Regulatory T Cells Ameliorate Cardiac Remodeling After Myocardial Infarction And Its Mechanistic Study

Part I Regulatory T cells ameliorate cardiac remodeling and improve cardiac function after myocardialObjective:Persistent inflammatory responses participate in the pathogenesis of adverse ventricular remodeling after myocardial infarction (MI), but the endogenous protective mechanism is unknown.CD4+CD25+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells that inhibit inflammatory response. The purpose of the study is to explore the effect of Treg on the adverse ventricular remodeling and cardiac function after MI.Method:MI was induced by ligation of the left anterior descending (LAD) coronaiy artery in Lewis rats. Sham-operated rats underwent the same procedure without ligation of coronary artery. Purified Treg or non-regulatory CD4+ T cells (Tconv) were isolated by magnetic sorting. MI rats were randomely allocated the the three experimental groups:1) MITreg, in which rats received 5×10 adopitvely transferred Treg; 2) MITreg, in which rats received 5×106 adopitvely transferred Tconv; 3) MI, in which rats received equal volumn of PBS.24 hours after the cell transfer, the hearts and lymph nodes were removed for tracking of transferred cells. The infiltration of Treg in the infarcted hearts was evaluated using immunohistochemistry on day 3 after cells transfer. The index of ventricular dilation and cardiac function including heart rate (HR), left ventricular end-systolic dimension (LVESD), LV end-diastolic dimension (LVEDD), fractional shortening (FS), LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and maximal positive and minimal negative first derivative of LV pressure (+dp/dtmax and-dp/dtmin) were assessed using echocardiography and hemodynamic analysis on day 28 after cell tranfer. Commitently, infarct size in the infarct zone (IZ) and fibrosis in the peri-infarct zone (PIZ) and non-infarct zone (NIZ) were evaluated using Masson’s trichrome. Matrix metalloproteinase (MMP)-2 activity in the NIZ was measured using gelatin zymography. Myocardial apoptosis in the PIZ and NIZ was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.Result:Infiltration of Treg was found in the peri-infarct zone (PIZ) on day 3 after MI, peaking on day 7, and then decreased on day 14. Transferred Treg recruited the infarted hearts and lymph nodes and Treg cell transfer resulted in an increased number of Treg in the PIZ of hearts from the MITreg group, relative to the MI or MITconv group. Compared with sham-operated rats, rats in the MI group showed marked LV dilation and cardiac dysfunction, as evidenced by increased LVEDD, LVESD and LVEDP, decreased LVSP, FS,+dp/dtmaxand -dp/dtmin. With the exception of infarct size, interstitial fibrosis, MMP-2 activity and cardiac apoptosis were attenuated in rats in the MITreg group as compared with the MI or MITconv group.Conclusion:Rats receiving adoptively transferred Treg demonstrated increased number of infiltrated Treg in the infarcted heart, ameliorated ventricular remodeling and improved cardiac function. Treg cells serve as an endogenous protective mechanismagainst adverse ventricular remodeling in myocardial infarction. PartⅡRegulatory T cells inhibit infarction-induced inflammatory response in vivoObjective:The purpose of the study is to explore the effect of Treg on infarction induced inflammatory response in the heart and the systemic cytotoxic response.Methods:Purified Treg or non-regulatory CD4+T cells (Tconv) were isolated by magnetic sorting. MI was induced by ligation of the left anterior descending (LAD) coronary artery in Lewis rats. Sham-operated rats underwent the same procedure without ligation of coronary artery. MI rats were randomely allocated the the three experimental groups:1) MITreg, in which rats received 5×106 adopitvely transferred Treg; 2) MITreg, in which rats received 5×106 adopitvely transferred Tconv; 3) MI, in which rats received equal volumn of PBS. Inflammatory cells including neutrophils, macrophages and T cells in the PIZ of hearts were detected using immunohistochemistry on day 3, day 7 and day 28 after cell transfer. Expression of inflammatory cytokines including TNF (tumor necrosis factor)-α, IL (interleukin)-1β, transforming growth factor (TGF)-β1and IL-10 were measured using RT-PCR. The killing effect of spleen lymphocyte on cardiomyocytes was tested in the cytotoxic activity assay, using crystal violet staining. Proliferation of CD8+ cytotoxic lymphocytes were evaluated using flowcytometric analysis.Results:Obvious infiltration of neutrophils, macrophages and T lymphocytes was observed in the PIZ after MI. The temporal pattern of this infiltration differed among inflammatory cells. The number of invading neutrophils reached a maximum as early as day 3 and dramatically declined to the baseline as in the sham-operated hearts on days 7 and 14. Macrophage infiltration showed a similar temporal pattern, but declined slowly in the infarcte heart. The infiltration of T lymphocytes peaked on day 7, and then gradually decreased. In the Treg cell transfer hearts the number of accumulated neutrophils in the PIZ was significantly decreased compared to MI or MITconv group on day 3 after MI, although no significant changes were observed on days 7 and 14. Infiltration of macrophages and lymphocytes was markedly decreased in MITreg group compared to MI or MITconv group at all tested timepoints. Pro-inflammatory cytokines TNF-αandIL-1βmRNA and anti-inflammatory cytokine TGF-β1 and IL-10 expression in the infarcted hearts was up-regulated on day 7 after MI compared to the sham-operated hearts. The expression of TNF-αand IL-1β, but not TGF-β1, was significantly decreased in MITreg group compared to MI or MITconv group. In contrast, a marked up-regulation of IL-10 was observed in group. Lymphocytes from MI group showed increased cytotoxicity against cardiomyocytes and increased CD 8+cytotoxic T lymphocyte proliferation compared with those from sham-operated rats. Treg transfer, rather than Tconv transfer, significantly inhibited the increased cytotoxic activity and CD8+ cytotoxic T lymphocyte proliferation.Conclusion:Adoptively transferred Treg decreased the inflammatory response in the infarted heart and inhibited systemic cytotoxic T lymphocyte after MI. PartⅢRegulatory T cells protect cardiomyocytes from apoptosis by direct cell-cell contact and IL-10 in vitroObjective:The purpose of the study was to examine the possibility that Treg cells provide direct benefit to stressed cardiomyocytes.Methods:Neonatal rat cardiomyocytes (NCM) were preincubated without T cells, with Treg cells or with Tconv cells, in the presence of anti-CD3, for 48 hours in a co-culture system and were then stimulated with LPS for an additional 24hours. Apoptosis of NCM was assessed using TUNEL. Inflammatory cytokines including TNF-α, IL-1β, TGF-β1 and IL-10 in the supernatant of the co-culture system were determined by ELISA. To analyze the effects of soluble factors, anti-TGF-β1 or anti-IL-10 blocking antibodies were added to the co-culture system in some cases. In some experiments, the role of direct cell-cell contact was evaluated in a 24-well transwell system with cardiomyocytes in the lower chamber and lymphocytes in the upper chamber.Results:NCM that were exposed to LPS showed marked apoptosis (18.1±1.2%,p<0.01) in comparison to NCM that were not exposed to LPS (5.2±1.2%). Co-culture with Treg cells (9.2±1.0%,p<0.01) significantly reduced the apoptosis of NCM in response to LPS, in comparison to co-culture with Tconv cells (18.7±2.5%).The concentrations of IL-10 and TGF-β1 were significantly higher in the supernatants from the co-culture system with Treg cells compared to either that with Tconv cells, or without T cells. Blocking IL-10 attenuated the protective effect of Treg cells. In contrast, blocking TGF-β1 did not affect the protection effect of Treg cells. The disruption of direct cell-cell contact between NCM and Treg cells in the transwell system resulted in a partial reversion of the protection. LPS stimulated the production of TNF-αand IL-1βby NCM, while Treg cells significantly reduced their release by NCM.Conclusion:Treg cells directly protect cardiomyocytes against LPS-induced apoptosis, and that this protection depends on the cell-cell contact and IL-10 expression. Furthermore, Treg cells inhibited proinflammatory cytokine production by cardiomyocytes.