Clinical Research On The Function And Immune Regulatory Mechanism Of HBV Antigen Specific Th17 Cell In Chronic Hepatitis B Patients

Hepatitis B virus (HBV) is a DNA virus and can cause hepatitis B in humans. Currently, HBV has infected more than 400 million people worldwide and HBV infection can result in chronic hepatitis B (CHB), eventually leading to the development of liver cirrhosis and hepatocellular carcinoma. During HBV infection, HBV antigens, such as HBsAg, HBcAg, induced cell-mediated immune responses, especially multispecific antiviral helper T cell (Th) and cytotoxic T lymphocyte (CTL) responses are essential for the control of virus infection. Unfortunately, patients often exhibit impairment of HBV-specific T cell activity during HBV infection thus developing into a persistent, chronic state. Indeed, little is known about the factors that impair the development of HBV-specific T cell immunity, particularly in CHB patients.Recently, the Th1/Th2 dichotomy has been revisited and the third member of the CD4+Th cell family, now widely known as Th17 cells, has substantially advanced our understanding of T cell-mediated immunity. The development of Th17 cells is regulated by IL-6 and TGF-βand dependent on STAT3-dependent expression of the transcription factors RORγt and RORα. Th17 cell responses have been demonstrated to be critical for inflammatory responses to tumors and contribute to the pathogenesis of many autoimmune diseases. Furthermore, antigen-specific Th17 cell responses have been associated with the viral clearance of HCV infection, and the high frequency of Thl7 cells in periphery was associated with disease progression and liver damage in CHB patients. However, the factors that regulate virus-specific Th17 cell responses during the development of CHB in humans are poorly understood.In the present study, we applied PBMC from CHB patients to analyze the frequency and cytokine production of Th17 cells in HBV infected patients, and also to evaluate the potential association between the frequency of Th17 cells and the levels of liver injury. Furthermore, we determined the role of IL-10 and CD4+CD25+Treg cells in HBcAg-specific Th17 cell responses and potential mechanism(s) underlying the action of IL-10/CD4+CD25+Treg cells in the development of HBcAg-specific Th17 cells in vitro. Thus understanding the factors that regulate the antiviral T cell responses is essential for devising rational strategies to prevent or combat this highly prevalent infection.This study includes three parts.Part I Clinical research of Th17 cell responses in Chronic Hepatitis B patientsObjectivesTh17 cells have been shown to mediate host defensive mechanisms in various infections, but their role in HBV infection in humans has not been well characterized. In this study, we analyzed the frequency and cytokines secretion of circulating Th17 cells in HBV infected patients with different statuses, and also evaluated the potential association of Th17 frequency with the levels of liver injury. MethodsThe study population consisted of 113 subjects, including 40 mild chronic hepatitis B (CHB) patients,37 severe CHB patients and 36 healthy controls. The frequency of circulating Th17 cells were carried out by intracellular cytokine staining analysis and serum IL-10 levels were measured by ELISA.Results1. Severe CHB patients had a significant increase of Thl7 cells frequency in peripheral blood compared with mild CHB patients and healthy control (both P<0.05).2. Tc17 cells frequency in peripheral blood from severe CHB patients was much higher than that in healthy control (P<0.05).3. The elevated prevalence of Thl7 cells is positively associated with the increased serum ALT levels in severe CHB patients (r=0.457, P=0.004).4. The serum IL-10 were negatively correlated with the frequency of Th17 cells in PBMC from patients with chronic HBV infection (r=-0.452, P<0.01).ConclusionsTh17 cells may contribute to the disease progression and pathogenesis of liver injury in HBV infected patients, and the induction of IL-10 may be one mechanism of constraining pro-inflammatory Th17 responses.PartⅡInterleukin 10 Inhibits HBcAg-specific Th17 Responses in Chronic Hepatitis B patientsObjectivesT helper (Th) 17 cells have been demonstrated to play an important role in host defense against viral infection. However, little is known about the regulation of Th17 cells in HBV infections. MethodsPeripheral blood mononuclear cells isolated from chronic hepatitis B (CHB) patients were simulated with anti-IL-10 antibody or recombinant IL-10, respectively. The frequency of hepatitis B core antigen (HBcAg)-specific Th17 cells was characterized and produced cytokines were determined by flow cytomix.Results1. A low frequency of HBcAg-specific Th17 cells and high frequency of Thl cells were detected in CHB patients.2. HBcAg stimulation promoted IL-17A, IL-22, IL-23, IL-6, TGF-βand IL-10 production by PBMC from CHB patients, but not from normal controls.3. Endogenous IL-10 inhibited HBcAg-stimulated production of IL-17A, IL-22, IL-6 and IL-23, but not TGF-p.4. Treatment with IL-10 inhibited the HBcAg-stimulated activation of Th17 cells while anti-IL-10 antibody significantly increased the frequency of Th17 and Thl cells, but not that of CD4+CD25+regulatory T cells, associated with up-regulating RORyt expression in CD4+T cells.ConclusionsHBcAg stimulated the production of IL-10, which negatively regulated HBcAg-specific Th17 cell responses in CHB patients. Our findings may represent one evasion strategy for HBV to subvert specific antiviral responses in humans.PartⅢCD4+CD25+regulatory T Cells Modulate Th17 cell Responses in Chronic Hepatitis B patientsObjectivesIL-17-secreting CD4+T (Th17) and CD4+CD25+regulatory T (Treg) cells have been demonstrated to associate with disease progression and liver damage in chronic hepatitis B (CHB) patients. However, little is known about the regulation role of CD4+CD25+Treg cells on IL-17-secrecting T cells in HBV infections.MethodsIn the present study, the frequencies of CD4+CD25+Treg/IL-17-secrecting T cells in CHB patients and healthy subjects were measured by flow cytometric analysis. Furthermore, the role of CD4+CD25+Treg cells on Ag-specific IL-17-secrecting T cell differentiation was determined by interfering Foxp3 gene expression or deleting CD4+CD25+Treg cells from peripheral blood mononuclear cells (PBMC) in CHB patients.Results1. Both frequencies of Th17 cells and CD4+CD25+Treg cells were increased in the peripheral blood of CHB patients compared with healthy controls (CD4+CD25+Treg cells:8.2±4.29% V.S 6.59±1.56%; P<0.01; Th17 cells:1.4±0.59% VS.1.16±0.36%, P<0.01 by the student T test).2. The frequency of CD4+CD25+Treg cells was significantly associated with Th17 cells in CHB patients (3.71±0.53% VS.6.13±0.52%, P=0.0057 by the student T test).3. Deletion of CD4+CD25+Treg cells or Foxp3 RNAi significantly enhanced cell proliferation of Th17 cells in PBMC following rHBcAg stimulation.ConclusionsThese results show that HBcAg-specific Th17 and Tcl7 cell responses are suppressed by CD4+CD25+Treg cells in CHB patients. Our data may demonstrate one strategy for HBV to regulate specific antiviral immune responses in HBV infection humans. 1. CHB patients had a significant increase of Th17 and CD4+CD25+Treg cells frequencies in peripheral blood compared with healthy control. Furthermore, the frequency of CD4+CD25+Treg cells was significantly associated with Th17 cells in CHB patients. Also, the elevated prevalence of Th17 cells is positively associated with the increased serum ALT levels in severe CHB patients2. HBcAg stimulation not only promoted Thl and Th17 cell responses in CHB patients, but also induced the secrection of IL-10. IL-10 negatively regulated HBcAg-specific Th17 cell responses in CHB patients through inhibiting HBcAg-stimulated production of IL-6, as well as reducing the expression of RORyt in CD4+T cells,3. CD4+CD25+Treg cells negatively regulated HBcAg-specific Th17 cell responses through inhibiting the expression levevl of Foxp3 in CHB patients.