Study On The Mechanism Of T-cell Pyroptosis And Its Effect On Immune Reconstitution After Treatment Of HIV Infection

Objective:Acquired Immune Deficiency Syndrome（AIDS）is a disease that endangers global human health caused by human immunodeficiency virus（HIV）infection,and can cause immunodeficiency and multiple complications.With the advent of Anti-retroviral Therapy（ART）,the morbidity and mortality of HIV infection have been greatly reduced.Most of the infected patients have good immune reconstitution after ART,but there are still10-40%of infected patients with poor immune reconstitution.Patients with poor immune reconstitution have persistent immune activation,immune depletion,immune differentiation and higher levels of inflammation,which lead to poor recovery of immune function and poor clinical outcome,high incidence of complications,and clinically relevant metabolic disorders and end-organ damage in severe cases.At present,effective interventions to promote immune reestablishment are very limited.In-depth research on the causes of immune failure in patients with poor immune reestablishment is an important issue urgently needed to be solved in the field of HIV.CD4⁺T cell count is an important indicator to predict and distinguish the level of immune reconstitution.Immune responders（IR）were defined as those whose CD4⁺T cell counts increased by more than 20%from baseline after two years of ART.Immunological Non-Responder（INR）was defined as the patients whose CD4⁺T cell count increased by less than 20%or less than 350 cells/μL after two years of ART.The mechanism of the failure of CD4⁺T cell number to recover in INR patients is still unclear.Existing studies suggest that it may be caused by decreased production of CD4⁺T cells and increased death in INR patients.After HIV infection,T cell death mainly included bystander cell pyroptosis,virus-infected cell apoptosis and bystander cell apoptosis,among which pyroptosis accounted for the highest proportion（95%）.Pyroptosis is a form of programmed cell death that is pro-inflammatory and has been studied mainly in the field of tumor and immune cells in untreated HIV infected patients.However,after receiving antiviral therapy,when the virus has been effectively suppressed,the relationship between the pyroptosis level of immune T cells and the immune response and the specific mechanism remains unclear.Clarifying the relationship between the number of CD4⁺T cells and pyroptosis in INR patients,exploring the mechanism of CD4⁺T cell pyroptosis in INR patients,and seeking effective interventions to promote immune recovery can provide new therapeutic strategies for improving prognosis.In this study,we identified the differences in pyroptosis levels and pyroptosis distribution characteristics of T cells between IR and INR patients by flow cytometry（thesis 1）,and further identified the key metabolic molecule ENO2（Enolase-γ）regulating pyroptosis by bioinformatics analysis of transcriptome data（thesis 2）.Subsequently,we explored the specific mechanism of ENO2 regulating CD4⁺T cell pyroptosis and found effective methods to interfere with pyroptosis by gene knockdown and enzyme inhibition（thesis 3）,which provided new clues and directions for improving the immune recovery and clinical outcome of HIV-infected patients after ART treatment.Methods:1.Study subjects.In this study,187 HIV-infected patients who received ART treatment for more than 2years were recruited,including 149 IR patients and 38 INR patients.The subjects were all from the First Affiliated Hospital of China Medical University,and had been approved by the Ethics Committee of China Medical University.All patients signed the informed consent form.2.Isolation peripheral blood PBMCs and the sorting of PD-1⁺and PD-1^-CD4⁺T cells.Peripheral blood mononuclear cells（PBMCs）were collected by density gradient centrifugation for further flow staining and sorting.CD3⁺,CD4⁺T cells were sorted using STEMCELL cell separation kit for magnetic bead negative selection and used in subsequent experiments.Sorting of PD-1⁺and PD-1^-CD4⁺T cells using flow antibodies CD4-APC-Cy7 and PD-1-FITC,the cells were divided into PD-1⁺and PD-1^-CD4⁺T cell subsets,and the two groups of cells were sorted by flow cytometry sorter.Sorted cells were used for PCR assays to verify differential expression of key glycolytic enzyme genes.3.Flow cytometry detection of peripheral blood cell pyroptosis markers Caspase-1and NLRP3.2 m L whole blood samples were collected from the subjects and peripheral blood mononuclear cells were extracted.After negative selection of T cells for Caspase-1 staining,the cells were redissolved in 50μL DMSO to form 150×storage solution,which was diluted to 1×PBS for use.Each tube was stained by adding 100μL of diluent,incubated at 37°C in the dark for 1 hour,and mixed by flicking every half hour.The mixture was gently mixed and washed once with 2 m L PBS containing 2%FBS.After centrifugation at200g for 10 minutes at room temperature,the supernatant was discarded,the surface was stained with CD4-APC-Cy7,and the machine was used for detection.PBMCs were extracted from 2 m L of whole blood.T cells were negatively selected for NLRP3 staining:CD4-APC-Cy7 was stained on the cell surface,and then 350μL membrane inhibitor was added and incubated at 4℃in the dark for 30 minutes to break the membrane.After membrane rupture,the cells were washed with 1 m L of membrane rupture wash,centrifuged at 500g of liter 5 and down 5 for 5 minutes,stained with intracellular NLRP3,98μL PBS plus 2μL NLRP3 dye,and incubated at room temperature in the dark for 30 minutes.Flow cytometry BD Canto II and Flowjo v 10.3 software were used to detect the pyroptosis markers Caspase-1 and NLRP3 in peripheral blood cells.4.Detection of pyroptosis subgroup distribution characteristics.2 m L whole blood was collected from the subjects and peripheral blood mononuclear cells were extracted.Negative selected T cells were stained with Caspase-1 and redissolved in 50μL DMSO to form 150×storage solution,which was diluted to 1×in PBS for use.Each tube was stained by adding 100μL of diluent,incubated at 37°C for 1 hour,and mixed by flicking every half hour.The mixture was gently mixed and washed once with 2m L PBS containing 2%FBS.Then,activation,exhaustion and differentiation surface markers were stained.The exhaustion markers PD-1 and TIGIT on CD4⁺and CD8⁺T cells were stained with CD4-APC-Cy7,CD3-PE-Cy7,Caspase-1-FITC,TIGIT-APC and PD-1-BV421 fluorescent antibodies.The activation markers CD38 and HLA-DR on CD4⁺and CD8⁺T cells were stained with CD4-APC-Cy7,CD3-PE-Cy7,Caspase-1-FITC,CD38-BV510 and HLA-DR-Per CP-Cy5.5 fluorescent antibodies.The differentiation markers CD45RA and CCR7 on the surface of CD4⁺and CD8⁺T cells were stained with CD4-APC-Cy7,CD3-PE-Cy7,Caspase-1-FITC,CD45RA-BV510 and CCR7-BV421fluorescent antibodies.After 20 minutes of staining in the dark at 4°C,after washing twice with PBS,the machine was used for detection.5.Transcriptome data analysis.The transcriptome data of PD-1⁺and PD-1^-CD4⁺T cells were analyzed using the GEO Datasets public database GSE17606.The differentially expressed genes were screened by GEO2R online tool,and the filter conditions were set as adj.P<0.05,and Fold Change>1.5.Unsupervised hierarchical clustering analysis and heat map of differentially expressed genes were performed by Me V（Multiple Experiment Viewer）4.9.0 software.DAVID website（https://david.ncifcrf.gov）was applied to differentially expressed genes in KEGG signal pathway enrichment analysis.The public GEO Datasets GSE18233 were used to analyze the differences in the expression of ENO2 and ENO3 in CD4⁺T cells of HIV negative controls（NC）,HIV-Infected individuals（HIV）who were not on treatment（HIV）,and HIV-Infected individuals who were on ART（ART）,and their relationship with CD4⁺T cell counts and the correlation analysis of viral load.At the same time,the correlation between ENO2,ENO3 and the pyroptosis indicators Caspase-1 and NLRP3 was analyzed.Based on the public GEO Datasets GSE18233,ENO2 was divided into ENO2^high and ENO2^low groups according to the m RNA expression level,and the filtering condition was set as adj.P<0.05.DAVID website（https://david.ncifcrf.gov）to get the GO analysis of differentially expressed genes.6.GSEA gene set enrichment analysis.Based on the public GEO Datasets GSE18233,the ENO2 datasets were divided into ENO2^high and ENO2^low groups according to their m RNA expression levels.The expression matrix and grouping were sorted out according to the input requirements of GSEA software.After sorting,import GSEA 4.1.0 software.The enrichment conditions were set according to the experimental requirements,and the database used for enrichment was selected to run the software for enrichment analysis.7.Cellular RNA extraction,reverse transcription,and real-time fluorescence quantitative PCR.Total cellular RNA was extracted by the RNeasy Plus Micro Kit according to the kit user manual.RNA reverse transcription was performed using Prime Script RT reagent Kit and real-time RT-PCR was performed using TB Green Premix Ex Taq II,and relative m RNA quantification was calculated using 2-^ΔΔCt.β-actin was used as an internal control.8.Oxidative phosphorylation of CD4⁺T cells was measured after inhibition of ENO2.CD4⁺T cells were divided equally into two fractions and cultured in 48-well plates.The experimental group was added with 10μM ENO2 inhibitor ENOblock and soluble CD3/CD28 tetramer stimulator,and the control group was added with equal volume of DMSO and soluble CD3/CD28 stimulator.After incubation at 37°C in the dark for 24 hours,mitochondrial Stress was detected by Seahorse XFp Cell Mito Stress Test Kit following the user’s manual.The following drugs were added at each dosing point:1.5μM Oligo,0.5μM FCCP,0.5μM rotenone and antimycin A（R&aa）.The cell culture plates were treated with cell adhesion agent（cell-tak）,then the cells after adjusting the number were added and equilibrated at 37°C for 20 minutes before detection.9.Flow cytometry detection of mitochondrial markers.Mito SOX ^TM Red was used to label mitochondrial Reactive Oxygen Species（ROS）and Mito Tracker ^TM Green FM was used to label Mitochondrial Mass according to the instructions.Mitochondrial Membrane Potential（MMP）was labeled with Mito Tracker ^TMOrange CMTMRos,and cytoplasmic ROS was labeled with Cell ROX?Deep red.10.Knockdown and inhibition of ENO2 experiments.Mitochondrial ROS and pyroptosis marker Caspase-1 were detected after knockdown of ENO2:CD4⁺T cells of HIV-infected patients after ART were electro-transfected with si ENO2 and si Negative control.After 6 hours,the solution was changed and CD3/CD28stimulation was added at the same time.The levels of mitochondrial ROS and Caspase-1were detected after incubation at 37℃for 48 hours in the dark.Mitochondrial ROS and Caspase-1 levels were measured after inhibition of ENO2:CD4⁺T cells from the negative selected study population were treated with ENO2 inhibitor ENOblock and CD3/CD28stimulator for 24 hours,and mitochondrial ROS and Caspase-1 levels were measured.11.Inhibiting and activating PI3K-AKT-m TOR-S6K signaling pathway to detect ENO2 expression.After negative selection of CD4⁺T cells,cells were seeded in 96-well culture plates at a density of 0.5×10⁶ per well.10μmol/L MHY1458,200 nmol/L Torin1,50μmol/L CMK,50μmol/L LY294002,20μmol/L SC79,and 10μmol/L MK-2206 were added into the corresponding wells,respectively.Cells were simultaneously cultured with CD3/CD28stimuli for 48 hours,and cells were lysed at the end of the culture for ENO2 PCR.12.Statistical analysis.Flowjo v 10.3 software was used to map the results obtained by flow cytometry,and Graph Pad Prism v 9.1.0 software was used for statistical analysis and mapping.Selection rules of statistical methods:（1）For comparison between the two groups,the data were in accordance with normal distribution and were paired,using Paired t test;the data were in accordance with normal distribution and were unpaired,using Unpaired t test.The data did not conform to the normal distribution and were paired,using Wilcoxon matched-pairs signed rank test.The data did not conform to the normal distribution and were unpaired,using Mann-Whitney test.（2）For comparison among the three groups,the data were in accordance with normal distribution and were paired,using Fisher’s LSD test;the data were in accordance with normal distribution and were unpaired,using Fisher’s LSD test.The data did not conform to the normal distribution and were paired,using Friedman test.The data did not conform to the normal distribution and were unpaired,using Kruskal-Wallis test.（3）For correlation analysis,Pearson correlation test was used for data conforming to normal distribution;Spearman’s correlation test was used for data that did not conform to a normal distribution.All tests were two-sided,and a P value of less than0.05 was considered to indicate a statistically significant difference.Results:1.The pyroptosis of CD4⁺T cells is increased in patients with INR and is associated with disease progression.Multi-color flow cytometry was used to detect the expression of Caspase-1 and inflammasome NLRP3 in CD4⁺and CD8⁺T cells in patients with INR and IR.It was found that the pyroptosis levels of CD4⁺and CD8⁺T cells in INR patients were significantly higher than those IR patients.The pyroptosis level of CD4⁺T cells is associated with disease progression.2.The pyroptosis of CD4⁺T cells in INR patients was found to be mainly distributed in terminal effector subsets,CD38⁺HLA-DR⁺double-positive subsets,PD-1⁺TIGIT⁺double-positive subsets.Further examination of the distribution characteristics of pyroptosis in T cells showed that the pyroptosis of CD4⁺T cells in INR patients was mainly distributed in end-effector subsets,CD38⁺HLA-DR⁺double-positive subsets,and PD-1⁺TIGIT⁺double-positive subsets.The pyroptosis of CD4⁺T cells in IR patients was mainly distributed in effector memory subsets,CD38⁺HLA-DR⁺double positive subsets and PD-1⁺TIGIT^-subsets.The pyroptosis of CD8⁺T cells in INR patients was mainly distributed in naive T cell subsets,CD38⁺HLA-DR⁺double positive subsets and PD-1⁺TIGIT^-subsets.Pyroptosis of CD8⁺T cells in IR patients was mainly distributed in effector memory subsets,CD38⁺HLA-DR⁺double positive subsets and PD-1⁺TIGIT^-subsets.3.We found that the expression of Caspase-1 was significantly positively correlated with PD-1 in CD4⁺T cells after ART treatment,and PD-1 could distinguish pyroptosis from non-pyroptosis CD4⁺T cells.Furthermore,the correlation between the expression of Caspase-1,the pyroptosis marker of CD4⁺and CD8⁺T cells,and the expression of differentiation CD45RA,CCR7,activation CD38,HLA-DR,exhausted PD-1 and TIGIT was analyzed.It was found that the expression of Caspase-1,the pyroptosis marker of CD4⁺T cells,was significantly positively correlated with the exhaustion marker PD-1.By performing a receiver operating characteristic（ROC）curve,we found that PD-1 could distinguish pyroptosis from non-pyroptosis CD4⁺T cells.4.ENO2 and ENO3 may be the key metabolic regulatory genes in regulating CD4⁺T cell pyroptosis.PD-1 could significantly distinguish pyroptotic from non-pyroptotic CD4⁺T cells.By analyzing the transcriptome data of PD-1⁺and PD-1^-CD4⁺T cells,we found that the down-regulated differentially expressed genes were enriched in metabolism-related signaling pathways including amino acid biosynthesis,glycolysis/glycogen synthesis and HIF-1signaling pathways.Furthermore,the intersection of genes involved in these three signaling pathways showed that ENO2 and ENO3 were involved in these three signaling pathways.In addition,PD-1⁺and PD-1^-CD4⁺T cells were sorted by flow sorter,and the expression levels of glycolysis-related enzymes were detected by PCR.The expression levels of glycolysis key enzymes ENO2 and ENO3 were significantly decreased in PD-1⁺T cells compared with PD-1^-CD4⁺T cells.5.A significant decrease in ENO2 in patients with INR and was associated with disease progression.We analyzed the expression of ENO2 and ENO3 in different populations,and found that the expression of ENO2 m RNA was significantly decreased after HIV infection,and returned to normal level after ART treatment.The expression level of ENO2 m RNA in INR patients was significantly lower than that in IR patients.ENO2 was significantly positively correlated with CD4⁺T cell count and negatively correlated with viral load.ENO3 m RNA level were significantly decreased after HIV infection and returned to normal level after ART treatment,but there was no significant difference in ENO3 between IR and INR.ENO3 was significantly positively correlated with CD4⁺T cell counts and negatively correlated with viral load.6.ENO2 was significantly negatively correlated with Caspase-1 and NLRP3,and inhibition of ENO2 significantly increased the pyroptosis level of CD4⁺T cells.ENO2 m RNA was significantly negatively correlated with the pyroptosis marker Caspase-1 m RNA and NLRP3 m RNA.There was no significant correlation between ENO3 m RNA and Caspase-1 m RNA,but a significant negative correlation between ENO3m RNA and NLRP3 m RNA.Further examination of the effect of ENO2 on CD4⁺T cell pyroptosis showed that the level of CD4⁺T cell pyroptosis was significantly increased after adding ENO2 inhibitor ENOblock.7.Inhibition of m TOR-S6K signaling inhibited ENO2 expression.PI3K-AKT-m TOR-S6K signaling pathway is a signaling pathway related to cell metabolism.Treatment of CD4⁺T cells with PI3K inhibitor LY294002,AKT inhibitor MK-2206 and activator SC79,m TOR inhibitor Torin1 and activator HY1485,and S6K inhibitor CMK for 48 hours showed that inhibition of S6K significantly suppressed ENO2expression in CD4⁺T cells.Inhibition of m TOR tended to suppress ENO2 expression in CD4⁺T cells,but had no significant effect on ENO3.Further study showed that S6K inhibitor CMK significantly increased the level of pyroptosis Caspase-1 in CD4⁺T cells,but the m TOR inhibitor Torin1 had no significant effect on CD4⁺T cell pyroptosis.8.Through transcriptome data analysis,it was found that ENO2 may regulate CD4⁺T cell pyroptosis by regulating mitochondrial metabolism.In order to further investigate the mechanism of ENO2 affecting CD4⁺T cell pyroptosis,we analyzed the transcriptome data of ENO2^low and ENO2^high,and found that the differentially expressed genes were mainly enriched in the mitochondrial pathway,and GSEA analysis showed that mitochondrial oxidative phosphorylation was mainly enriched in ENO2^high subgroup.9.Impaired mitochondrial oxidative phosphorylation in CD4⁺T cells after inhibition of ENO2.After the addition of 10μM ENO2 inhibitor ENOblock,it was found that inhibition of ENO2 significantly reduced the level of mitochondrial oxidative phosphorylation in CD4⁺T cells,and basal respiration,ATP production,maximum respiration,and reserve respiration were significantly reduced.10.The mitochondrial function of CD4⁺T cells was impaired after ENO2 inhibition.The addition of 10μM ENO2 inhibitor ENOblock revealed a significant increase in the percentage of depolarized mitochondria,a significant decrease in mitochondrial membrane potential,and a significant increase in mitochondrial cytosolic ROS in CD4⁺T cells.11.A significant increase in mitochondrial ROS in CD4⁺T cells after ENO2 inhibition.By electroporation of si ENO2 and addition of ENO2 inhibitor ENOblock,we found that the percentage of mitochondrial ROS and mean fluorescence intensity（MFI）were significantly elevated after knockdown or inhibition of ENO2.12.Phosphoenolpyruvate supplementation was found to restore mitochondrial oxidative phosphorylation,reduce mitochondrial ROS level,and reduce pyroptosis in CD4⁺T cells.After phosphoenolpyruvate（PEP）supplementation,we found that mitochondrial oxidative phosphorylation and ATP production in CD4⁺T cells were increased.Mitochondrial ROS level was significantly decreased when ENO2 was knocked down or inhibited while PEP was added.Knockdown or inhibition of ENO2 combined with PEP significantly reduced the pyroptosis level of CD4⁺T cells Caspase-1.Conclusion:1.In the first part of the study,the pyroptosis markers Caspase-1 and inflammasome NLRP3 in CD4⁺T cells were significantly increased in INR patients and correlated with disease progression;The pyroptosis of CD4⁺T cells in INR patients was mainly distributed in end-effector subsets,CD38⁺HLA-DR⁺double-positive subsets,PD-1⁺TIGIT⁺double-positive subsets.The pyroptosis level of CD4⁺T cells is significantly positively correlated with PD-1 expression,and PD-1 can distinguish pyroptosis from non-pyroptosis CD4⁺T cells.2.In the second part of the study,ENO2 expression was significantly reduced in patients with INR and correlated with disease progression;The key glycolytic enzyme ENO2 can regulate the pyroptosis of CD4⁺T cells.m TOR-S6K signaling pathway can increase the pyroptosis level of CD4⁺T cells by inhibiting the expression of ENO2.3.In the third part of the study,the mitochondrial function was impaired,the level of oxidative phosphorylation was decreased,the level of depolarized mitochondria,cytoplasmic ROS and mitochondrial ROS were increased after knockdown and inhibition of ENO2.PEP,the catalytic product of ENO2,could reduce mitochondrial ROS and pyroptosis in CD4⁺T cells.