The Relationship Between Gut Microbiota And Its Metabolites And Immune Recovery After Long-term Treatment In Primary HIV-1 Infetion Patients

Objective:Acquired immune deficiency syndrome（AIDS）is a severe infectious disease caused by human immunodeficiency virus-1（HIV-1）infection.Although combined antiretroviral therapy（cART）can significantly extend the lifespan of HIV-1patients,10-40 percent of HIV-1 patients still fail to recover their CD4⁺T cell counts and have high mortality rates.Moreover,HIV-1 infection leads to persistent immunoinflammation and immune activation,which significantly increases the incidence of non-AIDS complications and decreases the life quality of HIV-1 infected patients.In 2018,the World Health Organization（WHO）proposed the fourth“90%”goal,which refers that 90 percent of HIV-1 infected patients have good life quality.Therefore,investigating the risk factors for immune recovery after cART and exploring effective interventions to reduce immune activation and inflammation is an important research direction,which favours the life quality of HIV-1 infected patients.The intestinal tract is an important site for HIV-1 infection,the intestinal mucosal barrier will damage after HIV-1 infection,the intestinal permeability will increase and lead to continuous immune activation and inflammation.The intestinal flora of HIV-1infected patients is closely related to immune recovery,immune inflammation and activation after cART.HIV-1 infection can lead to decreased intestinal flora diversity and alter the composition of intestinal flora,which is related to immune recovery.Studies have shown that theαdiversity of gut microbiota of HIV-1 infected patients is positively correlated with CD4⁺T cell counts,and negatively correlated with microbial translocation markers after cART.HIV-1 infected patients charactered by increased level of Prevotella and low mean conunts of Bacteroidetes,Faecalibacterium prausnitzli,unclassified Subdoligranulum and Coprococcus,which were associated with poor immune recovery.With the widespride research on intestinal microecology,the metabolites of gut microbial communities are also considered to be important substances involved in the regulation of activities and metabolism.HIV-1 infection can cause severe imbalance of metabolites and affect immune recovery and cell function.The study showed that 139 bacteria metabolites were altered after HIV-1 infection.Short chain fatty acid（SCFAs）inhibited T cell activation in intestinal mucosal epithelium.In vitro studies showed that butyric acid can significantly affect the expression and function of cell genes through multiple ways.Early treatment can promote immune recovery;reduce immune activation and immune inflammation in HIV-1 infected patients.With long-term cART,the CD4⁺T cell counts in HIV-1 infected patiens may reach the normal level,and a few patients with long-term cART（more than 3 years）who initiated treatment in primary HIV-1infection（PHI）can achieve viremia control after treatment interruption.We speculate that intestinal flora may play a regulatory role in immune recovery after cART in PHI patients.Studies on simian immunodeficiency virus（SIV）infection animal models showed that the abundance of Akkermansia,Anaerovilbrio,Bacteroidales and Bifidobacterium decreased significantly in the early infection stage.In 2013,an American research team analyzed the intestinal microbiome of 10 PHI patients after cART,and found that the proportion of Lactobacillus was positively correlated with CD4⁺T cell counts.These studies indicate that the intestinal flora play an important role in the treatment efficacy and prognosis of PHI patients.In 2016,early treatment strategy was recommended in China and more and more PHI patients received treatment,but the influence of intestinal flora and its metabolites on the immune recovery after long-term cART in PHI patients is not clear.In recent years,Men who have sex with men（MSM）have become the main population of new HIV infections in large and medium cities in China,and the intestinal flora of MSM population differed from non-MSM population.Thus,we enrolled MSM as HIV-1 negative controls（HNC）to minimise bias.In this study,we investigated the diversities and compositions of fecal microbiota among PHI,chronic HIV-1 infection（CHI）and HNC to clarify the characteristics of fecal microbiota in PHI patients and determine the possible role of gut ecosystem in the immune recovery after long-term cART.Methods:1.SubjectsSubjects recruited for this study were all MSM,defined as having self-reported anal sex with a male partner within one year.A total of 97 subjects were collected in this study,including 45 PHI patients,37 CHI patients and 15 HNC.The eligibility criteria for PHI patients were as follows:（a）diagnosed with PHI infection,patients who accorded with one of the following definitions were defined as PHI:（1）a negative or indeterminate HIV-1 antibody test with HIV-1 viral load>10,000copies/m L;（2）confirmed HIV-1 positive western blot result with a negative testing evidence in the last six months.（b）age range is 18-50;（c）cART more than 3 years;（d）HIV-1 viral load<50 copies/m L.The inclusion criteria for CHI patients were as follows:（a）diagnosed with CHI infection;（b）age range is 18-50;（c）cART more than3 years;（d）HIV-1 viral load<50 copies/m L.The inclusion criteria for HNC were as follows:（a）a negative HIV-1 screening result;（b）age range is 18 to 50.The following exclusion criteria were applied to all the three groups:the use of any antibiotics,probiotics,prebiotics or immunotherapy within 30 days before the sample was taken;diagnosed with tumor,diabetes mellitus,or hepatitis B or C infections;with concurrent AIDS defining illness,or suffering from severe organ failures.All subjects signed the informed consent.This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.2.Measurement of CD4⁺T cell activationThe whole blood samples were stained with 3μL antibodies:anti-CD4-APC-cy7,anti-CD3-PE-cy7,anti-CD38-PE,anti-human leukocyte antigen DR-APC and anti-CD31-APC（all BD Biosciences,USA）.CD4⁺T cell activation markers were detected by FACS Calibur flow cytometry（BD,USA）and the data was analyzed by BD FACSDIVA software.3.Detection of microbial translocation markersThaw the frozen plasma.The original standard solution were diluted by multiple ratios and incubated at 37℃for 30 min.Added 50μL HRP-Conjugate reagent to each well,and incubated at 37℃for 30 min.Added 50μL chromogenic agent A and 50μL chromogenic agent B to each well,incubated at 37℃for 10 min.Added 50μL stop solution to stop the reaction.OD value was detected with enzyme label detector.4.Quantification of total HIV-1 DNA in peripheral blood mononuclear cells（PBMCs）and resting CD4⁺T cellsPBMCs were isolated by Ficoll density gradient centrifugation,and resting CD4⁺T cells were separated by CD4⁺T cell sorting kit.DNA was extracted by QIAamp DNA Micro Kit.Total HIV-1 DNA was amplified by the dd PCR（Bio-Rad,CA,USA）:dd PCR reaction solution for RPP 30 reference gene and LTR gene was added to each reaction tube.Added samples to the reaction tube and transferred to the droplet reaction card.The generated droplets were transferred to the reaction plate for PCR amplification.PCR amplification program were:95°C for 10 min,45 cycles of 94°C for 30 s,60°C for 1 min,and 98°C for 10 min.The expression level of the tested gene was calculated according to RPP 30 internal reference gene.5.16S r RNA gene sequencing of fecal microbiotaFresh fecal samples（approx.4 g）were collected using sterile fecal collection tubes and immediately frozen at-80°C.The main detection procedures were as follows:DNA was extracted from the stool samples using the QIAamp rapid DNA stool mini-kit（51604,QIAGEN,Germany）.The quality of total DNA was determined.The 16S r RNA gene in the V3-V4 region of bacterial DNA was amplified by PCR.The PCR amplification program was:95℃for 3 min,98℃for 20 s 30 cycles,58℃for 15 s,72℃for 20 s,72℃for 5 min.16S r RNA gene sequencing was performed on the Mi Seq platform.6.Detection of SCFAs in the facal and plasma samplesPretreatment of fecal sample:added 50μL 15%phosphoric acid,100μL internal standard（isohexanoic acid）solution with 125μg/m L concentration,400μL diethyl ether to 50 mg fecal sample,homogenated for 1 min,centrifuged at 12000 rpm for 10min at 4℃,seperated the supernatant and detected.Pretreatment of plasma sample:added 100μL plasma sample into 2 m L centrifuge tube,added 100μL 20%phosphoric acid solution and 500μL of 50 g/m L4-methylvaleric acid solution.After mixing and centrifugation（14000×g for 20 min）,1μL supernatant was taken for dectection.GC-MS detection condition:Agilent HP-INNOAX capillary column（30 m×0.25mm ID×0.25μm）was used.The injection volume was 1μL and the split ratio was10:1.The temperature of the inlet was 250℃,the temperature of the ion source was230℃,the temperature of the transmission line was 250℃,and the temperature of the quadrupole was 150℃.The initiation temperature was 90℃,then rised to 120℃at10℃/min,then to 150℃at 5℃/min,and finally to 250℃at 25℃/min for 2 min.The carrier gas was helium,and the gas flow rate was 1.0 m L/min.MS condition:electron bombardment ionization source,SIM scanning mode,electron energy 70 e V.7.CD4⁺T cell cultureCD4⁺T lymphocytes were resuspended with R10 medium containing different sodium butyrate concentrations to form different treatment groups.Immunocult^TMhuman CD3/CD28 T cell activator was added for stimulation.All cells were cultured in an incubator containing 5%CO₂ at 37℃for 24 h.8.Transcriptomics detectionThe experimental procedures included RNA extraction,sample quality testing,library construction and library quality control.The Illumina sequencing platform was used for sequencing and bioinformatics analysis was performed.9.Seahorse energy metabolism analysisThe probes were hydrated one day before the experiment carried out.Added the drugs and detected the probe plate.Added 25μL Cell-Tak adhesive solution to the96-well detection plate and incubated at room temperature for 20 min and washed.Added 50μL cells and 130μl medium to each well,incubated in a CO₂ free incubator at 37℃for 20 min and detected.10.Effects of sodium butyrate on differentiation,activation,exhaustion and metabolism of CD4⁺T cellsCD4⁺T cells were stained with 3μL antibodies:PE/Cyanine7 anti-human CD25,APC anti-human CD69,Brilliant Violet 421^TManti-human CD279（PD-1）,FITC anti-human CD57,APC/Cyanine7 anti-human CD45RA,BV786 anti-human CCR7,Pharringen 7-AAD,PE anti-human GLUT1.The cells were stained in dark for 30min.After washing,the cells were resuspended.BD LSR II Flow cytometry and Flow Jo software were used for detection and analysis.11.Statistical analysisSPSS 23.0 software was used for statistical analysis.Continuous variables were described by medians and quartiles.Classification variables were described by frequency and percentage.Students’T test,non-parametric Mann-Whitney U test,Chi-square test,Fisher test and one-way analysis of variance were used according to different types of variables,p<0.05 was considered statistically significant.We used R3.5.1 software to analyze the 16S r RNA gene sequencing data of intestinal flora.Operational taxonomic units（OTUs）were clustered according to 97%similarity,and OTUs were identified by Usearch software.Theαdiversities of the samples were evaluated using Qiime1.9.1 software.Principal component analysis（PCo A）combined with Adonis analysis was used for weighted and unweightedβdiversity analysis.Different bacterial taxa were identified by Kruskal-Wallis test and Linear discriminant analysis（LEf Se）（p<0.05 and LDA score>2.0 were defined as different）.Spearman correlation analysis was used to explore the correlation of intestinal flora and metabolites with immunological markers,p<0.05 was considered statistically significant.Results1.The characteristics of intestinal flora in PHI patients and its relationship with immune recovery after long-term cARTA total of 45 PHI patients,37 CHI patients and 15 HNC were included in this study.There were no statistical differences in age,ethnicity,BMI and cART durations among the three groups.The estimated durations of HIV-1 infection in PHI patients was significantly shorter than that of CHI patients.CD4⁺T cell counts,CD4/CD8ratios and HIV-1 viral load levels in PHI patients were significantly higher than those in CHI patients before cART.CD4⁺T cell counts and CD4/CD8 ratios of PHI patients were higher than those in CHI patients.Moreover,the percentages of HLADR⁺CD38^-CD4⁺T cells and HLADR⁺CD38⁺CD4⁺T cells in PHI patients were lower than those in CHI patients after long-term cART.The total DNA levels of PBMCs and resting CD4⁺T cells in PHI patients were significantly lower than those in CHI patients,suggesting that PHI patients obtained better immunological and virological responses after long-term cART.To compare the gut microbial communities of PHI patients,CHI patients and HNC,we evaluated theαandβdiversity of intestinal flora.Theαdiversity of microflora in PHI patients was higher than that of CHI patients,even reached the level of HNC,suggesting PHI patients had a richer intestinal microbial environment after long-term cART.Theβdiversity of microflora showed that the pattern of the intestinal flora in PHI patients was similar to that of HNC,which differed from that of CHI patients.PHI patients had enriched SCFAs producing bacteria,such as Roseburia,Lachnospiracea incertae sedis,Coprococcus,Blautia,Dorea,Ruminococcus,Anaerostipes and Eubacterium.Correlation analysis with clinical markers showed that the relative abundances of Coprococcus,Bifidobacterium,Butyricicoccus,Clostridium IV and Oscillibacter were significantly positively correlated with CD4⁺T cell counts.The results indicated that PHI patients had enriched SCFAs producing bacteria,which were positively correlated with CD4⁺T cell counts significantly.The intestinal flora of PHI patients were mostly involved in energy metabolism pathways,such as methane metabolism,glucose metabolism,amino acid metabolism,lipid metabolism and fatty acid synthesis.2.The relationship between fecal and plasma SCFAs levels and immune recovery after long-term cART in PHI patientsIn addition to intestinal flora,bacterial metabolites also play an important role in the immunological response of HIV-1 infected patients after cART.We detected the SCFAs levels in the fecal and plasma samples of PHI and CHI patients,and the results showed that the total intestinal SCFAs levels in PHI patients were significantly higher than those in CHI patients.Acetic acid and butyric acid levels in PHI infected patients were significantly increased,while other SCFAs levels were identical between two groups,such as propionic acid,isobutyric acid,iso-valeric acid,valeric acid and caproic acid.There were no significant differences in the plasma SCFAs levels between the two groups.In order to clarify the correlation between the SCFAs levels and immunological responses in HIV-1 infected patients,we analyzed the correlation between the levels of intestinal SCFAs and T cell counts,HIV reservoirs,intestinal mucosal injury indexes and immune activation markers in HIV-1 infected patients.The results showed that intestinal butyric acid level were positively correlated with CD4⁺T cell counts and the CD4/CD8 ratios,and negatively correlated with immune activation markers.It is suggested that butyric acid may be an important substance to promote immune recovery in HIV-1 infected patients after cART.Moreover,we analyzed the correlations between the relative abundance of SCFAs-producing bacteria enriched in PHI patients and butyric acid concentration.We found that the relative abundance of Roseburia（r=0.450,p<0.001）,Lachnospiracea＿incertae＿sedis（r=0.440,p<0.001）,Coprococcus（r=0.425,p<0.001）and Alistipes（r=0.511,p<0.001）were significantly positively correlated with intestinal butyric acid concentration.In conclusion,PHI patients had enriched butyric acid producing bacteria including Roseburia,Lachnospiracea＿incertae＿sedis,Coprococcus and Alistipes,which contribute to increased butyric acid level and may mediate immune response of HIV-1 infected patients after long-term cART.3.The effects of butyric acid on gene expression,energy metabolism and cell function of CD4⁺T cell in HIV-1 infected patientsOur previous study found that butyric acid,as a key metabolite of intestinal flora,may play an immunomodulatory role in the immune recovery of HIV-1 infected patients.Therefore,the effect of butyric acid on the function of CD4⁺T cells in HIV-1infected patients and the possible mechanism are worth further exploration.We incubated CD4⁺T cells with sodium butyrate for 24h in vitro.Transcriptome analyses were performed in both the sodium butyrate treated group（experimental group）and the control group without sodium butyrate.We identified a total of 3823 differentially expressed genes（DEGs）,Gene ontology（GO）enrichment analysis showed that most DEGs involved in biological processes including immune response,adaptive immune response,response to cytokines,cell membranes,T cell activation,immune regulation,etc.Molecular function analysis showed the main protein binding function may be related to cytokines.The DEGs were mainly concentrated in organelle lumen and nucleus.KEGG enrichment analysis showed that the DEGs were mainly concentrated in glucose metabolism,cell differentiation,senescence,PD-1 and other related signaling pathways.Based on the results of KEGG enrichment analyses of transcriptomics,we found that most DEGs were enriched in glucose metabolism pathways,suggesting that butyric acid may have an effect on the glucose metabolism of CD4⁺T cells.We detected oxidative phosphorylation and glycolysis of the cells.The results showed that butyric acid significantly reduced the maximum oxygen consumption capacity and residual respiratory capacity of CD4⁺T on a dose-dependent trend.Butyric acid also significantly reduced the glycolysis and maximal glycolysis of CD4⁺T cells.These results indicated that butyric acid can significantly reduce the energy consumption of CD4⁺T cells in HIV-1 infected patients.We detected the activation,differentiation,senescence function of CD4⁺T cells and the expression of Glut-1 on the cell surface.The results showed that butyric acid significantly reduced the expression of CD25 on CD4⁺T cells of HIV-1 infected patients in a dose-dependent manner.Therefore,we hypothesized that higher plasma butyric acid concentration might decrease immune activation in HIV-1 infected patients.Butyric acid had no effect on differentiation,senescence and glucose receptor expression of CD4⁺T cells in HIV-1 infected patients.Conclusions1.PHI patients had higher intestinal flora diversity than that of CHI patients after long-term cART.PHI patients had enriched SCFAs producing bacteria,which were significantly positively correlated with CD4⁺T cell counts.The intestinal flora of PHI patients was involved in energy metabolism pathways and played a role in the synthesis of fatty acids.2.We identified four kinds of butyric acid producing bacteria,including Roseburia,Lachnospiracea＿incertae＿sedis,Coprococcus and Alistipes,which were enriched in the PHI patients after long-term cART and lead to higher butyric acid concentration.The intestinal butyric acid level was significantly positively correlated with CD4⁺T cell counts and CD4/CD8 ratios,and negatively correlated with immune activation markers.3.Butyric acid significantly affected CD4⁺T cell gene expression in HIV-1infected patients,the down-regulated genes involved in adaptive immune response,T cell activation and immune regulation,and the DEGs were mainly concentrated in glucose metabolism pathway and cell differentiation,senescence,PD-1 and other related signaling pathways.Butyric acid significantly reduced oxidative phosphorylation and glycolysis of CD4⁺T cells in HIV-1 infected patients.Butyric acid significantly reduced the activation of CD4⁺T cells,but had no effect on the differentiation,senescence and glucose receptor expression of CD4⁺T cells in HIV-1infected patients.