Immunomodulatory Effect Of IL-21 In HBV Mouse Models

Background and AimsHepatitis B is a liver disease in the human body caused by the hepatitis B virus(HBV)through blood transmission,mother-to-fetus transmission,sexual transmission etc.,and that significantly affects the health of people around the world.By the statistics,about 2 billion people have a history of suffering from HBV infection around the world.In China,as a high-prevalence area of hepatitis B,the population with HBV infection has reached 5%to 7%,making it a medical issue necessitating our urgent attention[l].HBeAg seroconversion is a phenomenon that HBeAg disappears from and then HBeAb appears in the serum of a HBeAg-positive patient with chronic hepatitis B.It is found that better long-term prognoses and drastically decreased incidence of the complications of hepatitis B,including cirrhosis and hepatocellular carcinoma occur in HBeAg-positive patients with chronic hepatitis B achieving HBeAg seroconversion[2-5].Therefore,the major international guidelines for hepatitis B management by EASL and AASLD include HBeAg seroconversion as an indicator of drug withdrawal for HBeAg-positive patients with chronic hepatitis B in antivirus treatment[6-8].In our preliminary experiment,the cohort study showed a significant increase of serum IL-21 in the HBeAg seronconversion group using telbivudine at 12 weeks,indicating a certain relationship of IL-21 with HBeAg seroconversion[9].Interleukin-21(IL-21)is a cytokine produced by activated CD4+ T cells,mainly including T follicular helper cells(Tfh)and Th17 cells.As one of the IL-2 family,IL-21 contains the same gamma chain CD 132 as that of the receptors of the cytokines of this family,such as IL-2,IL-4,IL-7,IL-9 and IL-15[10-13].The wide distribution of IL-21R on various cells enables it to function in different aspects through these cells.It can accelerate the differentiation of B cells into plasmacytes to produce immune globulin and promote the immune globulin class switching and enhance the cytotoxic effect of NK cells and CD8+ T cells by boosting their production of perforin and granzyme[10,14,15].Research showed the significance of IL-21 in virological control over chronic virus infection.CD4+ and CD8+ T cells will be partially exhausted in the process of chronic virus infection,and IL-21 will be produced by CD4+ T cells in quantity at the later stage of the infection to act on memory CD8+ T cells,facilitating their proliferation,enhancing their killing effect,and therefore enabling virological control[16].Based on existing studies and our exploration of the relationship between IL-21 and HBeAg seroconversion,evidence shows IL-21 exerts immunoregulatory effect in the process of HBV infection and consequently facilitates disease outcome,and this is also a research hotspot over the past few years.Preliminarily,we observed that level of IL-21 was higher in chronic hepatitis B group than that in control group suggesting the phenomenon that chronic hepatitis B patients had high level of IL-21 probably was one kind of protection defensing HBV.Our vitro study also indicated that Tfh cells in circulation blood facilitate HBeAg seroconversion by generating IL-21.But clinical study have many limitation,such as disable to intervene in vivo and susceptible to personal gene background,different infected time and various pathogenicity of HBV.Therefore,it is necessary to utilize animal models that have genetic uniformity and can be intervened for further research.In present,studies about IL-21 in HBV infected mice models are not much.So we conducted this study utilising mouse models of HBV infection and focusing on the action mechanism of IL-21 in HBV infection.The mouse models of HB V infection mainly include the HBV-transgenic mouse,the human liver chimeric mouse model,plasmid-based transfection mouse model,virus-based transduction mouse model.The HBV-transgenic mouse is a model in which HBV genes are integrated into the genome of mouse fertilized eggs through embryo injection.The human liver chimeric mouse model is a model established by transplanting human fetal hepatocytes and/or immune cells into fetal mice and injecting the serum of HBV-infected patients into mice.The HBV transfection mouse model is a model established by injecting HBV gene-containing vectors through intravenous hydrodynamic transfection.The HBV transduction mouse model is conducted by commonly intravenously injecting HBV gene-containing hepatotropic virus vectors through viral vector transduction[17,18].With a view to the features of each of these models and for the purpose of this experimental study,rAAV8-HBV1.3 and pSM2 was selected to establish mouse models of hepatitis B.It is reported that IL-21 knockout mice and IL-21 R knockout mice served to establish chronic lymphocytic choriomeningitis virus(LCMV)infection models and found the mice with such defects are more vulnerable and sensitive to chronic virus infection and have a more protracted course of disease than normal mice[19,20].This background study enlightened us with a new way of thinking about our study.Therefore,based on the investigation into the action mechanism of IL-21 in HBV mouse models,this study was carried out along two lines.One is to build a mouse model of HBV infection by using IL-21R knockout mice and wild-type mice,the other line is to exogenously add IL-21 and rIgG control into the mouse models of HBV infection,so as to preliminarily investigate the mechanism of the immunoregulatory effect of IL-21 on HBV infection in virology and immunology.Methods1.To construct Mouse models of HBV infection.Forty-two wild-type mice were evenly divided into two groups that were given lOug pSM2 plasmids(experimental group)and 10ug pUC19 plasmids(control group)respectively via hydrodynamic tail vein injection.The process for the established models lasted for 28 days.The mice’s venous blood was collected every 2 days in the period from day 1 to 28 after model establishment.On day 28,the mice were sacrificed for liver tissue collection.1)The serum from all blood collection sites was detected by ELISA to observe the levels of HBsAg,HBsAb and IL-21;2)flow cytometry was employed to detect the frequencies of CXCR5+ CD4+ T cells and CD19+B cells in peripheral blood on days 0,7 and 28 after model establishment;3)real-time fluorescence-based quantitative PCR was used to detect the level of IL-21 mRNA in the liver;4)flow cytometry was used to detect the frequencies of CXCR5+CD4+T cells and CD19+B cells in the liver;5)immunohistochemical method was adopted to determine the expression of HBcAg in the liver;6)HE staining of liver tissue was conducted to observe intrahepatic inflammatory infiltration.2.IL-21 R knockout mice and pSM2 plasmids were used to establish mouse models of HBV.Dynamic observation was made of virological and immunological changes to explore,from the perspective of the absence of IL-21,the effect of IL-21 on HBV infection.IL-21 R knockout mice were given lOug pSM2 plasmids via hydrodynamic tail vein injection.Meanwhile,wild-type mice were used to establish models as the control group.Each of the two groups comprised more than 10 mice.The process for the established models lasted for 28 days.The mice’s venous blood was collected every 2 days in the period from day 1 to 28 after model establishment.The mice were sacrificed to collect their liver and spleen tissues respectively on days 7,14,21 and 28 after model establishment.1)The serum from all blood collection sites was tested by ELISA to observe the levels of HBsAg,HBsAb and IL-21;2)real-time fluorescence-based quantitative PCR was used to detect the level of IL-21 mRNA in the liver on day 28 after model establishment;3)lymphocytes were separated from the liver and spleen and co-cultured with HBV overlapping antigen peptide pools for 5h simulation,and then flow cytometry was employed to detect the function of HBV-specific T cells in the liver and spleen collected at the four time points of sacrifice.3.Mouse models of HBV were established and given mouse-derived IL-21 to observe whether IL-21 can facilitate virus removal.Twenty wild-type mice were given 200ul(2×1010 vector particles)recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome(rAAV8-HBV1.3)per mouse via routine tail vein injection to establish mouse models of HBV.At 4 weeks,they were divided into two groups based by serum HBsAg levels that were accordingly given mouse-derived IL-21 as experimental group and rIgG as control group by intraperitoneal injection once every other day.At 24 days after injection,they were observed for another 24 days.During the 48 days,venous blood was collected for testing of dynamic change in serum HBsAg every 6 days by ELISA.4.Statistical analysis.SPSS 20.0 and Graphpad Prism 5.0 were used for data analysis and drawing.Measurement data were expressed as mean ± standard deviation or interquartile range.If measurement data were in accord with normal distribution and homogeneity of variance,a parameter test was conducted;if not,a non-parametric test was performed.For intergroup comparison of means,independent-samples t-test or Mann-Whitney U test;spearman correlation was applied to the test of relation between two variables;all statistical analysis was based on a two-tailed hypothesis test,while a=0.05 was taken as significant level,and P<0.05 meant a statistically significant difference.Results1.Mouse models of HBV were successfully established with pSM2.The frequencies of CXCR5+CD4+T cells and CD19+B cells as well as the level of IL-21,in the peripheral blood and livers of the models,increased,and then HBsAg was removed and HBsAb appeared gradually.For the mouse models of HBV established with pSM2,serum HBsAg in all mice was removed on day 13,HBsAb began to be produced in the serum of several mice on day 7,and HBcAg expression was found in the intrahepatic immunohistochemical test on day 28.Compared with the control group,the level of serum IL-21 of the experimental group began to increase gradually on day 7 when serum IL-21 level was obviously higher than the baseline(P<0.05),and then peaked on day 28.Through real-time fluorescence-based quantitative PCR,it was found that the level of intrahepatic IL-21 of the experimental group was obviously higher than that of the control group(P<0.05).In further analysis,the frequencies of CXCR5+ CD4+ T cells and CD19+ B cells in periphery blood on days 7 and 28 and those in liver on day 28,were found higher than those at the baseline while the control group had no obvious changes(P<0.05).HE staining indicated that the mice given pSM2 by injection had intrahepatic lymphocyte infiltration compared with the control group.2.Compared with the wild-type mice,the virus removal delayed and no antibody could be produced in the mouse models of HBV established from IL-21Rknockout mice through the injection of pSM2 plasmids.Through the ELIS A of the mice’s serum HBsAg and HBsAb,it was found that in the IL-21R knockout mice,serum HBsAg continued to exist for a longer period of time,and from day 10,the level of HBsAg was significantly higher than that of the wild-type mice(P<0.05);the HBsAg expression in serum of several IL-21R knockout mice could last for more than 28 days.Compared with the wild-type mice,however,HBsAb in all IL-21R knockout mice could not be produced and on day 22,was obviously lower(P<0.05).The expression of IL-21 in the two groups was observed and no significant difference was found in the level of serum IL-21 between the two kinds of mice at the early stage,but after day 22,the level of serum IL-21 in the IL-21 R knockout mice was obviously lower than that in the wild-type mice(P<0.05);also the level of intrahepatic IL-21(P<0.05).Through a further analysis of the relation between HBsAg/anti-HBsAg and IL-21 in serum,IL-21 was found to be negatively correlated to HBsAg(r=-0.222,P=0.001)and positively correlated to HBsAb(r=0.355,P<0.001).After HBV specific lymphocytes was detected by flow cytometry on days 7,14,22 and 28,it was found that the function of HBV-specific T cells in liver and spleen peaked on day 28 and then gradually declined.Notably,except for there being no statistical difference in the frequency of intrahepatic CD4+IFN-γ+T cells between the two groups on day 28,the proportions of the CD4+ and CD8+ cell subgroups in secreting interferon gamma(IFN-γ)specific to HBV antigen peptide were higher in the liver and spleen of the wild-type mice than those in the IL-21 R knockout mice on days 22 and 28(P<0.05).3.Serum HBsAg in the mice gradually decreased,while the mouse models of HBV were given mouse-derived IL-21.On day 6 after the mouse models of HBV were given mouse-derived IL-21,serum HBsAg in the models significantly decreased compared with the control group(P<0.05),but subsequently changed little and remained at a low level until day 24 after discontinuation of rmIL-21,and during observation,the above-mentioned serum HBsAg from each blood collection site was obviously lower than that in the control group(P<0.05).Conclusions1.IL-21 can promote HBV antigen removal and antibody generation in mouse models injected with pSM2;2.Compared with the wild-type mice,HBV antigen removal delayed and HBsAb could not be produced in the IL-21 R knockout mice through hydrodynamic tail vein injection of pSM2 plasmids.3.Giving mouse-derived IL-21 can promote HBsAg removal in HBV mouse model established by tail vein injecting rAAV8-HBV1.3.