Correlation Between Gut Microbiota And Its Metabolites And Stages Of HIV Infection

Objective Although previous studies have shown that HIV infection is associated with disruption of intestinal flora,studies always focus on only microbiome or only metabolome,and the interaction between intestinal flora,metabolites and HIV infection was unknow.Fecal metabolites are modestly influenced by host genetics and can act as an intermediate phenotype reflect interactions between host and microbiome.In order to better understand the role of the composition and function of intestinal flora in the progression of AIDS disease,16s rDNA sequencing and untargeted metabolomics of stool samples was conducted,and interaction between intestinal flora,gut metabolites and disease indicators was analyzed by correlation analysis.Methods A cross-sectional study was carried out.From December 1,2017to December 30,2019,untreated HIV/AIDS patients and uninfected individuals were recruited from the Fourth People’s Hospital of Nanning and the First Affiliated Hospital of Guangxi Medical University by convenient sampling.According to CD4+T lymphocyte counts and clinical manifestations,untreated HIV-infectied individuals were divided into AIDS group(CD4+T cell counts<200,accompanied by or without AIDS indicative infection)and Pre-AIDS group(CD4+T cell count≥200 and without AIDS indicative infection).Meanwhile,uninfected individuals were used as control group.The epidemiological data,blood samples and stool samples of the subjects were collected respectively.The blood samples were tested for the levels of microbial translocation and immune activation,and the stool samples were tested for 16s rDNA sequencing and untargeted metabolomics.Analysis the changes in composition of intestinal flora and fecal metabolites at different stages of HIV disease progression,and explore correlation between intestinal flora,metabolites and disease indicators for further understanding the role of intestinal flora on HIV disease progression.Results A total of 115 subjects were recruited in this study,including 41 in the AIDS group,39 in the Pre-AIDS group,and 35 in the control group(C).There was no significant difference in age,sex and nationality among the three groups(P>0.05).(1)Higher level of immune activation and microbial translocation was associated with the HIV infection.The percentage of activated CD4+T cells in AIDS group was significantly higher than that in the Pre-AIDS group and the control group,and the percentage of activated CD4~+T cells in Pre-AIDS group was significantly higher than that in the control group(all P<0.010);The percentage of activated CD8~+T cells in AIDS group was significantly higher than that in the Pre-AIDS group and control group(all P<0.001);The level of plasma Endo CAb IgM in the AIDS group was significantly lower than that in the Pre-AIDS group and control group(all P<0.001).The level of plasma s CD14 in the AIDS group was significantly higher than that in the Pre-AIDS group and control group(all P<0.001).(2)The composition of intestinal flora and its functions are different in different stages of HIV infection.The alpha-diversity of intestinal flora in AIDS group was significantly lower than that in Pre-AIDS group and control group(all FDR p<0.1).Composition of Intestinal flora in different stages of HIV infection was aggregated into three cluster and significantly different(P=0.001,PERMANOVAR).The analysis of differences in intestinal flora among the three groups showed that AIDS group and the Pre-AIDS group were enriched in human pathogens or opportunistic pathogens,such as Enterococcus,Brevundimonas,Aeromonas and Pseudomonas at the genus level,or Enterococcus durans,Acinetobacter nosocomialis,Aeromonas caviae,Agrobacterium pusense at the species level;Butyrate-producing bacteria,such as Faecalibacterium,Lachnospira,Roseburia,Ruminococcaceae,Anaerostipes hadrus and Eubacterium ramulus,was decreased in AIDS group and Pre-AIDS group(all FDR p<0.1);Random forest analysis showed that human pathogens or opportunistic pathogens,such as Rhodococcus,Brevundimonas,Enterococcus,Aeromonas,Acinetobacter nosocomialis,Aeromonas caviae,Agrobacterium pusense were important in identification of HIV infection;KEGG Pathway enrichment analysis shows that disorder of pathways related to amino acid metabolism,and Immune system pathways,such as NOD like receptor signaling,Antigen processing and presenting,as well as pathways participated in inflammation inhibition,such as Vitamin B6 metabolism,Nicotinate and nicotinamide metabolism,and Thiamine metabolism were down-regulated in AIDS and Pre-AIDS group while pathways related to microbial translocation,such as Lipopolysaccharide biosynthesis proteins and Lipopolysaccharide biosynthesis,as well as the Bacterial invasion of epithelial cells,Staphylococcus aureus infection and Apoptosis pathway were up-regulated in the AIDS and Pre-AIDS group group(all FDR p<0.1).(3)Composition of metabolites in different stages of HIV infection was significantly differentOPLS-DA showed that composition of the metabolites among the three groups was significantly different(P=0.01);Differential metabolites analysis shown that amino acids such as L-tryptophan,L-phenylalanine was in disoder,and L-tryptophan was up-regulated in AIDS and Pre-AIDS group,while Ah R ligands of tryptophan metabolites which was related to intestinal immunity was down-regulated in AIDS and Pre-AIDS group;Polyamines such as putrescine,cadaverine,spermidine,phenylethylamine and N-Acetylputrescine was up-regulated in AIDS and Pre-AIDS group,while metabolites associated with anti-inflammatory and antioxidant,such as pyridoxine,pyridoxamine,cinnamaldehyde,salidroside,melatonin,fumaric acid,and ectoine etc,and phosphonoacetate,a DNA polymerase inhibitors,was decreased in AIDS and Pre-AIDS group(all FDR p<0.05 and VIP>1.0);ROC curve analysis showed that phenylalanine(AUC=0.905),tryptophan(AUC=0.887),d-glucuronic acid(AUC=0.802)and phosphoacylacetic acid(AUC=0.876)may be potential metabolic biomarkers associated with HIV infection.(4)Correlation between disease indicators and gut microbiota,differential metabolitesL-phenylalanine,L-tryptophan,D-glucuronic acid,putrescine,ect enriched in HSI and HNSI group were positively correlated with microbial translocation and immune activation.The down-regulated metabolites,such as phosphonoacetate,fumaric acid,niacinamide,pyridoxine,pyridoxamine,ect in AIDS and Pre-AIDS group were negatively correlated with the microbial translocation and immune activation.Enterococcus,Lactobacillus and other bacteria enriched in AIDS and Pre-AIDS group were positively associated with metabolites positively correlated with microbial translocation and immune activation.Rhodococcus,Faecalibacterium,Lachnospira,Dorea and other bacteria enriched in the control group were positively associated with metabolites negatively correlated with microbial translocation and immune activation;Among them,Rhodococcus and Rhodococcus erythropolis,the most important bacteria related to HIV infection,were positively associated with phosphonoacetate,while negatively associated with L-tryptophan,L-phenylalanine and D-glucuronic acid(all P<0.05).Conclusion By analyzing the changes of intestinal flora and fecal metabolites at different stages of HIV infection,our study found that the composition of intestinal flora and fecal metabolites was different at different stages of HIV infection,and changes began at the early stage of disease.After HIV infection,human pathogens and opportunistic pathogens were increased,butyrate-producing bacteria and bacteria related to inflammation inhibition was decreased.At the same time,amino acid metabolism disordered,and metabolites for HIV replication increased,while metabolites for inhibitiing HIV replication,anti-inflammatory and oxidation resistance were decreased.Fecal Metabolites were found to be related to microbial translocation and immune activation.Intestinal flora may affect bacterial flora translocation and immune system activation through the gut metabolites,consequently affecting the progression of AIDS disease.