Decreased Plasma IL-35 Levels Are Related to the Left Ventricular Ejection Fraction in Coronary Artery Diseases

Background Accumulating evidence shows that the novel anti-inflammatory cytokine IL-35 can efficiently suppress effector T cell activity and alter the progression of inflammatory and autoimmune diseases. The two subunits of IL-35, EBI3 and p35, are strongly expressed in human advanced plaque, suggesting a potential role of IL-35 in atherosclerosis and coronary artery disease (CAD). However, the plasma levels of IL-35 in patients with CAD have yet to be investigated. Methods Plasma IL-35, IL-10, TGF-β1, IL-12 and IL-27 levels were measured using an ELISA in 43 stable angina pectoris (SAP) patients, 62 unstable angina pectoris (UAP) patients, 56 acute myocardial infarction (AMI) patients and 47 chest pain syndrome patients as a control group. Results The results showed that plasma IL-35 levels were significantly decreased in the SAP group (90.74±34.22 pg/ml), the UAP group (72.20±26.63 pg/ml), and the AMI group (50.21±24.69 pg/ml) compared with chest pain syndrome group (115.06±32.27 pg/ml). Similar results were also demonstrated with IL-10 and TGF-β1. Plasma IL-12 and IL-27 levels were significantly increased in the UAP group (349.72±85.22 pg/ml, 101.75±51.42 pg/ml, respectively) and the AMI group (318.05±86.82 pg/ml, 148.88±68.45 pg/ml, respectively) compared with chest pain syndrome group (138.68±34.37 pg/ml, 63.60±22.75 pg/ml, respectively) and the SAP group (153.84±53.86 pg/ml, 70.84±38.77 pg/ml, respectively). Furthermore, lower IL-35 levels were moderately positively correlated with left ventricular ejection fraction (LVEF) in CAD patients (R = 0.416, P<0.01), whereas higher IL-27 levels were weakly negatively correlated with LVEF in CAD patients(R = −0.205, P<0.01). Conclusions The results of the present study show that circulating IL-35 is a potentially novel biomarker for coronary artery disease. Regulating the expression of IL-35 also provides a new possible target for the treatment of atherosclerosis and CAD.


Introduction
Coronary artery disease (CAD) is the leading cause of hospitalization and the predominant contributor to mortality in China. It is now widely spread accepted that atherosclerosis is not only merely a lipid disorder but also a chronic inflammatory disease [1]. Inflammatory cells and pro-inflammatory cytokines are found in both early lesions and advanced lesions, as well as following plaque rupture and during the thrombus formation stage. Anti-inflammatory cytokines, however, do not match the increase of pro-inflammatory cytokines; therefore, the imbalance of anti-inflammatory and pro-inflammatory cytokines results in the progression of atherosclerosis, plaque instability and the subsequent onset of acute coronary syndrome (ACS, including unstable angina pectoris and acute myocardial infarction) [2][3][4].
However, current information on the levels of plasma IL-35 and its potential role in coronary artery disease is still limited. In the present study, we measured the levels of plasma IL-35, IL-10 and TGF-b1 in CAD patients and their relationship to the other parameters of CAD, including plasma lipoproteins, C-reactive protein (CRP), coronary stenosis and left ventricular ejection fraction. Because IL-12, IL-27 and IL-35 belong to the IL-12 family and IL-35 interacts with both IL-12 through p35 and IL-27 through EBI3, the levels of plasma IL-12 and IL-27 were also measured in this study.

Study Subjects
We recruited 208 patients who underwent diagnostic coronary angiography between July 2011 and May 2012 in the People's Hospital of Guangxi Zhuang Autonomous Region, China. The patients were classified into 4 groups: 1: Stable angina pectoris (SAP) group (31 men and 12 women, mean age 61.6612.3). Inclusion criteria: typical exertion-induced chest discomfort associated with down-sloping or a horizontal STsegment depression .1 mm in an exercise test; 2: Unstable angina pectoris (UAP) group (40 men and 22 women, mean age 63.8610.2). Inclusion criteria: chest pain at rest with definite ischemic electrocardiographic changes: ST-segment changes and/or T-wave inversions; 3: Acute myocardial infarction (AMI) group (39 men and 17 women, mean age 64.3611.0). Inclusion criteria: myocardial infarction confirmed by significant rise of troponin I and creatine kinase MB levels; 4: Chest pain syndrome (CPS) group (31 men and 16 women, mean age 61.468.9) whose chest pain was not accompanied by ECG changes, coronary stenosis, or coronary spasm when an intracoronary injection of acetylcholine was given during coronary angiography.
Patients with AMI were treated with aspirin enteric-coated tablets 300 mg plus clopidogrel 300 mg one time in the emergency unit, followed with aspirin enteric-coated tablets 100 mg plus clopidogrel 75 mg daily. Those patients with SAP and UAP were treated with aspirin enteric-coated tablets 100 mg plus clopidogrel 75 mg daily. Clopidogrel is used alone when aspirin is absolutely contraindicated. Other medications such as b-blockers and angiotensin-converting enzyme inhibitors (ACEI) were treated according to the clinical condition of the patients.
Written informed consent was obtained from each patient. The study was approved by the Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, China. The permit number of this study is ''[2011] GS018.'' Patients with valvular heart disease, thromboembolism, collagen disease, disseminated intravascular coagulation, advanced liver disease, renal failure, malignant disease, septicemia or on steroid therapy were excluded from the study.

Blood Sample Collection and Processing
In the AMI group, blood samples were obtained upon the arrival of patients to the emergency unit. Fasting blood samples were obtained the morning following admission for the remainder of the study groups. Samples were collected into Sodium Heparin Vacutainers (Becton-Dickinson) and processed within 1 hour of collection by centrifugation for 15 min at 10006g. The supernatant was then aliquoted and stored at 280uC until analysis. All samples were thawed only once.

ELISA Detection of the Levels of Plasma Cytokines
The levels of plasma IL-35, IL-10, TGF-b1, IL-12 and IL-27 were measured using an enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions (Westtang Bio-tech, Shanghai, China). Intra-assay and inter-assay coefficients of variation for ELISA were ,5% and ,10%, respectively. All samples were measured in duplicate.

Effects of Aspirin and Clopidogrel on the Secretion of Cytokines
To investigate whether IL-35 production is rapidly affected by oral aspirin and clopidogrel in vivo, 60 health college students (aged from 19 to 25 years) from Guangxi Medical University were randomized into three groups (each containing 20 cases), receiving aspirin 100 mg, clopidogrel 75 mg or aspirin 100 mg plus clopidogrel 75 mg for once, respectively. Blood samples were collected before treatment and 24 hours after treatment. Plasma cytokines concentrations were measured using an ELISA.

Gensini Score
The severity of coronary stenosis in patients was estimated by the Gensini coronary score following coronary angiography. The Gensini score was computed by assigning a severity score to each coronary stenosis according to the degree of luminal narrowing and its geographic importance. Reduction in the lumen diameter and the roentgenographic appearance of concentric lesions and eccentric plaques were evaluated (reductions of 25, 50, 75, 90, and 99% and complete occlusion were assigned Gensini scores of 1, 2, 4, 8, 16, and 32, respectively).The score was then multiplied by a factor that incorporates the importance of the lesion's position in the coronary arterial tree as follows: 5 for the left main coronary artery; 2.5 for the proximal left anterior descending coronary artery (LAD) or left circumflex artery (LCX), 1.5 for the mid-LAD; and 1 for the distal LAD, the right coronary artery or the middistal LCX.

Left Ventricular Ejection Fraction assessment
We applied Biplane Simpson's method to quantify the left ventricular ejection fraction (LVEF) in 2-dimensional echocardiography. A GE ViVid E7 ultrasonography machine (GE Healthcare, America) with a transthoracic 1.5-4.3 MHz probe (M5S-D) was used for 2-dimensional echocardiography. Twodimensional echocardiographic examinations were performed on the subjects in the left lateral decubitus position. Standard 4chamber apical view and 2-chamber view were performed, and the dynamic 2-dimensional gray images were obtained at 60-100 frames per second.

Statistical Analysis
All data are given as the mean6SD. The data were analyzed by an ANOVA. Where significance was found, a Newman-Keuls test was performed for post-hoc analysis to detect the difference among groups. Spearman's correlation was used to calculate correlations between plasma cytokines levels and the other measured parameters. In all tests, a value of P,0.05 was considered to be statistically significant.

Basic Clinical Characteristics of Patients
There were no significant differences in age, gender, history of hypertension, diabetes or tobacco use among the four groups. The Gensini score and plasma CRP levels were significantly higher in CAD patients than in the chest pain syndrome group. The LVEF was lower in the SAP and AMI groups than in the chest pain syndrome group. The other parameters of each group, including lipid and lipoprotein fractions, fasting glucose, and pre-hospital medications are listed in Table 1.

Plasma Cytokines Concentrations in CAD Patients
As shown in Table 2 and Figure 1, plasma IL-35, IL-10, TGF-b1, IL-12 and IL-27 levels were detected in each group. The IL-35 concentrations in patients with AMI, UAP, and SAP were significantly decreased compared with those in patients with chest pain syndrome. The IL-10 concentrations in patients with AMI and UAP were significantly lower than those in patients with SAP and chest pain syndrome. The TGF-b1 concentrations in patients with AMI, UAP and SAP were significantly lower than those in patients with chest pain syndrome. IL-12 and IL-27 levels in patients with AMI and UAP were significantly higher than those in patients with SAP and chest pain syndrome. To exclude potential inflammation from the chest pain syndrome group, 30 healthy college students (aged from 21 to 25 years) from Guangxi Medical University were also studied. There were no differences between the chest pain syndrome group and the healthy students (IL- 35

Plasma Cytokines Concentration Effected by Aspirin and Clopidogrel
As shown in Figure 2, the plasma IL-35 levels did not significantly change after treatment with aspirin and clopidogrel. In addition, there was also no significant difference in plasma levels of IL-10, TGF-b1, IL-12 and IL-27 (data not shown).

Plasma Cytokines Concentration and other Measured Parameters
We assessed whether the plasma cytokine levels were associated with the Gensini score used to quantify the severity of coronary artery stenosis in CAD. There was no significant correlation between the plasma cytokine levels and the Gensini score (data not shown). We further assessed whether plasma cytokine levels were associated with lipid and lipoprotein fractions (triglycerides, highdensity lipoprotein cholesterol and low-density lipoprotein cholesterol), fasting glucose, CRP and LVEF in patients with CAD. The results showed that lower IL-35 levels were moderately positively correlated with LVEF in CAD patients (R = 0.416, P,0.01) whereas higher IL-27 levels were weakly negatively correlated with LVEF in CAD patients (R = 20.205, P,0.01) (Figure 3) but not with other parameters (data not shown).There was no significant correlation between the levels of other cytokines and other parameters mentioned above (data not shown).

Discussion
In this study, the plasma levels of the novel anti-inflammatory cytokine IL-35, IL-10, TGF-b1, and two other IL-12 family members, IL-12 and IL-27, were investigated in CAD patients. The results showed that the levels of plasma IL-35, IL-10 and TGF-b1 were dramatically decreased, whereas plasma IL-12 and IL-27 levels were significantly increased in patients with UAP and AMI compared with chest pain syndrome patients. Some studies found that aspirin could efficiently regulate the secretion of cytokines in vitro, whereas some showed contrary results [27][28][29]. In this study, we did not find a significant change in plasma levels of IL-35 and other cytokines after treatment with aspirin and clopidogrel, suggesting that a longer time is required to observe the effect of aspirin and clopidogrel on IL-35 production in vivo. The relationship between the levels of each cytokine and the severity of coronary arteray stenosis was measured by Spearman's correlation. We found that the levels of each cytokine were not related to the Gensini score, suggesting that changes in cytokine levels are  Evidence from atherosclerosis-prone models shows that the development and progression of atherosclerosis is related to the imbalance of anti-inflammatory and pro-inflammatory cytokines [2,3]. Pro-inflammatory cytokines, including IFN-c and TNF-a, are mainly Th1-type cytokines and can be found even in the early stage of the lesion. These cytokines are significantly increased in the shoulder of vulnerable plaques, and constitute a large network system with inflammatory cells that amplify the inflammatory response, leading to plaque rupture and thrombosis. IL-12 is abundant in atherosclerotic plaques and is the principal cytokine that promotes the development of Th1 cells, which secrete IFN-c and play critical pathogenic role in atherosclerosis [30]. The treatment of ApoE knockout mice with recombinant IL-12 aggravated atherosclerosis progression, and higher plasma IL-12 levels were also confirmed as a biomarker in CAD patients [4,31]. Similar to IL-12, IL-27 positively induces Th1-type responses and negatively regulates Th2-type responses [32,33]. Though the exact role of IL-27 in atherosclerosis is unknown, it is reasonable to hypothesize that IL-27 may participate in atherosclerosis and that higher plasma IL-27 levels could be the new biomarker of CAD [34]. However, the changes in serum anti-inflammatory cytokine levels are still controversial. Some studies suggest that the levels of serum anti-inflammatory cytokines were increased in ACS patients, while some studies observed that the levels of serum anti-inflammatory cytokines were significantly decreased [5][6][7][8][9][10][11]35,36]. These studies indicate that the levels of serum antiinflammatory cytokines or the ratio of anti-inflammatory to pro-inflammatory cytokines can be used to predict the incidence of cardiovascular events and the prognosis of ACS patients. Evidence from the CAPTURE study showed that elevated serum IL-10 levels not only at baseline but also before discharge are associated with a favorable outcome in ACS patients [9]. In our previous study, we observed that plasma TGF-b1 levels in ACS patients were lower than those in control and SAP patients, while there was no notable difference in plasma TGF-b1 levels between chest pain syndrome patients and SAP patients [11]. A possible reason for this difference is that the SAP patients in the previous study were mostly Canadian Cardiovascular Society (CCS) class I-II, but the SAP patients treated with drug therapy were considered CCS class III-IV in this study. Therefore, the follow-up of these patients may be very meaningful. The data from this study demonstrate for the first time that the levels of plasma IL-35, a novel antiinflammatory cytokine, are positively correlated with LVEF in patients with CAD, which is an independent predictor of recurrent coronary events and prognosis in CAD patients. Therefore, decreased plasma IL-35 levels not only represent insufficient antiinflammatory activity in vivo but also hold promise as a novel biomarker to assess the prognosis of CAD patients.
IL-35 is an IL-12 family member cytokine composed of an a chain p35 and a b chain EBI3 [12,13].The two subunits also form components of other cytokines. The p35 subunit binds with a p40 subunit to form IL-12 while the EBI3 sub-unit binds with a p28 subunit to form IL-27, another member of the IL-12 family. EBI3 induced in B-lymphocytes by EBV infection encodes a 34-kDa glycoprotein homologous to the p40 subunit of IL-12. EBI3 is expressed at high levels in placental trophoblast cells, activated dendritic cells and lymphocytes and at lower levels in macrophages and endothelial cells, but not in normal resting CD3+ T cells in humans [14,20]. On the other hand, the p35 gene is constitutively expressed at low levels in many cell types. It has been found that a large number of p35 subunits are co-expressed with EBI3. Both Ebi3 and p35 knockout mice show overt autoimmunity or inflammatory disease, suggesting that the EBI3/p35 heterodimer may be an important immunomodulator [14,15,37]. The EBI3/ p35 heterodimer, which is currently designated as IL-35, has been  confirmed to suppress Teff cell activity, expand the effect of Treg cells and attenuate established collagen-induced arthritis [12]. Collision et al. found that both EBI3 and p35 are highly expressed and constitutively secreted by mouse Foxp3+ Treg cells but not by activated Teff cells [13]. Furthermore, the regulatory activity of Treg cells from Ebi3 or p35 knockout mice was significantly reduced compared to that of wild-type Treg cells in vivo and in vitro, suggesting that IL-35 is critical for the regulatory activity of Treg cells [13,38,39]. The role of IL-35 in human Treg cells, however, is more complicated. Studies performed by Devergne et al. and Allan et al. showed that IL-35 is not constitutively expressed by human Treg cells but is instead expressed by activated Teff cells and macrophages, indicating that IL-35 may not be related to the suppressive mechanism of human Treg [20,21]. However, when stimulated by anti-CD3 and anti-CD28, the expression of both EBI3 and p35 in human Treg cells was significantly higher than that in Teff cells. A neutralizing anti-IL-35 antibody completely abolished the suppression of human Treg cells, suggesting that the difference in the role of IL-35 in human Treg cells observed by these studies may be due to the timing of the analysis, the purification techniques, and/or the stimulation used [22]. Furthermore, IL-35 was shown to efficiently induce the conversion of suppressed target Teff cells into the Foxp3-independent Treg population, namely iTr35, in both human and mice [22,23,40,41]. When co-cultured with dendritic cells activated by human rhinovirus (R-DC), iTr35 can also be induced to secrete IL-35. This effect could be reversed by blocking of inhibitory receptors B7-H1 and sialoadhesin on R-DC, suggesting an important mechanism in regulating the IL-35 expression [40]. In addition to inducing the generation of iTr35 cells and suppressing the proliferation of Teff cells, IL-35 performs its biological effect via up-regulating the expression of anti-inflammatory cytokines such as IL-10 and IL-35 and directly inhibiting the activity of other target cells.
Recent studies on IL-35 have focused on disease-prone models and healthy populations, but the role of IL-35 in coronary artery disease has yet to be understood. Liu et al. found that EBI3 and p35 are highly expressed in CD4+T cells from chronic hepatitis B patients, which may contribute to the immune escape of HBV [42]. However, they did not measure the plasma IL-35 levels in their patients. It has been shown that EBV-specific T lymphocytes can be frequently observed in human atherosclerotic plaques, suggesting the ability of these T lymphocytes to secrete IL-35 or IL-27 [43]. In fact, the expression of p35 was only found in the brain, intestine, and spleen, while the expression of EBI3 was found in the placenta, eye, lymph node, and pancreas; however, neither subunit was expressed in the heart or vessels of healthy human subjects [44]. However, IL-35 could be up-regulated following induction in human tissue [26]. A recent study revealed that EBI3 and p35 are expressed in almost all advanced plaque lesions and are co-expressed in atheroma vascular smooth muscle cell, indicating that IL-35 may be secreted by vascular smooth muscle cells [26]. Stimulated by the pro-inflammatory cytokines TNF-a or IFN-c or both, the expression of EBI3 and p35  increased and this effect was attenuated by pretreatment with peroxisome proliferator-activated receptor-c (PPARc) agonist rosiglitazone, suggesting a potential role of IL-35 in the progression of atherosclerosis. When we considered this observation together with our research, it raised a new question: why is the expression of IL-35 up-regulated in plaques but down-regulated in peripheral blood? We speculate that plasma IL-35 is mostly secreted by Treg cells and that the suppressive function of peripheral Treg cells is significantly reduced in acute coronary syndromes [45].
To the best of our knowledge, our study is the first to measure the plasma IL-35 levels in CAD. In conclusion, the results of this study show that the levels of plasma IL-35 are dramatically decreased and positively correlated with LVEF in patients with CAD. Our study also creates two new areas of investigation, namely, the potential of IL-35 as a novel biomarker to assess the onset and prognosis of CAD, and second, IL-35 gene regulation as a highly effective therapeutic tool for the treatment of atherosclerosis and CAD. In prospective studies, we will recruit more patients to observe the changes of plasma IL-35 and confirm the relationship between these changes and the prognosis of CAD. However, the exact role of IL-35 in the atherosclerosis process and in the onset of CAD has yet to be elucidated. Further studies are required to investigate the precise effect and the signaling transduction mechanisms of IL-35 in the atherosclerosis process.

Author Contributions
Conceived and designed the experiments: QWJ. Performed the experiments: YZL YH CL YS LL XYW. Analyzed the data: YZL YH ZDL CL QTZ. Wrote the paper: YFC QWJ.