Heme Oxygenase-1Restores Impaired GARP~+CD4~+CD25~+Regulatory T Cells From Patients With Acute Coronary Syndrome By Upregulating GARP And LAP Expression On Activated T Lymphocytes

Part Ⅰ The Frequency and Function of GARP+CD4+CD25+Regulatory T Cells Are Impaired in Patients with Acute Coronary SyndromeAim glycoprotein A repetitions predominant (GARP) is a transmembrane proteins which identified recently. GARP is specifically expressed on activated human natural regulatory T lymphocytes (nTregs) and is not induced on non-nTregs after T cell receptor (TCR). Moreover, GARP+CD4+CD25hi Foxp3+Tregs showed greater suppressive function than GARP"CD4+CD25hi Foxp3+Tregs. Thus, GARP+CD4+CD25+Tregs may be more suitable for distinguishing bona fide activated human nTregs with high suppressive capacity from Tresp and induced Tregs. In this part, we will explore the frequency and suppressive function of GARP+CD4+CD25+Tregs in each group.Methods After rigorous screening, a total of121patients from Wuhan Union Hospital were recruited to the study. All the volunteers were divided the three group:(1) Healthy volunteers (control;21men and9women)(2) Patients with stable angina (SA;28men and12women)(3) Patients with acute coronary syndrome (ACS) including30cases of acute myocardial infarction (AMI) and21cases of unstable angina (UA). Blood samples were collected from all patients upon admission and within24h of the onset of symptoms. After centrifugation, peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient and isolated T lymphocytes. After TCR stimulation, The frequencies of T-helper1(Th1), Th2, Th17and the expression of GARP by CD4+CD25+T cells were then assessed by measuring flow cytometry. The suppressive function of activated GARP+CD4+CD25+was measured by thymidine uptake. The levels of transforming growth factor-1(TGF-β11) in the plasma were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of the genes encoding these proteins were analyzed by real-time polymerase chain reaction. Result The frequencies of CD4+CD25+T cells were detected by flow cytometry (Fig.1A), and no significant differences were detected among the three groups (P=0.36). However, after measuring the expression of GARP in CD4+CD25+T cells, we found that the expression of GARP was sharply reduced in patients with ACS (6.02±1.05%) compared with patients in the SA and control groups (9.09±1.23%,9.58±1.51%, respectively P<0.01) after stimulation in vitro, However no significant differences were observed between the SA and control groups (P=0.39). In addition, the suppressor function of Tregs from patients with ACS was significantly impaired in different ratios compared with Tregs isolated from the SA and control group (P<0.01). At the same time, the frequencies of Th1and Th17were clearly higher in the ACS group than in patients with SA or control subjects (P<0.01).Conclusion the attenuated number of GARP+CD4+CD25+Tregs was responsible for the impaired suppressive function and the destructive immune response characterized by the hyperproliferation of Th1and Th17cells in patients with ACS. The pro-inflammatory cytokines that are secreted by Th1and Thl7destabilized the fibrous cap leading to plaque rupture. Part Ⅱ Heme Oxygenase-1Restores Impaired GARP+CD4+CD25+Regulatory T Cells from Patients with Acute Coronary Syndrome by Upregulating GARP and LAP Expression on Activated T LymphocytesAim:Recent research further demonstrated that HO-1is involved in the regulation of adaptive immunity by up-regulating the expression of Foxp3on CD4+CD25+Tregs. As a receptor controlling the expression of Foxp3, We found that GARP+CD4+CD25+Tregs were impaired in the frequency and biological function in patients with ACS. Thus, the current study sought to investigate the effect of HO-1on the levels and immunosuppressive function of GARP+CD4+CD25+Tregs in different groups of patients and explore the potential mechanisms by which HO-1regulates adaptive immunity.Methods:After rigorous screening, Blood samples were collected from121volunteers. After centrifugation, peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient and isolated T lymphocytes. After treatment with hemin and TCR stimulation, The frequencies of T-helper1(Th1), Th2, Thl7, latency-associated peptide (LAP)+CD4+T cells and the expression of GARP and Foxp3by CD4+CD25+T cells were then assessed by measuring flow cytometry. The suppressive function of activated GARP+CD4+CD25+was measured by thymidine uptake. The expression levels of the genes encoding these proteins were analyzed by real-time polymerase chain reaction.Results:HO-1has effectively restored the biological function of impaired GARP+CD4+CD25+Tregs. With the recovery of impaired GARP+CD4+CD25+Tregs, the hyperproliferation of Th1and Th17cells were restrained in patients with ACS. In addition, the up-regulation of HO-1also promotes the expression of LAP and Foxp3.Conclusion:HO-1can effectively restore impaired GARP+CD4+CD25+Tregs by promoting LAP and GARP expression on activated T cells and inhibit the hyperproliferation of Th1and Th17cells in patients with ACS.