Mengyang Liao

The Th17/Treg functional imbalance during atherogenesis in ApoE−/− mice

OBJECTIVE Atherosclerosis is a chronic inflammatory disease regulated by T lymphocyte subsets. Recently, CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells and Th17 cells have been described as two distinct subsets and have the opposite effects on autoimmunity. Clinical observation has revealed that the Th17/Treg imbalance exists in patients with acute coronary syndrome. We investigated whether the Th17/Treg functional imbalance existed during atherogenesis in ApoE(-/-) mice. METHODS AND RESULTS Th17/Treg functions at different levels including cell frequencies, related cytokine secretion and key transcription factors were investigated comparatively between ApoE(-/-) mice and their age-matched C57BL/6J mice. The results demonstrated that ApoE(-/-) mice revealed significantly increased secretion of Th17 related cytokines (IL-17 and IL-6) and expression of transcription factor (RORgammat) levels and obviously decreased number in Treg cells, secretion of Treg related cytokines (TGF-beta(1)) and expression of transcription factor (Foxp3) levels as compared with age-matched C57BL/6J mice. Th17 related mediators reached their maximum expression values at the early stage (8-16weeks of age) in ApoE(-/-) mice, and then followed by continuous depression of their expression levels. Meanwhile, the expression of Treg related mediators was much lower in ApoE(-/-) mice than in their age-matched wild-type littermates. CONCLUSIONS Th17/Treg functional imbalance exists during atherogenesis in ApoE(-/-) mice, suggesting a potential role of Th17/Treg imbalance in the formation and progression of atherosclerosis.

Regulatory T cells ameliorate cardiac remodeling after myocardial infarction

Persistent inflammatory responses participate in the pathogenesis of adverse ventricular remodeling after myocardial infarction (MI). We hypothesized that regulatory T (Treg) cells modulate inflammatory responses, attenuate ventricular remodeling and subsequently improve cardiac function after MI. Acute MI was induced by ligation of the left anterior descending coronary artery in rats. Infiltration of Foxp3+ Treg cells was detected in the infarcted heart. Expansion of Treg cells in vivo by means of adoptive transfer as well as a CD28 superagonistic antibody (JJ316) resulted in an increased number of Foxp3+ Treg cells in the infarcted heart. Subsequently, rats with MI showed improved cardiac function following Treg cells transfer or JJ316 injection. Interstitial fibrosis, myocardial matrix metalloproteinase-2 activity and cardiac apoptosis were attenuated in the rats that received Treg cells transfer. Infiltration of neutrophils, macrophages and lymphocytes as well as expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were also significantly decreased, and the CD8+ cardiac-specific cytotoxic T lymphocyte response was inhibited. Expression of interleukin (IL)-10 in the heart, however, was increased. Additional studies in vitro indicated that Treg cells directly protect neonatal rat cardiomyocytes against LPS-induced apoptosis, and this protection depends on the cell–cell contact and IL-10 expression. Furthermore, Treg cells inhibited proinflammatory cytokines production by cardiomyocytes. These data demonstrate that Treg cells serve to protect against adverse ventricular remodeling and contribute to improve cardiac function after myocardial infarction via inhibition of inflammation and direct protection of cardiomyocytes.

Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: Initiated by hypoxia inducible factor 1α, presented by exosomes

Excessive tumor necrosis factor-α (TNF-α) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-α is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-α release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-α expression of cardiomyocytes, the role of hypoxia inducible factor-1α (HIF-1α) in TNF-α regulation and potential secretory pathway of TNF-α. Elevated TNF-α expression and HIF-1α activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-α mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1α small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1α protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-α gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-α promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-α within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1α initiates expression of TNF-α, mediated by exosomes in cardiomyocytes.

Inhibition of IL-17A in atherosclerosis

OBJECTIVE To determine the effects of interleukin (IL)-17A inhibition on experimental atherosclerosis. METHODS AND RESULTS ApoE(-/-) mice were treated with either rat anti-mouse IL-17A, mouse anti-mouse IL-17A or isotype-matched control antibodies for 12 weeks (n=8-10 per group). Ldlr(-/-) mice were transplanted with IL-17A-deficient or wild type bone marrow (n=8 per group). Rat anti-mouse IL-17A treatment obviously reduced plaque size by 43% (p<0.01) without evidence of reduced IL-17A signaling. In contrast, mouse anti-mouse IL-17A treatment and IL-17A deficiency in bone marrow cells did not alter lesion size despite significant reduction of IL-17A production. CONCLUSIONS Inhibition of IL-17A signaling does not alter lesion development in Th1-biased C57BL/6 ApoE(-/-) and Ldlr(-/-) mice with already low levels of IL-17A production. Alteration of lesion development after repeated injections of rat anti-mouse IL-17A antibody in ApoE(-/-) mice could not be attributed to blockade of IL-17A signaling.

The altered expression of inflammation-related microRNAs with microRNA-155 expression correlates with Th17 differentiation in patients with acute coronary syndrome

MicroRNAs (miRNAs) are a novel class of small, non-coding RNAs that play a significant role in both inflammatory and cardiovascular diseases. Immune cells, especially T helper (Th) cells, are critical in the development of atherosclerosis and the onset of acute coronary syndrome (ACS). To assess whether inflammation-related miRNAs (such as miR-155, 146a, 21, 125a-5p, 125b, 31) are involved in the imbalance of Th cell subsets in patients with ACS, we measured the expression of related miRNAs in patients with acute myocardial infarction (AMI), unstable angina (UA), stable angina (SA) and chest pain syndrome (CPS); analyzed the relationship between miRNA expression and the frequency of Th cell subsets; and observed the co-expression of miR-155 and IL-17A in peripheral blood mononuclear cells (PBMCs) of patients with ACS. The results showed that the expression of miR-155 in the PBMCs of patients with ACS was decreased by approximately 60%, while the expression of both miR-21 and miR-146a was increased by approximately twofold. The expression patterns of miRNAs in plasma correlated with those in PBMCs, except for miR-21, which was increased by approximately sixfold in the AMI group and showed no significant difference between the UA group and the CPS group. We also found that the expression of miR-155 inversely correlated with the frequency of Th17 cells (r=−0.896, P<0.01) and that miR-155 was co-expressed with IL-17A in patients with ACS. In conclusion, our study revealed the expression patterns of inflammation-related miRNAs in patients with ACS and found that miR-155 may be associated with Th17 cell differentiation.

Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis.

In this study, we investigated the hypothesis that regulatory T cells (Treg) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. Treg numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of Treg were determined in vitro. Our data revealed that the suppressive function of Treg from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, Treg numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of Treg from primary T cells and enhanced preexisting Treg function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of Treg by AT. In conclusion, AT significantly increased Treg numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Defective Circulating CD4+CD25+Foxp3+CD127low Regulatory T-cells in Patients with Chronic Heart Failure

Aims: Increasing evidences confirm the role of immune activation in the pathogenesis of chronic heart failure (CHF). Regulatory T cells appear central to the control of immune homeostasis. We assessed the hypothesis that the circulating frequency and function of CD4+CD25+ Foxp3+CD127low T regulatory cells (Tregs) would be deranged in patients with CHF. Methods: Ninety-nine CHF patients due to non-ischemic (NIHF) or ischemic etiology (IHF) and 24 control donors were enrolled in the study. Frequency of circulating Tregs was evaluated by flow cytometry. Foxp3 in peripheral blood mononuclear cells (PBMCs) was assayed at the mRNA level by real-time PCR. Functional properties of Tregs to suppress proliferation and pro-inflammatory cytokines secretion of activated CD4+CD25- T cells were measured by proliferation assay and ELISA. Results: The results demonstrated that CHF patients had significantly lower frequency of circulating Tregs and reduced Foxp3 expression in PBMCs compared with control donors. Moreover, Tregs from CHF patients showed compromised function to suppress CD4+CD25- T cells proliferation and pro-inflammatory cytokines secretion. A similar pattern with reduced Tregs frequency and compromised function was found in both NIHF and IHF patients. Correlation analysis suggested that Tregs frequency and function positively correlated with LVEF, whereas negatively correlated with LVEDD and NT-proBNP in patients with CHF. Conclusions: Our data are the first to demonstrate that frequencies of circulating Tregs in patients with CHF are reduced and their suppressive function compromised independently of the etiology. Defective Tregs may be an underlying mechanism of immune activation in CHF patients.

Atorvastatin Modulates Th1/Th2 Response in Patients With Chronic Heart Failure

BACKGROUND The T-helper (Th)1/Th2 imbalance has been demonstrated to be involved in chronic heart failure (CHF). We sought to determine whether atorvastatin exhibited any effect on CHF through modulating the Th1/Th2 response. METHODS AND RESULTS We measured serum concentrations of interleukin (IL)-12, -18, interferon (IFN)-gamma, IL-4, and IL-10 from 20 controls and 72 patients with nonischemic CHF by enzyme-linked immunosorbent assay. To investigate the effect of atorvastatin in vivo, CHF patients were either classified into a usual therapy group (n = 35) or usual therapy plus atorvastatin (10 mg/day) group (n = 37). Patient serum levels of IFN-gamma and IL-4 were measured at time of admission and 2 weeks after treatment. Peripheral blood mononuclear cells from patients of CHF group were cultured in the presence or absence of atorvastatin (0, 0.4, 1, and 4 micromol/L) in vitro, and IFN-gamma and IL-4 levels were detected. Serum levels of IL-12, IL-18, and IFN-gamma were significantly higher in the CHF group than in the control group. The levels of IFN-gamma and the ratios of IFN-gamma:IL-4 were significantly decreased with atorvastatin treatment both in vivo and in vitro, whereas levels of IL-4 did not differ significantly. CONCLUSIONS Th1 polarization exists in patients with CHF, and atorvastatin can modulate the Th1/Th2 response through inhibiting Th1 cytokine production.

Impaired thymic export and increased apoptosis account for regulatory T cell defects in patients with non-ST segment elevation acute coronary syndrome.

Background: The mechanisms of Treg cell defects in NSTACS patients remain unclear. Results: The frequency of RTE-Treg and TREC content were markedly lower, and apoptosis of Treg cells in NSTACS patients was markedly increased. Conclusion: Impaired thymic output and enhanced apoptosis were responsible for Treg cell defects in NSTACS patients. Significance: Our findings explain the mechanisms of Treg cell defects in NSTACS patients. Regulatory T (Treg) cells play a protective role against the development of atherosclerosis. Previous studies have revealed Treg cell defects in patients with non-ST elevation acute coronary syndrome (NSTACS), but the mechanisms underlying these defects remain unclear. In this study, we found that the numbers of peripheral blood CD4+CD25+CD127low Treg cells and CD4+CD25+CD127lowCD45RA+CD45RO− naive Treg cells were lower in the NSTACS patients than in the chronic stable angina (CSA) and the chest pain syndrome (CPS) patients. However, the number of CD4+CD25+CD127lowCD45RA−CD45RO+ memory Treg cells was comparable in all of the groups. The frequency of CD4+CD25+CD127lowCD45RO−CD45RA+CD31+ recent thymic emigrant Treg cells and the T cell receptor excision circle content of purified Treg cells were lower in the NSTACS patients than in the CSA patients and the CPS controls. The spontaneous apoptosis of Treg cells (defined as CD4+CD25+CD127lowannexin V+7-AAD−) was increased in the NSTACS patients compared with the CSA and CPS groups. Furthermore, oxidized LDL could induce Treg cell apoptosis, and the oxidized LDL levels were significantly higher in the NSTACS patients than in the CSA and CPS groups. In accordance with the altered Treg cell levels, the concentration of TNF-α was increased in the NSTACS patients, resulting in a decreased IL-10/TNF-α ratio. These findings indicate that the impaired thymic output of Treg cells and their enhanced susceptibility to apoptosis in the periphery were responsible for Treg cell defects observed in the NSTACS patients.

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.