Immunological Effect Of Follicular Dendritic Cells In Chronic HBV Infection

BackgroundChronic hepatitis B virus(HBV)infection is an immune-mediated disease.The clearance of HBV in the host is mainly dependent on the immune function of cellular immunity and antibody-mediated humoral immunity.In recent years,the role of antibody-mediated humoral immunity in HBV clearance has gained increasing attention.Previous research show that there are ectopic germinal center formation and maturation of follicular dendritic cells(FDCs)in the liver after HCV infection.In the process of HBV infection,cellular immunity directly kills liver cells and deposition of HBsAg-anti-HBs immune complexes can lead to increased intrahepatic inflammation which can promote the maturity of FDCs.FDCs can recognize immune complexes independent of T cells and present antigens to B cells,promoted B cell proliferation,differentiation and antibody secretion.The germinal center(GC)is a complex,highly dynamic microenvironment formed after infection.GC is closely related to the production of high-affinity antibodies,plasma cells and differentiation of memory B cells.FDCs are non-bone marrow-derived antigen-presenting cells present in secondary,tertiary lymphoid follicles.As the skeleton of the GC,the FDCs network participates in the composition of the germinal center and is necessary for the formation of the germinal center and the maintenance of the reaction.FDCs can present antigens,load immune complexes,secrete IL-6,IL-7 and other cytokines,secrete CXCL13 and other chemokines,and express adhesion molecules ICAM-1,VCAM-1 and B cell nutrition molecules BAFF.In the dark zone of GC,FDCs promote B cell proliferation and somatic hypermutation.In the light zone of GC,FDCs can make high affinity selection for B cells.FDCs promote the production of memory B cells and plasma cells,regulate immunoglobulin conversion,play an important role in autoimmune diseases and lentivirus infections.At present,the immune function of FDCs in viral infection is mainly about the HIV infection.FDCs have been shown to enhance HIV infection.FDCs can directly interact with the HIV envelope protein gp120 through the C3 complement pathway,and endocytosis of HIV into the cells for a long time to escape the immune surveillance of the body.But because the number of FDCs is extremely small and brittle,there are relatively few studies on the immune effects of FDCs in other viral infections,especially in chronic HBV infection.ObjectiveIn this study,the study subjects were HBV-infected patients who were not treated with antiviral drugs and patients with HBV-related liver cancer or splenomegaly who needed surgery.We analysis the characteristics of FDCs in chronic HBV infection.The immunological effects of FDCs in chronic HBV infection were investigated by studying the differences in the frequency of FDCs in different tissues and organs in patients with HBV infection,combined with in vitro experiments to study the interaction between FDCs and B cells and T cells.Methods1.Differences in the frequency of FDCs in different tissues and organs of HBV infection1.1 Patients with chronic HBV infection who did not take antiviral drugs were selected and PBMC were isolated after peripheral blood was drawn.Patients with HBV-infected liver cancer or splenomegaly who require surgery should receive liver tissue and spleen tissue,and tissue lavage,tissue lymphocytes were isolated,and FDCs,memory B cells,plasmablasts,etc.were phenotypically labeled.The frequency of relevant cells in peripheral blood,liver and spleen was detected by flow cytometry,and statistical analysis was performed according to different stages of HBV infection.1.2 ELISA method to detect the expression of IL-6,IL-7,IL-15,CXCL13,BAFF and other cytokines in plasma of HBV-infected patients,combined with clinical information and related serum and viral indicators(ALT,AST,HBV DNA,HBeAg,HBsAg,etc.),statistical analysis of FDCs and their correlation.2.In vitro stimulation of FDCs secreting cytokinesPatients with spleen resection with chronic HBV infection-related cirrhosis were selected,spleen tissue blocks were collected,and FDCs cells were isolated and cultured and verified by immunofluorescence experiments.Then,different concentrations of IL-2,IL-4,IL-10,IL-21,LPS,IFN-2α,PMA,CpG,CD40L,LTα1β2,TNF-α were used to stimulate the FDCs cultured in vitro,and the upper layer of the cell culture was collected.The expression of IL-6,IL-7,IL-15,BAFF and CXCL13 was detected by ELISA.3.Study on the immunomodulatory effects of FDCs on T cellsCD4~+T cells and CD8~+T cells were sorted by magnetic beads.The sorted T cells were co-cultured with FDCs,and the corresponding stimulators were added for 3 days.After 3 days,the expression levels of IL-21 and IFN-γ in CD4~+ T cells were detected by ELISA.The magnetic beads were sorted into CD4~+T cells and CD8~+T cells.The sorted T cells were co-cultured with FDCs,and the corresponding stimulators were added for 3 days.After 3 days,the expression levels of IL-21 and IFNy in CD4~+T cells and CD8~+T cells were detected by ELISA.4.Study on the interaction between FDCs and B cells4.1 CD19~+B cells were sorted by magnetic beads.The sorted B cells were co-cultured with FDCs,and the corresponding stimulators were added for 3 days.After 3 days,the expression levels of IL-6,IL-7,IL-15,BAFF and CXCL13 in FDCs were detected by ELISA.4.2 CD19~+ B cells were sorted by magnetic beads.B cells were labeled with CFSE,and then co-cultured with FDCs for 7 days.The proliferation of B cells was detected by flow cytometry.Results1.Differences in the frequency of FDCs in different tissues and organs of HBV infection1.1 FDCs are not only found in lymphoid organs,but also very small amounts of FDCs are found in peripheral blood and liver.The frequency of FDCs in the spleen was significantly higher than that in the liver and peripheral blood(p<0.005,p<0.0005).The frequency of FDCs in the liver was significantly higher than that in the peripheral blood(p<0.0005).The frequency of FDCs in peripheral blood of HBV infected patients was higher than that in healthy people.The frequency of FDCs in the IC group was significantly higher than that in the IT group(p<0.0005).1.2 The frequency of FDCs in HBV-infected patients was positively correlated with serum levels of ALT(p=0.0024,r=0.7359)and AST(p=0.0024,r=0.7365),and HBsAg(p=0.782,r=0.075).There was no correlation between e antigen HBeAg(p=0.714,r=0.103)and e antibody HBeAb(p=0.1202,r=0.4528),and positive correlation with plasmablast frequency(p=0.001,r=0.8233),and naive There was no significant correlation between B cells(p=0.2894,r=0.3335)and memory B cells(p=0.1521,r=0.4402).1.3 In HBV infection,the plasma levels of IL-7 in CHB group were significantly higher than in the IC group(p=0.039),but there is no significant differences were observed in different stages of HBV infection about the plasma levels of IL-6,IL-15,CXCL13 and BAFF.There is a positive correlation between the plasma levels of IL-15 and the frequency of FDCs in patients with HBV infection(r=0.36,p=0.02).2.In vitro stimulation of FDCs secreting cytokinesIsolation and culture of FDCs in the spleen of HBV patients,stimulated with different cytokines in vitro,found that in the autocrine of FDCs,high expression of IL-6,can secrete CXCL13 and IL-7,but BAFF and IL-15 expression is extremely low.Under cytokine stimulation,LPS can stimulate FDCs to express IL-6,IL-4,IL-10 and TNFα can also promote the expression of IL-6 in FDCs,but IL-21 can inhibit the expression of IL-6 in FDCs.In addition,FDCs can produce a small amount of CXCL13 under the stimulation of LTαlβ2.3.Study on the immunomodulatory effects of FDCs on T cellsFDCs were isolated from the spleen of HBV-infected patients were co-cultured with T cells.It was found that FDCs can promote the secretion of IL-21 and IFNγ by T cells.Among them,in the co-culture system without any irritant,T cells expressed higher IL-21,and after the addition of the stimulant,the secretion of IL-21 by T cells was inhibited.In addition,the levels of IFNγ in the FDCs and T cell co-culture groups were significantly higher than those in the T cells alone culture group.4.Study on the interaction between FDCs and B cellsFDCs were isolated from the spleen of HBV-infected patients were co-cultured with B cells.It was found that FDCs interacted with B cells in vitro,and B cells could not promote FDCs fun ction.FDCs may not be able to stimulate without antigen stimulation.Promote the proliferation and differentiation of B cells.Conclusions1.FDCs were found in the liver and peripheral blood of HBV-infected patients,and the frequency of intrahepatic FDCs was significantly higher than that in peripheral blood.The frequency of FDCs was positively correlated with ALT,AST level,plasmablast frequency and the plasma levels of IL-15 which suggesting that FDCs are involved in chronic HBV infection.Play a certain immune role.HBV infection may promote intrahepatic ectopic germinal center formation and FDCs maturation through intrahepatic inflammation,which in turn affects humoral immune responses,especially intrahepatic immunity.2.FDCs in the spleen of HBV-infected patients can express other cytokines such as IL-6 and CXCL13 in vitro stimulation.In vitro co-culture of FDCs and T cells can promote the secretion of IL21 and IFNγ by CD4~+T cells,suggesting that in chronic HBV infection,FDCs may promote T cell antiviral effects by secreting cytokines.3.In vitro co-culture of FDCs and B cells in spleen of patients with HBV infection showed no significant interaction between FDCs and B cells,suggesting that FDCs promote B cell proliferation,affinity selection and immune regulation of B cells during HBV infection.It may depend on the presence of antigenic stimulation or the structure of the lymphoid follicular germinal center.