Abnormal Platelet Kinetics In Patients With Chronic HBV Infection And The Immune Mechanism For The Associated Thrombocytopenia

Part I Abnormal Platelet Kinetics Are Detected Before the Occurrence of Thrombocytopenia in HBV-related Liver DiseaseBackground and objective:Thrombocytopenia is a frequent feature in patients with HBV-liver disease. Its underlying mechanism is not fully understood. Multiple factors might contribute to the development of the thrombocytopenia. In this study, we investigated the reticulated platelets (RP), glycocalicin (GC), serum thrombopoietin (TPO) and platelet glycoprotein (GP) in the different stages of the disease. Materials and methods:One hundred and fourteen patients with HBV-related liver disease (30 with chronic hepatitis B,20 patients in Child A without thrombocytopenia,19 patients in Child A with thrombocytopenia,45 in Child B or C with thrombocytopenia) and 25 normal controls (NC) were enrolled. Liver cirrhosis (LC) was classified according to modified Child-Turcotte-Pugh (CTP) score. Serum TPO levels and GC were measured by ELISA. RP and platelet glycoprotein (GP) CD41a and CD42a expression were detected by flow cytometry. Results:The TPO levels of patients with LC were significantly lower than that of controls, even in patients of Child A without thrombocytopenia group. Serum TPO level was positively correlated (r= 0.65, p< 0.01) with serum albumin in Child B/C group. Both the percentages of RP and the levels of GCI were significantly higher in patients groups including CHB and Child A without thrombocytopenia than that of normal controls. A negative correlation existed in HBV DNA copies and the percentage of platelet (CD41a+ CD42a+) in patients with CHB and Child A without thrombocytopenia. Conclusion Abnormal platelet production, destruction and platelet specific glycoproteins were detected before the occurrence of thrombocytopenia in HBV-related liver disease, indicating that multiple mechanisms might play roles on thrombocytopenia in HBV-infected patients, and mechanisms for thrombocytopenia potentially include impaired platelet production and increased platelet destruction.Part II Regulatory B Cells in the pathogenesis of Chronic Hepatitis B virus infectionBackground and objective:The mechanism of HBV persistence is not fully understood but is likely multifactorial including HBV-specific immune suppression, persistence of stable forms of HBV. It is now widely accepted that CD8+CTL immune response mediate the clearance of the HBV. However, patients with chronic hepatitis B show a severer impairment of HBV-specific -T-cell function. The mechanism for the T-cell dysfunction in chronic hepatitis B infection is not fully understood. Successful clearance of the virus as well as the establishment of liver disease is largely driven by a complex interaction between the virus and the host immune response. A regulatory subset of B cells has been found to modulate immune responses in autoimmunity, infection, and cancer, but it has not been investigated in the setting of human persistent viral infection. IL-10 is elevated in patients with chronic hepatitis B virus infection (CHB), but its cellular sources and impact on antiviral T cells have not been addressed. We investigated the role regulatory B cells in the pathogenesis of CHB. Materials and methods:Peripheral blood mononuclear cells from 48 chronic HBV infection including 25 patients with CHB and 23 patients with LC were were stained with CD5/CD19 antibodies for flow cytometry analyses. The HBV-specific-CTL was detected by the Pro5(?)MHC Pentamer (A*02:01/ FLPSDFFPSV Pro5(?) MHC Pentamer staining of HBV core 18-27-specific T cells) and flow cytometry. After 24 h culture in the presence of PMA, ionomycin and Brefeldin A, cells were permeabilized and stained with APC-IL-10/IFN—γ antibody to investigate intracellular IL-10 expression. Supernatant and serum IL-10/IFN-y concentration were detected by ELISA. Functional B cell assays were assessed by the magnetic cell isolation by Miltenyi Biotec. Results:The percent of CD5+B cells was elevated in patients with CHB and liver cirrhosis than the normal control. The level of the IFN-γ, IL-10 concentration were increased in the patients with CHB. Percentage of IL-10+ cells and intracellular IL-10 in CD5+B cells from CHB and LC patients were significantly higher than that in controls. IL-10-producing B cells were enriched in patients. Among the 48 patients, there are 13 patients with HLA-A2 positive and there were 7 patients with detected HBV-specific-CTL. After sorting the CD8+cell and CD5+CD19+cell and culturing them together, CD5+CD19+ cells suppressed HBV-specific CD8+ T cell and non-HBV-specific CTL responses in the 5 patients out of the 7 patients with detected HBV-specific-CTL. Conclusion:These data reveal a novel IL-10-producing subset of B cells may be able to regulate the function of non-HBV-specific and HBV-specific CTL.Part Ⅲ The Immune Mechanism for the Associated Thrombocytopenia in the Patients with Chronic HBV infectionBackground and objective:Thrombocytopenia is the frequent feature of the HBV-related liver disease. About 37% patients with HBV-related liver disease present thrombocytopenia. Abnormal platelet kinetics were detected before the occurrence of thrombocytopenia. The mechanisms for abnormal platelet kinetics in the CHB and Child A without thrombocytopenia are not fully understood. We aim to investigate the role of cellular immunity and the CD5+B cells on the thrombocytopenia associated chronic HBV infection. Materials and methods: Peripheral blood mononuclear cells from 88 cases including 25 patients with CHB and 12 Child A without thrombocytopenia and 13 Child A with thrombocytopenia and 18 patients with Child B/C and 20 normal control. Patients were stained with CD5/ CD 19 antibodies for flow cytometry analyses. The HBV-specific-CTL was detected by the Pro5(?) MHC Pentamer (A*02:01/FLPSDFFPSV Pro5(?) MHC Pentamer staining of HBV core 18-27-specific T cells). After 24 h culture in the presence of PMA, ionomycin and Brefeldin A, cells were permeabilized and stained with APC-IL-10/PE-IFN-γ antibody to investigate intracellular IL-10/IFN-γ expression. Supernatant and serum IL-10 and IFN-γ concentration was detected by ELISA. Functional B cell assays were assessed by the cell sorting method. Results:Platelet count presented the negative association with the percent of the IFN-γ+CD8+cell in the CHB patients (r=-0.51, p= 0.01). Regardless of the CHB or LC group, platelet count showed the negative association with the percent of the CD8+CD107a (CHB, r=-0.57, p= 0.03; LC, r=-0.50, p= 0.03). The percentage of CD5+B cells was elevated in patients with CHB and liver cirrhosis than the normal control. Further comparison among the patients group, the patients in Child B/C showed a significant increase compared with the CHB and Child A with/without thrombocytopenia. Intracellular IL-10 in CD5+B cells from all the group patients were significantly higher than that in controls. In contrast, patients with Child B/C showed lowest IL-10 concentration in supernatants among the patients groups. Conclusion:The mechanisms of the associated thrombocytopenia in chronic HBV infection involve multifactors. The CTL cell plays a role for thrombocytopenia in stage of CHB. The increased percentage of CD5+B cells in which IL-10 is accumulated with decreased IL-10 concentration in supernatants in patiens with Child B/C suggests that the ability of CD5+B cells to secret IL-10 is impaired in patients with decompensated liver cirrhosis,which may be involved the mechanism for the thrombocytopenia in the Child B/C.