Study On The Role Of ME1 In CD38-mediated T Cell Senescence In HIV Infection

Objective:Acquired immunodeficiency syndrome（AIDS）is an infectious disease caused by human immunodeficiency virus（HIV）infection.Antiretroviral therapy（ART）is effective in suppressing human immunodeficiency virus（HIV）replication,but it can not completely restore HIV induced chronic immune activation and subsequent immune senescence.T cell senescence is one of the main features of immune senescence,currently,the mechanisms of T cell senescence in HIV infection have not been fully elucidated and effective interventions are lacking.It has been long known that CD38 expression on T cells is used to effectively assess the prognosis of HIV infection,as measured by survival time,time to onset of opportunistic infections,and lower level of CD4+T cells,and ART can not restore CD38 expression to normal levels.Our previous study found that high CD38expression on T cells correlated with T cell immune senescence by affecting mitochondrial homeostasis in HIV infection,but the latent molecular mechanism of CD38-induced mitochondrial dysfunction and cellular senescence remains unknown to the best of our knowledge.The transcriptome of CD38+/CD38-T cells revealed that malic enzyme 1（ME1）,a tricarboxylic acid（TCA）cycle-associated enzyme,is significantly downregulated in CD38+T cell.Emerging studies have demonstrated that silencing the expression of ME1 leads to a strong induction of senescence in tumor cell lines and mouse models.Blockage of ME1 leads to the aberrant production of mitochondrial ROS level and loss of mitochondrial membrane potential in uterine stromal cells.However,the physiological role of ME1 in CD38-induced mitochondrial impairment and T cell senescence is still unexplored.This study is the first to detect the effect of ME1 in CD38-mediated T cell senescence,and to study the latent mechanism responsible for the downregulation of ME1 in CD38+T cells and the CD38 increase on T cells after ART.Methods:1.Study participantsHIV infected patients were recruited at The First Hospital of China Medical University.The pertinent inclusion criteria for ART-treated patients were duration of successful ART>2 years,CD4+T cell counts>350 cells/mm³ and had viral load less than 20 copies/m L.Studies have already been approved by the local Ethics Review Committee.2.Cell cultureHuman primary CD3+T cells were isolated from human peripheral blood mononuclear cells（PBMCs）using the Easy Sep TM Human T Cell Isolation Kit（STEMCELL Technologies）.To explore whether CD38 led to cellular senescence by modulating ME1,after 24h ME1 inhibitor（6.25μM,MCE）pre-treatment,T cells were co-cultured with Dynabeads Human T-Activator CD3/CD28 or anti-CD3（1μg/ml,Stemcell）for 48h to examine cellular senescence and mitochondrial homeostasis.To investigate whether ME1 was regulated by p53,T cells were treated with pifithrin-α（p53 inhibitor;10μM,Santa Cruz Biotechnologies）in the presence of Dynabeads Human T-Activator CD3/CD28 for 24h and ME1 mRNA levels were then detected.To determine whether SIRT controls p53 activity,T cells were treated with EX-527（Sirt1-specific inhibitor;1μM,Cayman）in the presence of Dynabeads Human T-Activator CD3/CD28 for 24h to examine acetylated levels of p53 by flow cytometry analysis.To test whether the effect of CD38 on cellular senescence could be reduced,T cells were treated with quercetin（3.75μM,MCE）for 36h.To investigate the effect of IP-10 on the expression of CD38,T cells were treated with recombinant human IP-10（5ng/m L;R&D）and stimulated with Dynabeads Human T-Activator CD3/CD28（Thermo Fisher Scientific）for 48h.3.Flow cytometric analysisFor phenotypic staining,cells were stained with surface antibodies for 30minutes at 4°C,then washed with cold fluorescence-activated cell sorting（FACS）buffer（PBS and 1%FBS）.To measure the levels of p53 acetylation,cells were fixed and permeabilized with a Cytofix/Cytoperm Fixation/Permeabilization Solution Kit（BD Biosciences）.Then,cells were stained with Anti-p53（acetyl K382）antibody（1mg/m L,Abcam）followed by Alexa 488-conjugated chicken anti-rabbit antibody（2mg/m L,Invitrogen）.To determine mitochondrial function,cells were incubated with Mito Tracker Green（50n M;Thermo Fisher Scientific）and Mito Tracker Orange（25n M;Thermo Fisher Scientific）in RPMI 1640 supplemented with 2%FBS for 30min at 37℃prior to staining,then,CD4-APC-Cy7 and CD8-APC were added for surface staining.For assessment of mitochondrial reactive oxygen species（mROS）,cells were incubated at 37℃for 10 min in PBS containing Mito SOX^TMRed Mitochondrial Superoxide Indicator（5μM;Thermo Fisher Scientific）,and then CD4-APC-Cy7 and CD8-APC were added for surface staining.Senescence-associatedβ-galactosidase（SA-β-Gal）was determined using a Senescenceβ-Galactosidase Activity Assay Kit（Cell Signaling Technology）.Briefly,cells were incubated with 100n M Bafilomycin A1 for 1 hour at 37℃followed by,and then incubation with 33μM SA-β-Gal for 3 hours at the same temperature.Finally,CD4-APC-Cy7 and CD8-APC were added for surface staining.Data were analyzed by flow cytometry using a BD LSR II（BD Biosciences）with Flow Jo software（Ashland）.4.qRT-PCR AnalysisTotal RNA was extracted using the RNeasy Plus Mini Kit（Qiagen）.The Primp Script RT reagent kit（TAKARA）was used for RNA reverse transcription.Data were normalized to GAPDH expression.The mRNA expression level for each gene was evaluated using the 2-^ΔΔCT approach.5.Statistical AnalysisStatistical analysis was performed using Graph Pad Prism v8.0 software and Microsoft Excel.Statistical analyses were carried out using Spearman correlation test for the correlation between CD38 and variables as appropriate.Unless otherwise indicated,Wilcoxon signed-rank test or paired t test was used for comparison of matched pairs.P<0.05 was considered statistically significant.Result1.Decreased ME1 activity leads to cellular senescence.As previously reported,silencing the expression of ME1 leads to a strong induction of senescence in tumor cell lines and mouse models.However,the physiological role of ME1 in CD38-induced T cell senescence is still unexplored.Treatment with a potent ME1 inhibitor,we found that SA-β-Gal expression and CD28-CD57+senescent CD4+and CD8+T cells were increased compared to the control group（Figure 1）.2.Decreased ME1 activity leads to mitochondrial dysfunction.Emerging studies have demonstrated that blockage of ME1 leads to the loss of mitochondrial membrane potential in uterine stromal cells.However,the physiological role of ME1 in CD38-induced T cell mitochondrial impairment is still unexplored.The mitochondrial membrane depolarization in CD4+and CD8+T cells were higher in ME1-inhibited T cells compared to control cells.Mitochondrial membrane potential（MMP）,as evaluated in mitochondrial function,was higher in ME1-inhibited CD8+T cells and showed an upward trend in ME1-inhibited CD4+T cells（Figure 2）.3.Decreased ME1 activity elevates mitochondrial ROS levels.Blockage of ME1 leads to aberrant production of mitochondrial ROS level.We found that the mitochondrial ROS levels in CD4+and CD8+T cells were higher in ME1-inhibited T cells compared to control cells（Figure 3）.4.Activation of p53 represses the expression of ME1.After confirming the function of ME1 in sustaining mitochondrial homeostasis and T cell senescence in HIV infection,we explored the underlying mechanism responsible for the downregulation of ME1 in CD38+T cells.A previous study has indicated that p53 activation suppressed ME1 mRNA expression and enzymatic activity.To test whether p53 could downregulate ME1,we treated T cells with Pifithrin-α（PFTα）,an inhibitor of p53,resulting in a significant increase in mRNA levels of ME1（Figure 4A）.p53,the first nonhistone substrate,is acetylated by SIRT1 in a NAD-dependent manner.Previous studies showed that the function of p53 is repressed through deacetylation by SIRT1.To verify the implication of the SIRT1 signaling cascade in regulating ME1 through p53 activation,we gauged the p53 acetylation level at K382 in T cells treated with a SIRT1 inhibitor EX527 and found that,p53 acetylation level was elevated by treatment with EX527,suggesting that inhibition of SIRT1 activity could induce p53 acetylation（activated form of p53）in T cells（Figure 4B-C）.Together,these data demonstrate that ME1 is suppressed through SIRT1-p53 axis,which is controlled by CD38 expression.5.Quercetin reverses mitochondrial impairment and T cell senescence.SIRT1-related senolytic quercetin is a naturally occurring flavonoid,which currently has been used in the first clinical trial.We found that quercetin,a SIRT1-related senolytic drug,significantly reduced the expression of CD57 and depolarized mitochondria on CD4+and CD8+T cells.The mitochondrial membrane depolarization and mitochondrial mass（MM）in CD4+and CD8+T cells were lower in quercetin-treated T cells compared to control cells（Figure 5）.6.IP-10 induces CD38 expression on T cells.Senescent cells contribute to the secretion of senescence-associated secretory phenotype（SASP）that delivers senescence signals to the surrounding,inducing the mitochondrial dysfunction and senescence to neighboring cells.IP-10 is a newly identified SASP factor.IP-10 plasma levels are tightly associated with HIV disease progression and can be reduced,but not to normal levels,by administration of ART.We found that IP-10 correlated positively with the frequency of CD38 on CD4+and CD8+T cells,and induced CD38 expression on CD4+and CD8+T cells（Figure 6）.Conclusion:Our results unveil that ME1 participates in regulating mitochondrial function and cellular senescence in T cell.Specifically,inhibition of ME1 can induce cellular senescence and mitochondrial dysfunction in T cells.Moreover,our data confirm the acetylated level of p53 is regulated by SIRT1 and inhibition of p53 could increase ME1 expression.The link between SIRT1 and ME1 complements and explains the mechanism of downregulated ME1 in CD38+T cells.In addition,IP-10,one of the SASP factors,contributes to CD38 expression on T cells.