Association Between Th17 Cell And Related Cytokines In The Progression Of HBV Infection Related Liver Disease

【Background】Hepatitis B virus(HBV) infection is major threat to human heath. It has been recognized that dysregulation of the immune system is a major cause of chronicity of HBV infection, and associated with the progression of HBV infection-related disease. T cell subgroups and cytokines play important roles in the homeostasis of the immune response. T cells can be subgrouped into Th1, Th2, Th17 and Treg cells. Among them, Th17 is mainly involved in the proinflammatory response. IL-23 is the key cytokine that promotes the differentiation of na?ve Th cells to Th17 cells. Mature Th17 cells secret regulatory cytokines such as IL-17, IL-21 and IL-22. It has been established that Th17 and related cytokines are involved in the progression of a variety of liver diseases. Our previous studies found that IL-17 was elevated in the peripheral blood in the decompensated cirrhotic patients with HBV. IL-17 level decreased after treatment with peripheral stem cell autograft transplantation. Futher animal study suggested that IL-17 exerbated the liver damages induced by CCl4, and neutralization of IL-17 with monoclonal antibody could reduce the liver damage; exogenous IL-17 antagonized the therapeutic effect of stem cell transplantation. Therefore, it is indicated that IL-17 is major regulatory facot in liver damage and repair. Since IL-17 is mainly produced by Th17 cells, it would be interesting to elucidate the roles of Th17 cell and related cytokines including Il-23, Il-21 and IL-22 in progression of HBV infection-related liver disease and investigate whether stem cell autograft transplantation could affect this pathway. These important issues are still yet to be addressed. Objectives1. To elucidate the roles of Th17 cells and related cytokines in the progression of HBV-related liver dieases.2. To investigate the effects of stem cell autotransplantation on Th17 cells and related cytokines in HBV-related cirrhosis patients. Methods1. Study SubjectsThis study is approved by the institutional review board and ethical committee. Written informed consent was given by all subjects. The first part of this study included healthy volunteers, patients with chronic HBV infection and patients with compensated and decompensated liver cirrhosis due to HBV(50 in each group). Chronic HBV infection and HBV-related liver cirrhosis was diagnosed according to the national guidelines. The second part of the study included 20 patients with decompensated liver cirrhosis, who underwent peripheral stem cell autotransplantation in our hospital. The patients were followed up for at least one year, and had both complete baseline and follow-up information.2. Clinical samplesFor the first part of the study, 20 ml blood was drawn from each subjects under fasting condition. For the second part of the study, blood samples were collected before, 3 months, 6 months and 12 months after stem cell auto-transplantation. All samples were collected with heparin anti-coagulation tubes and were centrifuged immediately for serum. All serum samples were put into liquid nitrogen and then transferred into-80℃ freezer for future use. Mononuclear cells were isolated by Ficoll-Hypaque solution and frozed in liquid nitrogen.3. Detection methodsFor cytokine, commercial ELISA kits were used and manufacturer protocol was followed to examine the levels of IL-17, Il-21, IL-22 and Il-23 in the serum samples.For T cell subgroups, frozen cells were recovered and cultured. PMA, Inomycin and Monensin were used to treat the cells. Then, cells were labeled with CD4, IL-17, IL-22 and IFN-γ. Flow cytometry was used to study the Th17 and Th22 percentages.4. Statitistical analysisSPSS softwere(v17.0) was used. Count data were presented as mean±sd, and differences between groups were analysed with t-test. Measurement data were analysed by Kruskal-Wallis H test. p<0.05 was considered significant. Results1. Comparing with heathy volunteers, patients with chronic HBV infection and HBV-related liver cirrhosis had significant higher levels of IL-17, IL-21, IL-22 and IL-23. Patients with cirrhosis had higher levels of these cytokines than chronic HBV infection patients. They were also significantly higher in decompensated cirrhosis patients as to compensated cirrhosis patients.2. The percentage of Th17 and Th22 cells are higher in patients with chronic HBV infection and HBV-related liver cirrhosis than health volunteers. Patients with cirrhosis had higher Th17 and Th22 percentages comparing with chronic HBV infection patients. Th17 and Th22 percentages were also found to be elevated in decompensated cirrhosis patients as to compensated patients. The differences between each 2 groups are all significant.3. During follow-up, indexes of liver functions were normalized in patients receiving stem cell auto-transplantation 3 months after the treatment(including serum albumin, aminotransferases toal billirubin and MELD score).4. 3 months after the stem cell auto-transplantation, serum levels of IL-17, IL-21, IL-22 and IL-23 decreased significantly and reached the lowest at 6 month. IL-23 was positively correlated with serum levels of IL-17, IL-21, IL-22 and IL-23. ConclusionsAfter HBV infection, Th17 and the related cytokines increased as disease progresses. Perihperal stem cell autograft transplantation could significantly improve the liver functions of patients with decompensated cirrhosis induced by HBV infection. Th17 cells and related cytokines also decreased as the patient condition improved. Taken together, our findings suggest that Th17 and related cytokine may play important roles in progression of HBV-induced liver diseases. Peripheral stem cell auto-transplantion may promote the recover of liver function by downregulating the Th17 and its related proinflammatory cytokines.