Yudong Peng

miR-146a in PBMCs modulates Th1 function in patients with acute coronary syndrome

The upregulation of Th1 cells has been suggested to have an essential function in the development of atherosclerosis (AS). Recent studies indicate that miR‐146a is a microRNA specifically and highly expressed in Th1‐driven autoimmune disease. The aim of the study was to investigate the possible mechanisms of the miR‐146a in the onset of acute coronary syndrome (ACS). The results showed that the expression of miR‐146a in peripheral blood mononuclear cells (PBMCs) was significantly increased in patients with ACS. We showed that overexpression of miR‐146a in PBMCs could significantly upregulate the function of Th1 cells. Furthermore, we showed that miR‐146a treatment could modulate the Th1 differentiation through posttranscriptional enhancing the T‐bet pathway in PBMCs. In addition, this study also provided evidence that miR‐146a treatment in vitro could induce the protein expression of TNF‐α, MCP‐1, NF‐κB p65, which are key pro‐inflammatory cytokines and critical transcription factor in AS. In contrast, miR‐146a inhibitor could attenuate these phenomena significantly. The results support the concept that miR‐146a may be a novel regulatory factor in Th1 differentiation and a new therapeutic target for AS and ACS.

Downregulation of T Helper Cell Type 3 in Patients with Acute Coronary Syndrome

BACKGROUND AND AIMS There is an imbalance between Th1 and Th2 in the development and progression of atherosclerosis and in patients with acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina. T helper cell type 3 (Th3), which primarily secretes transforming growth factor beta-1 (TGF-beta1), has been shown to inhibit both Th1 and Th2 cells. The present study was designed to investigate whether Th3 cells are involved in plaque destabilization and the onset of ACS. METHODS Ninety one patients who underwent diagnostic catheterization were classified into four groups (AMI group, unstable angina group, stable angina group and chest pain syndrome group). The cell frequencies of Th1, Th2 and Th3 were detected using flow cytometry, and the concentrations of their related cytokines IFN-gamma, IL-4 and TGF-beta1 were studied by ELISA. RESULTS Apart from the imbalance between Th1 and Th2, results revealed a significant decrease in peripheral Th3 number and levels of TGF-beta1 in patients with ACS as compared with those in patients with stable angina and chest pain syndrome (p<0.01). CONCLUSIONS Downregulation of Th3 cells in patients with ACS may play a potential role in plaque destabilization and the onset of ACS.

Expansion of myeloid-derived suppressor cells in patients with acute coronary syndrome.

Aim: The aim of this study was to explore whether the circulating frequency and function of myeloid-derived suppressor cells (MDSCs) are altered in patients with acute coronary syndrome (ACS). Methods: The frequency of MDSCs in peripheral blood was determined by flow cytometry, and mRNA expression in purified MDSCs was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The suppressive function of MDSCs isolated from different groups was also determined. The plasma levels of certain cytokines were determined using Bio-Plex Pro™ Human Cytokine Assays. Results: The frequency of circulating CD14+HLA-DR-/low MDSCs; arginase-1 (Arg-1) expression; and plasma levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-33 were markedly increased in ACS patients compared to stable angina (SA) or control patients. Furthermore, MDSCs from ACS patients were more potent suppressors of T-cell proliferation and IFN-γ production than those from the SA or control groups at ratios of 1:4 and 1:2; this effect was partially mediated by Arg-1. In addition, the frequency of MDSCs was positively correlated with plasma levels of IL-6, IL-33, and TNF-α. Conclusions: We observed an increased frequency and suppressive function of MDSCs in ACS patients, a result that may provide insights into the mechanisms involved in ACS.

Inhibition of IFN Regulatory Factor-1 Down-Regulate Th1 Cell Function in Patients with Acute Coronary Syndrome

BackgroundThe crucial role of T helper (Th) cells and chronic inflammation in atherosclerosis and coronary artery disease is no longer controversial. Evidence has revealed that Th cell type 1 (Th1) is closely associated with the pathogenesis of acute coronary syndrome (ACS). But the mechanisms involved in the generation of Th1 cells have not been fully elucidated. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor involved in innate immunity and chronic inflammation disease. The study was undertaken to investigate the potential effect of IRF-1 on the Th1 cell function in patients with ACS in vitro.MethodsPatients with clinical presentation of chest pain, stable angina, unstable angina, and acute myocardial infarction were enrolled in this study. Circulating CD4+ T cells were enriched and analyzed for mRNA and protein expression of IRF-1. Silencing IRF-1 gene with small interfering RNA in CD4+ T cells from patients with ACS was performed to explore the possible mechanisms involved in ACS.ResultsThe results demonstrated that the expression of IRF-1 in CD4+ T cells was significantly increased in patients with ACS and positively correlated with plasma Th1 cytokine profile. Inhibition of IRF-1 in CD4+ T cells from patients with ACS prevented the induction of the frequencies and cytokines expression of Th1 cells. In addition, this study also revealed that IRF-1 modulate Th1 differentiation through establishing IL-12 responsiveness by acting on IL-12 receptor β1.ConclusionThe present data demonstrate that inhibition of IRF-1 obviously decrease the function of Th1 cells and may be a novel participator in the progress of ACS.

Genome-Wide Analysis of DNA Methylation and Acute Coronary Syndrome.

Rationale: Acute coronary syndrome (ACS) is a leading cause of death worldwide. Immune functions play a vital role in ACS development; however, whether epigenetic modulation contributes to the regulation of blood immune cells in this disease has not been investigated. Objective: We conducted an epigenome-wide analysis with circulating immune cells to identify differentially methylated genes in ACS. Methods and Results: We examined genome-wide methylation of whole blood in 102 ACS patients and 101 controls using HumanMethylation450 array, and externally replicated significant discoveries in 100 patients and 102 controls. For the replicated loci, we further analyzed their association with ACS in 6 purified leukocyte subsets, their correlation with the expressions of annotated genes, and their association with cardiovascular traits/risk factors. We found novel and reproducible association of ACS with blood methylation at 47 cytosine-phosphoguanine sites (discovery: false discovery rate <0.005; replication: Bonferroni corrected P<0.05). The association of methylation levels at these cytosine-phosphoguanine sites with ACS was further validated in at least 1 of the 6 leukocyte subsets, with predominant contributions from CD8+ T cells, CD4+ T cells, and B cells. Blood methylation of 26 replicated cytosine-phosphoguanine sites showed significant correlation with expressions of annotated genes (including IL6R, FASLG, and CCL18; P<5.9×10−4), and differential gene expression in case versus controls corroborated the observed differential methylation. The replicated loci suggested a role in ACS-relevant functions including chemotaxis, coronary thrombosis, and T-cell–mediated cytotoxicity. Functional analysis using the top ACS-associated methylation loci in purified T and B cells revealed vital pathways related to atherogenic signaling and adaptive immune response. Furthermore, we observed a significant enrichment of the replicated cytosine-phosphoguanine sites associated with smoking and low-density lipoprotein cholesterol (Penrichment⩽1×10−5). Conclusions: Our study identified novel blood methylation alterations associated with ACS and provided potential clinical biomarkers and therapeutic targets. Our results may suggest that immune signaling and cellular functions might be regulated at an epigenetic level in ACS.

Heme Oxygenase-1 Restores Impaired GARP+CD4+CD25+ Regulatory T Cells from Patients with Acute Coronary Syndrome by Upregulating LAP and GARP Expression on Activated T Lymphocytes

Background: Accumulating evidence shows that the pathological autoreactive immune response is responsible for plaque rupture and the subsequent onset of acute coronary syndrome (ACS). Naturally occurring CD4+CD25+regulatory T cells (nTregs) are indispensable in suppressing the pathological autoreactive immune response and maintaining immune homeostasis. However, the number and the suppressive function of glycoprotein-A repetitions predominant (GARP) + CD4+ CD25+ activated nTregs were impaired in patients with ACS. Recent evidence suggests that heme oxygenase-1 (HO-1) can regulate the adaptive immune response by promoting the expression of Foxp3. We therefore hypothesized that HO-1 may enhance the function of GARP+ CD4+ CD25+Tregs in patients with ACS and thus regulate immune imbalance. Methods: T lymphocytes were isolated from healthy volunteers (control, n=30) and patients with stable angina (SA, n=40) or ACS (n=51). Half of these cells were treated with an HO-1 inducer (hemin) for 48 h, and the other half were incubated with complete RPMI-1640 medium. The frequencies of T-helper 1 (Th1), Th2, Th17 and latency-associated peptide (LAP) +CD4+ T cells and the expression of Foxp3 and GARP by CD4+CD25+T cells were then assessed by measuring flow cytometry after stimulation in vitro. The suppressive function of activated Tregs was measured by thymidine uptake. The levels of transforming growth factor-1 (TGF-β1) 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. Results: Patients with ACS exhibited an impaired number and suppressive function of GARP+ CD4+ CD25+Tregs and a mixed Th1/Th17-dominant T cell response when compared with the SA and control groups. The expression of LAP in T cells was also lower in patients with ACS compared to patients with SA and the control individuals. Treatment with an HO-1 inducer enhanced the biological activity of GARP+ CD4+ CD25+Tregs and resulted in increased expression of LAP and GARP by activated T cells. Conclusions: The reduced number and impaired suppressive function of GARP+ CD4+ CD25+Tregs result in excess effector T cell proliferation, leading to plaque instability and the onset of ACS. HO-1 can effectively restore impaired GARP+ CD4+ CD25+Tregs from patients with ACS by promoting LAP and GARP expression on activated T cells.

Impairment of Circulating CD4 + CD25 + GARP + Regulatory T Cells in Patients with Acute Coronary Syndrome

Background: Atherosclerosis (AS) is an inflammatory and immune disease. Regulatory T cells (Tregs) suppress the activation of T cells and have been shown to play a protective role during the pathogenesis of AS. However, specific markers for Tregs are lacking. Recently, glycoprotein A repetitions predominant (GARP) was discovered as a specific marker of activated Tregs, and we therefore utilized GARP as a specific surface marker for Tregs in the current study. Methods: To assess whether GARP+ Tregs are downregulated in patients with acute coronary syndrome (ACS), we examined CD4+CD25+GARP+ T cell frequencies as well as their associated cytokines and suppressive function. Additionally, we compared GARP expression to that of FOXP3, which may be more sensitive as a marker of activated Tregs in patients with ACS. Results: Patients with ACS demonstrated a significant decrease in circulating CD4+CD25+GARP+ Tregs. Moreover, the suppressive function of Tregs and levels of related cytokines were also impaired in ACS patients compared to those with stable angina (SA) or normal coronary artery (NCA). Additionally, after TCR stimulation, peripheral blood mononuclear cells (PBMCs) from patients with ACS exhibited a decrease in CD4+CD25+GARP+ Tregs. Conclusions: These fnding indicate that circulating CD4+CD25+GARP+ Tregs are impaired in patients withACS. Thus, targeting GARP may promote the protective function of Tregs in ACS.

Thymic Stromal Lymphopoietin-Induced HOTAIR Activation Promotes Endothelial Cell Proliferation and Migration in Atherosclerosis

Endothelial cells’ (EC) injury is a major step for the pathological progression of atherosclerosis. Recent study demonstrated that thymic stromal lymphopoietin (TSLP) exerts a protective role in atherosclerosis. However, the effect of TSLP and the exact molecular mechanism involved in EC remains unknown. In the present study, we found that long noncoding RNA (lncRNA) HOTAIR was much lower in EC from atherosclerotic plaque. Functional assays showed that HOTAIR facilitated cell proliferation and migration, and suppressed apoptosis in EC. Moreover, we demonstrated that TSLP functions upstream of HOTAIR. We found that serum level of TSLP was decreased in atherosclerosis patients and serum TSLP level positively correlated with HOTAIR expression in EC. Further investigation demonstrated that TSLP activated HOTAIR transcription through PI3K/AKT-IRF1 pathway and then regulates the EC proliferation and migration. TSLP-HOTAIR axis also plays a protective role in low-density lipoprotein (ox-LDL)-induced EC injury. Taken together, TSLP-HOTAIR may be a potential therapy for EC dysfunction in atherosclerosis.

Regulatory T Cells Protect Fine Particulate Matter-Induced Inflammatory Responses in Human Umbilical Vein Endothelial Cells

Objective. To investigate the role of CD4+CD25+ T cells (Tregs) in protecting fine particulate matter (PM-) induced inflammatory responses, and its potential mechanisms. Methods. Human umbilical vein endothelial cells (HUVECs) were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm2) of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4+CD25− T cells (Teff), or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined. Results. Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and inflammatory cytokines, such as interleukin (IL-) 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1) to endothelial cells was increased and NF-κB activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF-κB activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors. Conclusions. Tregs could attenuate fine particles-induced inflammatory responses and NF-κB activation in HUVECs.

Increased IL-37 concentrations in patients with arterial calcification

BACKGROUND Our previous study indicates that IL-37 plays a critical role in both atherosclerosis and arterial calcification. However, whether IL-37 concentrations are significantly changed in patients with arterial calcification has not yet been investigated. METHODS Anterior tibial arterial wall specimens were obtained from 8 patients with type 2 diabetes mellitus and 8 patients who experienced a traffic accident. IL-37 expression was measured by immunohistochemistry in the calcified and the normal samples. In addition, plasma IL-37 concentrations were measured in 75 patients with coronary artery calcification (CAC) and 50 patients without coronary artery calcification (NCAC). RESULTS High concentrations of IL-37 were detected in calcified samples, whereas low concentrations of IL-37 were detected in the normal arteries. Macrophages and vascular smooth muscle cells were the main source of IL-37. Plasma IL-37 concentrations were significantly increased in CAC patients compared with NCAC patients. A correlation analysis showed that IL-37 was positively correlated with age, fasting glucose, alkaline phosphatase, IL-6, TNF-α, C-reactive protein and Agatston scores. Binary logistic regression analyses demonstrated that fasting glucose and IL-37 were independently associated with the presence of CAC. CONCLUSIONS Increased IL-37 concentrations are associated with the onset of arterial calcification.