HBV-DNA. IL-21 was positively correlated with AST, TB, DB, and was negative correlated with HBV-DNA .Conclusion: The function of Th17 cells in peripheral blood of Chronic HBV infection patients was in active state, and to a certain extent, was positively correlated with the severity of disease. Treg cells, which may lead to immune tolerance, were negatively correlated with serum HBV-DNA loads. Th17 cells in peripheral blood were negatively correlated with Treg cells. And considering the generation mechanism of these cells, we inferred that there may exist an immune balance of the two different types of cells. There was no relationship between Th17-derived IL-17 and liver cell injury, while IL-21 was positively correlated with the severity of chronic HBV infection. Our study suggested that Th17 cells may exert immune response via IL-21 in the host.