The Mechanism Of Gut Microbiota Facilitating The Clearance Of Hepatitis B Virus

Hepatitis B virus(HBV)infection can cause hepatitis,liver cirrhosis,and hepatoma formation.Approximately 240 million people are estimated to be chronically infected with HBV worldwide.The International Agency for Research on Cancer within the World Health Organization has classified HBV as a "rominent human carcinogen".Persistent infection with HBV is related to the age at which HBV exposure occurs.Maternal-neonatal transmission of HBV and acquisition of HBV infection in childhood can both contribute to the chronicity of infection.More than 90%of HBV-exposed neonates and approximately 30%of children who acquire HBV infections between the ages of 1 year and 5 years cannot clear HBV,whereas approximately 95%of adult-acquired HBV infections are cleared spontaneously.This difference has been postulated to be caused by differences in immunity between young and adult humans.Compared with infants and young children,adults have a fully developed immune system that is more likely to resolve HBV infection.The human gut is populated by trillions of microbes.Commensal microbiota play an important role in human health,including modulating metabolic phenotypes,regulating epithelial development,instructing the mucosal immune system,regulating the immune response of mucosal immune system such as the gut tract and lung as well as non-mucosal immune system such as liver.Located in a unique systemic circulatory system,the liver receives blood from the hepatic artery and portal vein,making it an important site for metabolism and detoxification and immune cells to function.A large number of intestinal commensal microbial products and metabolites enter the liver through the intestinal blood supply,which contributes to a special immune microenvironment of the liver and plays an important role in regulating the antiviral immune response of the liver.A recent study has revealed that the establishment of the commensal microbiota is required for the age-related immune clearance of HBV.However,how commensal microbiota modulate the immune response to HBV remains unclear.In this regard,our study is trying to elucidate this topic by using a well-known hydrodynamic HBV transfection mouse model and treatment with antibiotics(Atb).Our major results are shown as follows:1.Establishment of microbiota-depleted mouse modelCommensal microbiota were depleted using a well-established Atb protocol.Five weeks old C57BL/6(B6)mice received 1 g/L metronidazole(Metro),ampicillin(Amp),and neomycin sulfate(Neo)as well as 0.5 g/L vancomycin(Vanco)in their drinking water for 4 weeks(Atb group)to deplete the gut microbiota.The body weight of Atb-treated mice decreased in the first week but recovered thereafter.There was no significant difference in body weight between mice receiving combined Atb in drinking water and mice drinking Atb-free water after four weeks of Atb treatment.And Atb treatment alone had no influence on liver inflammation.2.Establishment of HBV transfection mouse modelFive weeks old B6 mice were hydrodynamic injectied with 6 ?g of HBV plasmids to contruct the HBV-carrie mouse model.Five weeks old B6 mice were hydrodynamic injectied with 20 ?g of HBV plasmids to contruct the HBV-clearance mouse model.3.Establishment of microbiota-maintained anti-HBV mouse modelFive-week-old B6 mice were divided into two groups:Atb-free and Atb-treated.Mice underwent HDI(hydrodynamic injection)with 6 ?g or 20?g of HBV plasmids after drinking Atb-containing water or Atb-free water for 4 weeks.Microbiota depletion delayed HBV clearance in adult B6 of low-dose HBV transfection.4.CD4+ T cells play a critical role in microbiota-maintained anti-HBV immunityThe percentage and number of splenic and hepatic CD44hi CD62L-effector CD4+T cells,the percentages of splenic and hepatic HBsAg(126-138)+CD4+T cells and the percentages of splenic and hepatic IFN-?+CD4+ T cells after HDI of HBV plasmids were significantly increased compared to controls of pAAV plasmids.Howevre,the condition in these mice could not be maintained if they were treated with Atb.Indispensable role of CD4+T cells in microbiota-maintained anti-HBV was confirmed by utilizing Ragl-/-mice and CD4-/-mice to construct HBV transfection mouse model with or without Atb treatment.5.Impaired humoral immune response after deleption of microbiotaAdult B6 mice are able to produce the anti-HBS at 4wpi(weeks post-injection)and serum levels of anti-HBs increased with time.The percentage of GC B cells,Tfh cells in adult B6 mice HDI with HBV plasmids were significantly increased compared to control pAAV mice.However,the production of anti-HBS is impaired after depletion of the commensal microbiota,accompanying with lower percentage of GC B cells,Tfh cells than Atb-untreated B6 mice HDI with HBV plasmids.These results suggest that commensal microbiota support the differentiation of GC B cells through CD4?Tfh cells,and thereby promote the anti-HBV humoral immunity.6.Global microbial load is essential for CD4+ T cell-dependent HBV clearanceIndividual antibiotic treatment showed no significant influence on the HBV clearance compared to combined antibiotic treatment.Supplementation with TLR agonists(TLR2,TLR4,TLR9)fails to reverse the delayed clearance of HBV in adult Atb-treated mice.Deficiency of TLR2/TLR4/TLR9 pathway does not influence the promotion of HBV clearance mediated by commensal microbiota.These data suggest that depletion of the global microbiota impairs HBV clearance possibly through influencing the total load of microbiota without specifically affecting antibiotic-sensitive species of commensal bacteria.Conclusion:In adult B6 mice,the ability to clear HBV was impaired significantly after depletion of microbiota.This impairment was due to a reduction in the number of splenic and hepatic effector CD4+ T cells,lower percentage of HBV-specific CD4+ T cells as well as functional impairment of CD4+ T cells.In addition,the absence of CD4+ T cells abolished the process by which commensal microbiota facilitate HBV clearance.We found that the humoral immune response was impaired after depletion of the commensal microbiota,which was accompanied by inhibition of germinal center(GC)formation and impaired production of anti-HBS.This led to reduced efficiency of HBV clearance.Our results suggest that commensal microbiota play an important role in maintaining efficient CD4+ T-cell responses against HBV in the hydrodynamic HBV transfection mouse model.