Metagenomics And Metabonomics-based Study On The Association Between Specific Gut Microbiota And The Dysfunction Of NK Cells In The Immune-tolerant Phase Of Chronic Hepatitis B Virus Infection

Objective:To reveal the association between specific gut microbiota and the dysfunction of NK cells in the immune-tolerant(IT)phase of chronic HBV infection(CHBI)bsed on metagenomics and metabonomics.Methods:1.After stool samples being collected and all microbial DNA being extracted from the stool samples,a metagenomic library was constructed.By using high-throughput sequencing technology,the genetic composition of all gut microbiota contained were analyzed.Besides,the diversity and specificity of the gut microbiota were analyzed to find out the special gut microbiota in the IT phase of CHBI.2.Based on the Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer(UHPLC-QE-MS),the metabolomic data of stool specimens in the IT phase of CHBI were obtained.Through univariate and multivariate statisticl analysis of the qualitative and quantitative results of the metabolomic data,the significantly differentiated metabolites were screened out.A series of bioinformatics analyses were performed on the significantly differentiated metabolites,such as KEGG analysis,Metabolite pathway analysis and ROC analysis.3.The Person analysis of microbiome and metabolome was used to to find out the significantly differentiated metabolites related with the significantly differentiated gut microbiota in the IT phase of CHBI.4.In vitro intervention experiments were used to explore the effects of special metabolites on the composition and proportion of PBMCs and the effect on the function of NK cells of patients in the IT phase of CHBI.Results:1.Compared with the healthy control group and the IA group,the fecal microbial community structure of IT phase presents a significantly different composition at the level of phylum,class,order,family,and genus.The abundance of harmful bacteria and opportunistic pathogenic bacteria increases,while the abundance of beneficial bacteria decreases.2.At phylum level,the significant dominant gut microbiota in IT phase of CHBI is Bacteroides;at family level are Prevotaceae(Bacteroides-Bacteroides),Bath Drechneriaceae(Proteobacteria-Pasteurellales)and Alcaligenes families(Proteobacteria-Burkholderia);at genus level are Prevotella and Alloprevotella(Bacteroides-Prevotella family),Sutterella(Proteobacteria-Alcaligenes family),Haemophilus(Degenerative-Pasteurella family),Senegalimassilia(actinomycetes-Coriobacteriaceae).3.Compared with healthy controls,a total of 85 differentiated metabolites were screened out in the stool of patients in the IT phase of CHBI,of which 57 were down-regulated and 28 were up-regulated.Among the 85 differential metabolites,45 were second-order exact-matched metabolites,of which 16 were down-regulated and 29 were up-regulated.differentiated metabolites were mainly involved in protein digestion and absorption metabolic pathways,mineral absorption metabolic pathways,glycine,serine,threonine metabolic pathways,aminoacyl t RNA biosynthetic pathways,central carbon metabolism in cancer,amino acid biosynthesis,2-oxocarboxylic acid metabolism pathway,valine,leucine and isoleucine biosynthesis.4.1-(3-Aminopropyl)-4-aminobutyraldehyde,mesna and 7-methyladenine were most closely related to the significantly differentiated gut microbiota.Among them,1-(3-aminopropyl)-4-aminobutyraldehyde and mesna were positively correlated with the special gut microbiota of patients with IA phase and healthy controls,but were negatively correlated with the special gut microbiota of patients with IT phase.However,7-methyladenine was positively correlated with the special gut microbiota of patients with IT phase,but was negatively correlated with the significant dominant gut microbiota of healthy control group and IA group.5.Mesna promoted the proportion of NK cells(CD16~+CD56~+)in PBMCs,which was prominently manifested as an increase in the proportion of NKT cells(CD3~+CD16~+CD56~+).However,7-methyladenine inbibited the proportion of NK cells(CD16~+CD56~+)in PBMCs.The reduction of the ratio of NK cells was prominently manifested as the reduction of the ratio of NKT cells(CD3~+CD16~+CD56~+).Mesna promoted the expression of IFN-γ,TNF-α,Perforin and Granzyme B in NK cells,while 7-methyladenine inhibited the expression of IFN-γ,TNF-α,Perforin and Granzyme B in NK cells.Conclusions:1.There was a disorder of gut microbiota in patients with IT phase of CHBI,which was manifested as an increase in the abundance of harmful bacteria and opportunistic pathogenic bacteria,while the abundance of beneficial bacteria decreased.2.Alloprevotell and Prevotella＿2may participate in HBV immune tolerance by producing 7-methyladenine.3.Mesna increasde the proportion of NK cells in PBMCs and enhanced the antiviral ability of NK cells.