Study On The Metabolic Markers And Their Effects On Oxidative Stress And Inflammatory Response In HIV-infected Patients

Objective: Human immunodeficiency virus(HIV)is the main cause of Acquired Immune Deficiency Syndrome(AIDS).With the application of highly active anti-retroviral therapy(HAART),the HIV viral load in HIV-infected patients can be reduced to below the detectable level,and the CD4+T lymphocytes can be increased.After antiviral treatment for a period of time,the plasma viral load of most HIV-infected patients can reach the detection limit.However,with the deepening of study and the extension of the treatment time,it was found that on the premise of ensuring good compliance,although the plasma viral load can be controlled at the undetectable level for a long time after receiving HAART,there are about 30% of the patients whose immune cells did not increase significantly,which is called poor immune reconstruction.Poor immune reconstruction can induce HIV related complitications,which become an urgent problem to be solved in HAART era of AIDS.There are many factors leading to poor immune reconstitution,including gender,age,antiviral drugs,virus and immune factors,genetic factors and so on.At present,metabolic disorder was thought to be closely related to immune reconstitution.Previous studies showed that T lymphocyte dysfunction was related to oxidative stress,and the number of CD4+T lymphocytes was negatively related to reactive oxygen species(ROS).In addition,the increase of T lymphocyte aerobic glycolysis caused by HIV infection can make immune activation,increase the release of inflammatory cytokines,and the excessive inflammatory response can not be recovered even through HAART treatment.At present,although studies have shown that HIV infection can bring about oxidative stress and excessive aerobic glycolysis,there is no systematic study to explore the metabolic changes of HIV-infected patients before and after antiviral treatment in the context of WHO recommendations of treatment rate expansion,so it is impossible to evaluate the impact of antiviral treatment on metabolic disorders of HIV-infected patients.Therefore,it is urgent to identify the metabolic changes of HIV-infected patients before and after antiviral treatment,and to find out the metabolites related to the immune reconstitution.In addition,for oxidative stress and inflammation caused by glycolysis,there is no specific antioxidantthat can be combined with HAART to improve immune reconstitution,and the therapeutic potential of reducing inflammation by inhibiting excessive glycolysis in T cells is unclear.Therefore,to explore the anti-inflammatory and anti-oxidative effects of metabolites in infected patients can provide a new strategy for improving the immune reconstruction of HIV-infected patients.Metabolomics is the study of an organism(including cells,tissue or individual)changes in metabolites generated under different conditions.When the disease occurs,it will inevitably lead to pathological and physiological abnormalities in the body,and then induce changes in the level of metabolites,so as to meet cell activation,differentiation and play a specific immune effect.Metabolomics plays an important role in the occurrence,development and prognosis of cancer,neuropsychiatric diseases,metabolic diseases,infectious diseases and other pathophysiological processes.In addition,as metabolomics has the advantages of small damage,high throughput,easy detection,it has been used to find new markers of some diseases.At present,it has made important achievements in the screening and early diagnosis of cancer,Alzheimer’s disease,cardiovascular disease and some infectious diseases.However,the study of metabolomics in HIV-infected patients is very limited.Although previous studies have focused on the metabolite profiles of patients after HIV infection or HAART,there is still a lack of systematic research on the characteristics of metabolite changes in HIV-infected patients before and after HAART,especially the metabolites related to inflammation and oxidative stress have not been fully defined.So it is important to comprehensively evaluate the metabolic characteristics of HIV-infected patients vertically and systematically,especially analyze the metabolites associated with oxidative stress and inflammation.Therefore,this study intends to use metabolomics to study the plasma metabolites profile of HIV-infected patients before and after antiviral treatment,systematically analyze the influence of antiviral treatment on metabolites of HIV-infected patients,further clarify the characteristics of metabolism changes in HIV-infected patients before and after antiviral treatment,and screen out key metabolites related to inflammation and oxidation which were correlated with immune reconstruction.In order to provide experimental basis for discovering the treatment strategy of improving immune reconstitution,the effects of metabolites on monocyte,T-lymphocyte inflammatory response and oxidative stress werethen studied in vitro.Methods:1.Study Subjects1.1 In the first part of this paper,the plasma samples of 19 HIV-infected patients before and after antiviral treatment were collected.There was no dyslipidemia before antiviral treatment,and after antiviral treatment for at least 3 months,the viral load decreased to below 400copies/mL.Plasma samples from 18 healthy controls(HC)were also collected.1.2 In the second part of the paper,CD4+T lymphocytes and CD8+T lymphocytes were extracted from fresh blood samples of HIV-infected patients.1.3 In the third part of the paper,monocytes,CD4+T lymphocytes and CD8+T lymphocytes were extracted from fresh blood samples of HIV-infected patients.2.Experimental methods2.1 Metabolon Platform2.1.1 Sample pretreatmentTake 100μL samples or other quality control samples,including water,methanol,mixed samples,healthy blood samples,and transfer them to 96 deep hole plate;then add450μL methanol extraction solution(containing internal standard)to each hole(already containing 100μL liquid),seal it with sealing film;shake it up and down manually for2 mins,and centrifuge the 96 deep hole plate on the desktop centrifuge at 2000 rpm for5mins;then transfer 110μL extract liquid to LC-POS/LC-NEG plate,spare plate and GC plate respectively;dry the sample in each plate on the nitrogen blower,and store it at-80℃for use.2.1.2 Metabolite detectionMetabolomic profiling was performed by Metabolon using ultra high performance liquid chromatography and tandem mass spectrometry(UPLC/MS/MS2)and gas chromatography(GC)/MS.The resulting extract was divided into five fractions: one for analysis by UPLC-MS/MS with positive ion mode electrospray ionization,one for analysis by UPLC-MS/MS with negative ion mode electrospray ionization,one for LC platform,one for analysis by GC-MS,and one sample was reserved for backup.Samples were placed briefly on a TurboVap?(Zymark)to remove the organic solvent.For LC,the samples were stored overnight under nitrogen before preparation for analysis.For GC,each sample was dried under vacuum overnight before preparation for analysis.2.2 CD4+T cell countThe peripheral venous blood of the subjects was collected by EDTA.The mixed dye of TriTEST CD4-FITC/CD8-PE/CD3-Percp was added to the flow tube,then the whole blood was added.The whole blood was stained at room temperature without light,then the hemolysin was added and kept away from light at room temperature.The number and percentage of CD3+/CD4+,CD3+/CD8+T subsets were calculated by MultiSET software.2.3 HIV-RNA viral loadThe whole blood of the subjects was collected and the plasma was separated for detection.The viral load was determined by real-time quantitative PCR using Cobas Amplicor automatic load analyzer provided by Roche.2.4 Sorting of primary cellsPeripheral blood mononuclear cell(PBMC)was extracted by density gradient centrifugation.Firstly,the whole blood PBMC was extracted,and the CD14+ monocytes and CD4+T,CD8+T lymphocytes in PBMC were selected by magnetic bead method.2.5 Detection of ROS in T cellsThe primary CD4+T and CD8+T lymphocytes were stimulated with anti-CD3/CD28,and the metabolic marker was added to the cells and incubated for 18-24 hours.After incubation,5μM ROS detection reagent(CellRox? Deep Red Reagent C10042,Thermo Fisher Scientific)was added into the tube and then incubated at 37℃ shed from light.BD LSR II flow cytometer and Flow Jo software were used to analyze.2.6 Detection of intracellular mitochondrial functionThe primary CD4+T and CD8+T lymphocytes were stimulated with anti-CD3/CD28,and the metabolic marker was added to the cells and incubated for 18-24 hours.After incubation,100-500 nM mitochondrial membrane potential(MMP)detection reagent(MP7510,Thermo Fisher Scientific)labeled with PE,20-200 nM mitochondrial mass(MM)detection reagent(MP7511,Thermo Fisher Scientific)labeled with FITC were added and mixed,incubated in dark for 30 mins at 37℃.BD LSR II flow cytometer and Flow Jo software were used to analyze.2.7 Detection of CD4+T and CD8+T lymphocyte activationThe primary CD4+T and CD8+T lymphocytes were stimulated with anti-CD3/CD28,and the metabolic marker was added to the cells and incubated for 18-24 hours.After incubation,FITC labeled mouse anti-human CD25,PE labeled mouse anti-human CD69 and APC labeled anti-HLA-DR were added to the experimental tube;after incubation in dark for 15 minutes at room temperature,2mL of PBS was added,washed twice,then added 200μL of PBS to suspend cells.At last,BD LSR II flow cytometer and Flow Jo software were used to analyze.2.8 Detection of cytokinesThe primary monocytes were stimulated with lipopolysaccharide(LPS),and the metabolic marker was added to the monocytes,and incubated for 18-24 hours.After incubation,the supernatant is collected by centrifugation and put into-80℃ refrigerator for detection.On the day of the experiment,the cryopreserved samples were taken out,and the standards,magnetic beads and antibodies were also diluted;then added 50μL magnetic beads to 96 well plates.After 3 times of plate washing,50μL standard and supernatant samples were added.After 3 times of plate washing,25μL antibody was added to the plates,which were incubated in high frequency shaking table at room temperature in dark for 1h.After 3 times of plate washing,50μL SA-PE was added.Finally,125μL assay buffer was added to the plates,which were incubated in dark for detection.2.9 Statistical analysisSPSS l9.0 and Graphpad Prism 6 were used for statistical analysis.Data conforming to normal distribution were analyzed by independent sample t test or paired t test.Data not conforming to normal distribution were analyzed by non-parametric test.The correlation between metabolites and CD4+T lymphocyte counts was analyzed by Spearman rank correlation test;the metabolites with significant difference were screened according to the label of fold change(FC)≥1.0 and p<0.05,which was statistically significant,0.05≤p<0.1was of marginal significance.In addition,principal component analysis(PCA),cluster analysis and random forest analysis(RFA)were also used in the metabolon execution system.Results1.Screening out metabolic markers related to immune reconstitution in HIV-infected patients1.1 The metabolites profile changed significantly in HIV-infected patientsThrough the metabolomics detection,we found that the overall metabolic profile of HIV-infected patients was significantly different from that of normal people.Among the 331 metabolites detected,HIV infection can cause 67 metabolites change,of which the proportion of lipids is the largest(59.7%),followed by amino acids(26.9%).The main metabolites were tryptophan,kynurenine,lysine,N-acetylcarnosine,and ketone body,long-chain fatty acid and acyl carnitine,which were related to inflammation,oxidative stress and mitochondrial function.Among them,histidine was thought to be involved in both inflammation and oxidative stress.1.2 Histidine can be restored by effective antiviral treatmentOf the 67 abnormal metabolites in HIV-infected patients,only 19 metabolites,including histidine can return to normal level after effective antiviral treatment.1.3 Histidine can be used as a metabolite related to immune reconstitution in HIV-infected patientsAmong the 19 metabolites which returned to normal levels,6 were corraleted with CD4+T lymphocyte counts,including histidine(r=0.512,p=0.001),1-oleoyl-GPE(18:1)(r=0.325,p=0.046),1-palmitoyl-GPE(16:0)(r=0.375,p=0.021),1-arachidonoyl-GPI(20:4)(r=0.37,p=0.020),N-acetylcarnosine(r=0.335,p=0.040)and cholesterol(r=0.382,p=0.018),which may be a group of metabolic markers related to immune reconstitution.Among them,histidine,which is believed to have both anti-inflammatory and anti-oxidative effects,may play an important role in the immune reconstruction of HIV-infected patients.2.No significant effect of histidine on oxidative stress of T lymphocytes was observed in vitroCD4+T and CD8+T lymphocytes were extracted from untreated HIV-infected patients and incubated for 18-24 hours with anti-CD3/CD28 stimulation and histidine at doses of2 mM,20mM,50 mM and 100 mM respectively.The results showed that the addition of histidine in vitro had no significant improvement in mitochondrial membrane potential,mitochondrial mass and ROS production.3.Histidine can reduce inflammation in HIV-infected patients in vitroHistidine was used to incubate T cells and monocytes from untreated HIV-infected patients.The results showed that histidine could reduce the expression of CD25 on thesurface of CD4+T and CD8+T lymphocytes and the release of IP-1,interlukin(IL)-6 and tumor necrosis factor(TNF)-α from monocytes.Conclusion1.Many metabolic pathways are abnormal after HIV infection;2.Histidine,which is related to inflammation and oxidation reaction,changed after HIV infection and can be restored by HAART.As histidine has a positive correlation with CD4+T lymphocyte countes,it can be used as a metabolic marker related to immune reconstitution of HIV-infected patients;3.Histidine did not significantly improve the oxidative stress of T cells in vitro;4.Histidine can reduce the release of proinflammatory cytokines/chemokines from monocyte and the expression of CD25 on T lymphocytes in vitro.