Abnormal Bile Acid Levels Induced By Gut Microbiota Dysbiosis Inhibits The Anti-HBV Effect Of NK Cells Of HBe-positive CHB

Background and aims Chronic HBV persistent infection leads to liver damage,which can progress to liver cirrhosis,liver failure and even liver cancer.It has been reported that bile acid can promote the transcription and expression of HBV gene in liver cell lines,and bile acid can promote the biosynthesis of HBV in animal experiments,which suggest that bile acid can affect the replication of HBV in patients with chronic HBV infection,and then affect the prognosis of the disease.Moreover,it has been clear that the metabolism of bile acid is realized through enterohepatic circulation,and there is a close relationship between intestinal flora and bile acid.In addition,studies have shown that the activation of NK cells and the secretion of IFN-γ are strongly inhibited in patients with chronic hepatitis B(CHB),but the mechanism is unknown.Therefore,the purpose of this study is to explore the relationship between intestinal flora and bile acid and the immune regulation mechanism of bile acid on NK cells in patients with chronic HBV infection,so as to provide new ideas for improving the outcome and prognosis of patients.Methods1.The serum bile acid components of 20 healthy controls,20 chronic HBV carriers and30 HBeAg positive CHB patients were quantitatively detected by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)to reveal the bile acid spectrum characteristics of CHB patients.two。.The fecal samples of 15 healthy controls,15 chronic HBV carriers and 15 HBeAg positive CHB patients were analyzed by 16SV34 Miseq PE300 sequencing to reveal the distribution characteristics of intestinal flora in CHB patients.3.Flow cytometry((FCM))was used to detect the percentage and absolute value of NK cells,surface activated receptors NKG2 D,CD244 and cytokines / cytotoxic particles secreted by NK cells in 30 cases of HBeAg positive chronic HBV infection with normal serum total bile acid(TBA)level and 20 cases of HBeAg positive chronic HBV infection.The percentage of NK cells and the level of cytokines / cytotoxic granules secreted by peripheral blood mononuclear cells(PBMC)stimulated by chenodeoxycholic acid(CDCA)were detected by FCM technique to confirm the effect of CDCA on the immune function of NK cells in vitro.HepG2.2.15 cells were co-cultured with bile acidstimulated NK cells by Transwell assay,and the expression levels of HBsAg,HBeAg and HBV DNA in HepG2.2.15 were detected to evaluate the effect of bile acid on HBV replication in HepG2.2.15 cells,and FCM technique was used to detect the apoptosis of NK cells stimulated by CDCA for 24 h.FCM technique was used to detect the percentage of peripheral blood NK cells and the level of secreting cytokines / cytotoxic granules in C57BL/6 mice(carrying rAAV8-1.3HBV)after intragastric administration of CDCA for2 weeks,in order to evaluate the effect of CDCA on the immune function of NK cells in vivo.Results1.Abnormal bile acid level in HBeAg positive CHB patients caused by imbalance of intestinal flora.Our bile acid spectrum analysis showed that the level of primary bile acid increased significantly in HBeAg positive CHB patients.Although the absolute concentration of secondary bile acid increased,the percentage of secondary bile acid decreased significantly,and the ratio of primary bile acid to secondary bile acid also increased significantly.A significant decrease in the abundance of related bacteria producing 7-αdehydroxylase in the intestinal flora caused changes in the primary and secondary bile acids.2.CDCA inhibits the anti-HBV effect of NK cells.The percentage of NK cells,surface activated receptors NKG2 D,CD244 and cytokines/ cytotoxic granules secreted by NK cells were significantly decreased in HBeAg positive chronic HBV infections with elevated total bile acid levels.It was confirmed in vitro that CDCA could damage the immune effect function of NK cells secreting cytokines / cytotoxic particles and promote the replication of HBV in HepG2.2.15 cells.In vivo experiments confirmed that CDCA could reduce the percentage of mouse NK cells and damage the immune effect function of NK cells secreting cytokines / cytotoxic particles,thus promoting the replication of HBV.ConclusionsThe imbalance of intestinal flora leads to the abnormal level of bile acid in HBeAg positive CHB patients.In vivo and in vitro experiments have confirmed that CDCA promotes HBV replication by damaging the immune function of NK cells.This study suggests that the regulation of intestinal flora and bile acid levels is expected to become a new target for CHB therapy.