Activated Regulatory T-cells Attenuate Myocardial Ischemia/Reperfusion Injury Through A CD39-dependent Mechanism

Part I Adoptive transfer of in vitro-activated Tregs protects against MIRI Objectives The inflammatory response plays an important role in myocardial ischemia/reperfusion injury(MIRI).Regulatory T-cells cells(Tregs)are generally regarded as key immunomodulators that participate in the occurrence and development of various inflammatory diseases.However,its role in myocardial ischemia/reperfusion injury(MIRI)remains unknown.The purpose of the present study was to determine the effect of Tregs on mouse MIRI.Methods Mice were subjected to 30 minutes of ischemia by ligating the left anterior descending coronary artery followed by varying periods of reperfusion,then we detected the infiltration of Tregs in the heart and spleen at different time points(5 minutes,6 hours,and 24 hours after reperfusion)by flow cytometry.Foxp3-GFP knockin mice were subject to ischemia and executed at 6h after reperfusion,then we used immunofluorescence to detect the infiltration of Tregs in mouse heart.Immunomagnetic beads combined with flow cytometry was used to sort CD4+CD25+GFP+ Tregs from the spleen of Foxp3-GFP knockin C57BL/6 mice which were activated by anti-CD3/CD28 and IL-2 in vitro for 3 days or directly used for adoptive transfer,and Tregs were injected intravenously into mice 5 minutes before reperfusion.We used an anti-mouse CD25 monoclonal antibody 72h before the operation which could partially deplete Tregs or DEREG mice which allowed to selectively remove Tregs,1 day after reperfusion MIRI-related indicators were detected,including serum troponin and Evan's blue and TTC double staining which were used to detect the area of infarct and risk.Echocardiography was used to measure left ventricular ejection fraction(EF)and fractional shortening of the left ventricular short axis(FS)to evaluate the cardiac function.Results The percentage of CD4+CD25+Foxp3+ Tregs among CD4+ T-cells within the myocardium had a tendency to increase as early as 5 min following MIRI,but there was no significant difference at any time point,even 24 h,compared with sham-operated mice.However,the absolute number of Tregs infiltrated in the myocardium showed a significant increase,rising after 5 min,peaking at 6 h and remaining elevated at 24 h.Compared with the wild type mice,the infarct size of DEREG group increased and the cardiac function was further deteriorated after MIRI,as both ratio of the infarct/area at risk(I/AAR)and serum troponin were increased,while EF and FS were decreased.However,the myocardial infarct size and cardiac function of the anti-CD25 monoclonal antibody group had no significant change compared with the isotype control group.In addition,compared with the vehicle group,mice which were adoptive transferred with in vitro-activated Tregs showed a smaller infarct size and better cardiac function,but there was no significant changes in the mice which were given exogenous freshly isolated Tregs.Conclusion The infiltration of Tregs increased after reperfusion in mice.Anti-CD25 monoclonal antibody or adoptive transfer of freshly isolated Tregs had no effect on MIRI.Selective removal of Tregs exacerbates MIRI in DEREG mice,while given Tregs activated in vitro,MIRI was reduced,indicating that activated Tregs in MIRI play a protective role.Part II Tregs protect against MIRI through a CD39-dependent mechanism Objectives Tregs convey suppressive action through cell-to-cell contact and/or secretion of soluble factors such as IL-10 and TGF-?1.Additionally,CD39 and CD73 which are highly expressed on the surface of Tregs can sequentially degrade extracelluar ATP to AMP and AMP to adenosine respectively.Moreover,adenosine is considered as an important immunoregulatory mediator of Tregs.The purpose of the present study was to determine the effect molecules of Tregs in MIRI.Methods IL-10-/-mice with a C57BL/6 background were hybridized with Foxp3-GFP knockin mice to generate IL-10-/-Foxp3-GFP knockin mice.Then the vitro-activated IL-10-/-Tregs,CD39-/-Tregs and WT Tregs were isolated and stimulated according to the methods mentioned above.Anti-TGF-?1 antibody and Tregs were injected intravenously 5 minutes before reperfusion,and then we detected the MIRI related indexes 24 hours after reperfusion.Results Compared with WT Tregs,intravenous injection of WT Tregs with anti-TGF-?1 antibody or IL-10-1-Tregs had comparable protective effect on MIRI,manifested as decreased infarct size and improved cardiac function.However,CD39-/-Tregs had no protective effect on MIRI.Conclusion In vitro-activated Tregs protect MIRI through a CD39-dependent mechanism.Part III Tregs attenuated cardiomyocyte apoptosis and inhibited neutrophil infiltration in MIRIObjection:Cardiomyocyte apoptosis and neutrophil infiltration were both well-known participants in the pathophysiology of MIRI.The aim of this study was to observe the effect and mechanisms of Tregs on cardiomyocyte apoptosis and neutrophil infiltration after myocardial reperfusion.Methods:C57BL/6 mice were randomly assigned to four groups:1)mice subjected to a sham operation;2)mice injected i.v.with 200?l PBS;3)mice were injected i.v.with in vitro-activated Tregs from WT mice;4)mice were injected with in vitro-activated Tregs from CD39-/-Foxp3-GFP knockin mice.Then we detected the cardiomyocyte apoptosis,activity of the caspase-3,neutrophil infiltration,activity of MPO and the expression of chemokines(KC,LIX and MIP-2).Additionally,C57BL/6 mice wre randomly assigned to two groups:Sham and MIRI group(The MIRI model was established as described above).The activation of RISK signaling pathway was detected at 30 min,1 hour and 3 hours after reperfusion by Western Blot.The phosphorylation of Akt and ERK1/2 in neonatal mice cardiomyocytes were measured at different time points including 30min,1h and 3h after H2O2 stimulation.Moreover,primary cultures of neonatal mice cardiomyocytes were incubated with pre-activated WT Tregs or CD39-/-Tregs in a co-culture system with stimulation of H2O2 for different duration,and then the following experiment were performed:1)Apoptosis was detected by TUNEL staining after 4 h stimulation;2)The phosphorylation of Akt and ERK1/2 was detected by Western Blot 30 minutes later;3)After 24 h,KC and LIX were quantified using a commercial ELISA kit according to the manufacturer's instructions and the migration of neutrophils was detected by transwell.Results:In vivo data demonstrated that WT Tregs transfer reduced apoptosis of cardiomyocytes,as revealed by terminal deoxynucleotidyltransferase mediated dUTP nick-end labeling(TUNEL)staining and caspase-3 activity,whereas this reduction was abolished by a CD39 deficiency,indicating the impaired function of CD39-/-Tregs.Moreover,this effect was also apparent in vitro.Pre-incubation with WT Tregs significantly decreased cardiomyocyte apoptosis,but CD39 deficiency partially blocked this antiapoptosis effect of Tregs.Additionally,pre-incubation with adenosine could also reduce cardiomyocyte apoptosis.The phosphorylation of Akt and ERK1/2 increased significantly after reperfusion,with the peak of activation occurring at 30 min and decreasing at 3 h after reperfusion.The WT Tregs transfer further increased both Akt and ERK1/2 phosphorylation at 30 min of reperfusion.However,the further increased phosphorylation of ERK1/2 and Akt was not observed in CD39-/-Treg-transferred mice.The treatment of cardiomyocytes with WT Tregs significantly increased the phosphorylation of Akt and ERK1/2,whereas a CD39 deficiency significantly impaired this effect of Tregs.Moreover,such an effect of WT Tregs could be simulated by adenosine.Neutrophil infiltration was prominent at 3h in the PBS group,as demonstrated by myeloperoxidase(MPO)activity and FACS analysis.The transfer of Tregs from WT mice remarkably attenuated neutrophil infiltration,whereas the transfer of Tregs derived from CD39-/-mice had no such effect.Indeed,quantitative PCR performed at the site of ischaemic myocardium revealed that WT Tregs,but not CD39-/-Tregs,blunted the rise of the neutrophil-attracting chemokines KC and LIX,whereas the expression of MIP-2 was not significantly affected.The production of KC and LIX by cardiomyocytes and neutrophil migration were significantly reduced in the presence of Tregs.However,this effect was reversed by CD39 deficiency.Moreover,pre-incubation with adenosine achieved comparable effects with WT Tregs,indicating the pivotal role of CD39/adenosine in Tregs.Conclusion:Tregs attenuated cardiomyocyte apoptosis and activated RISK pathway involving Akt and ERK1/2.Besides,Tregs inhibited neutrophil infiltration in MIRI through down-regulation of the cardiac production of the chemokines KC and LIX.Part IV The frequency of circulating Tregs decreased,whereas the expression of CD39 by Tregs increased,after primary PCI in patients with AMIObjectives:After the above investigation of Tregs in MIRI model was conducted,we sought to determine the regulation of Tregs in the acute reperfusion of ST-elevation myocardial infarction(STEMI)in humans.Methods:Our study focused on 15 patients undergoing primary percutaneous coronary intervention(PCI).Blood samples were taken before re-opening the infarct-related artery,during predilation and 1 h,6 h,12 h,24 h and 72 h after stent implantation.Fifteen age-matched individuals with angiographically normal arteries were enrolled as the control group.The proportion of Tregs and the CD39 expression were detected by flow cytometry.Results:We found that the percentage of Tregs in the CD4+ T-cell population did not show significant differences between the control group and the baseline of the STEMI group.However,compared with the baseline,Tregs decreased after reperfusion of the STEMI group,which began at pre-dilation.A significant reduction occurred at 1 h,reaching a minimum at 6 h and this reduction lasted for at least 72 h after PCI.The fraction of CD39+Tregs was not decreased along with the proportion of total Tregs at each time point after PCI.By contrast,the relative frequency of CD39+ cells within the Tregs population increased after reperfusion and reached statistical significance at 12 h and 24 h following PCI.Conclusion:The frequency of circulating Tregs decreased after reperfusion within the STEMI group while the expression of CD39+ cells of Tregs increased.