Comparisons Of Gut Microbiota Profiles Between Wild-type C57BL/6J Mice And HLA-DRB1*1202 Mice

Intestinal flora plays a key role in maintaining homeostasis in the host.Intestinal tract is the important place of human physiology,immune system development,digestion,fat storage,regulation of angiogenesis,behavior,development and detoxification.It has a far-reaching impact on human health.Intestinal microflora disorders are associated with a variety of intestinal diseases.Recent studies have found that intestinal microflora is closely related to many autoimmune diseases,including autoimmune hepatitis,spondylitis,multiple sclerosis and so on.Previous studies have shown that genetic factors can influence the composition of intestinal flora.Williams Turpin et al.found that one third of the intestinal flora was heritable.They identified 58 host SNPs associated with the abundance of 33 bacterial flora.Bonder et al.studied 1514 samples of Dutch population and identified the loci affecting human intestinal microflora.It was found that there were some SNPs related to intestinal microflora in MHC loci,such as those located near the MUC22 gene,suggesting that HLA-II molecules could regulate intestinal microflora and lead to structural and functional changes.In many HLA transgenic mice models,it has been confirmed that HLA is closely related to intestinal microflora.For example,studies have shown that in HLA-DR3_AIH model,intestinal microflora has changed significantly,but not in wild-type mice,which means that different HLA alleles may lead to different composition and structure of microflora.Several GWAS studies based on a large sample of patients have found that the genetic variation of HLA-II molecule determines the infection/clearance of HBV in the global scope of genetic factors.Our previous GWAS study of HLA-DR in patients with HBV-related ACLF showed that HLA-II molecule DR/DQ was the key molecule to determine the severity of hepatitis B injury,haplotype DRB1*1202 played an important role in the individual immune injury of chronic HBV infection,and genotype DRB1*1202 was the immune injury type.Bacterial infection can induce or aggravate renal failure,hepatic encephalopathy and multiple organ failure through systemic inflammatory response synDRome,which is one of the common complications and important causes of death in ACLF patients.Our previous studies of intestinal flora characteristics in patients with HBV-related chronic and acute liver failure based on high-throughput sequencing showed that intestinal flora structure and abundance evolved in different severe stages(mild hepatitis,severe hepatitis,chronic and acute liver failure)caused by acute exacerbation of chronic hepatitis B,and pathogenic bacteria showed a significant increase in patients with ACLF.Therefore,combining our previous research findings we suspect that haplotype DRD1*1202 has some unknown relationship with intestinal floraThe purpose of this study was to investigate the difference of intestinal microflora structure between wild C57BL/6J mice and HLA-DRB1*1202 transgenic mice.DRB1*12:02 transgenic mice are mice models with HLA-DRA1-DRB1 1202 gene knocked into at chromosome 6 in the background of C57BL/6Jmice.Both genotypes of mice were raised under the same conditions.And in order to remove the effect of different cages,we designed the co-housing experiment.Microbial analysis was performed with Illumina MiSeq and the intestinal microbial components were compared and correlated.By analyzing the composition and structure of the microflora,we revealed the different characteristics of intestinal microflora between wild C57BL/6JBL/6J mice and HLA-DRB1*1202 transgenic mice.Our results are as follows:1.In the separate-housing experiment,(1)Intestinal flora composition and structure analysis(PCoA)showed that: DRB1*1202 mice intestinal flora composition and structure were significantly different from C57BL/6J wild-type mice(Adonis R2 = 0.32,P = 0.001);(2)Community richness and diversity analysis showed that DRB1*12:02 transgenic mice were significantly different.(3)Species composition of intestinal flora showed that the abundance of Bacteroidetes and Deferribacteres in intestine of DRB1*1202 mice was significantly lower than that of C57BL/6J wild mice(P=0.046;P=0.0002),while the abundance of Cyanobacteria increased(P=0.0404);family level analysis showed that the abundance of Bacteroidetes and Deferribacteres in intestine of DRB1*1202 mice was significantly lower than that of C57BL/6J wild mice(P=0.046;P=0.0002).The abundance of Prevotellaceae,Alcaligenaceae and Veillonellaceae was significantly lower than that of C57BL/6J wild-type mice(P=0.0001;P=0.046;P=0.00001),Rikenellaceae and Gastranaphileroales increased(P=0.019;P=0.04).Genus level analysis showed that the abundance of Prevotellaceae UCG-001,Parasutterella,Desulfovibrio and Mucispirillum was significantly lower than that of C57BL/6J wild mice(P=0.00003;P=0.046;P=0.0001;P=0.00002),while the abundance of Alipistes and Gastrophila increased(P=0.00002).= 0.0108;P=0.0404;2.Through the analysis of the contribution degree of the main factors affecting the difference of intestinal flora,we found that there were differences in intestinal flora between the separate-housed two genotypes of mice.The total explanations of genotype,cage and mother were 0.66(Adonis R2 = 0.66,P = 0.001),and the explanations of DRB1*12:02 allele were 0.32(Adonis R2 = 0.32,P = 0.001).3.Further co-housed experiments were carried out in order to strictly control the factors such as different cages and different mother,and to highlight the influence of genotypes on intestinal flora.The results showed that:(1)at phylum level the abundance of Cyanobacteria in intestinal tract of DRB1*1202 mice was significantly higher than that of wild C57BL/6J mice(P=0.0375),and the Deferribacteres was significantly lower than that of wild C57BL/6J mice(P=0.0375).(P=0.0325);at family level,the abundance of Alcaligenaceae and Gastranaerophilales was significantly lower than that of wild C57BL/6J mice(P=0.026;P=0.03251;P=0.035);at genus level,the abundance of Parasutterella and Mucispirum in DRB1*1202 mice was significantly lower than that of wild C57BL/6J mice(P=0.026;P=0.035);= The abundance of Lachnospiraceae and Gastranaerophilales increased(P = 0.0404;P = 0.035).4.Researching the characteristic intestinal flora of DRB1*1202 mice,we found that after co-housed for 4 weeks,LEfSe and Wilcox analysis confirmed that the abundance of Lachnospira and Gastraophilales in DRB1*1202 mice(LEfSe analysis,LDA threshold 2.0;)was significantly higher than C57BL/6J.5.Further,we predicted intestinal flora function by PICRUSt and found that there were 15 different functional gene pathways in DRB1*1202 mice compared with C57BL/6J mice,including Adipocytokine signaling pathway,Biotin metabolism,Cell division,Geraniol degradation,Glycan biosynthesis and metabolism,Lipopolysaccharide biosynthesis proteins,Metabolism of cofactors and vitamins,Phosphatidylinositol signaling system,Phosphotransferase system(PTS),Porphyrin and chlorophyll metabolism,Primary bile acid biosynthesis,Retinol metabolism,Ribosome,Toxoplasmosis,Ubiquitin system,indicate that the intestinal flora affected by DRB1*1202 may affect the above metabolic pathways and related diseases.In conclusion,the composition and structure of intestinal flora in HLA-DRB1*1202 transgenic mice and wild-type C57BL/6J mice were analyzed based on 16 SrDNA high-throughput sequencing.Our results showed that the intestinal flora of HLA-DRB1*1202 transgenic mice was significantly different from that of wild-type C57BL/6J mice;When the two groups mice were separate-housed,the changes of intestinal flora of HLA-DRB1*1202 transgenic mice were affected by gene factors,different cage feeding factors and mother effect;after co-housed,although was affected by fecal and feeding environment with wild-type C57BL/6J mice,the intestinal flora of HLA-DRB1*1202 transgenic mice still showed the effect of HLA-DRB1*1202 gene on intestinal flora.This study revealed the effect of HLA-DRB1*1202 gene on intestinal flora of C57BL/6J mice,and provided a basis for exploring the relationship between HLA-II molecule DRB1*1202,intestinal flora and participating in the occurrence and development of diseases such as HBV-ALCF.