Coronary artery tissue (n=10) was collected from the coronary arteries of patients with CAD who underwent heart transplant surgery. The control samples (n=7) were obtained from deceased donors who had had traffic accidents or strokes and were declared brain-dead by experienced clinicians. Donors had no history of cardiovascular disease, and the samples showed no signs of pathology. All the coronary tissues were collected by surgeons during heart transplant procedures at the People's Hospital of Guangxi Zhuang Autonomous Region (China). Written informed consent was obtained from families, and the study protocol was approved by the Medical Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region.

Coronary samples were lysed, and total protein was collected. After quantification using a bicinchoninic acid (BCA) Protein Assay Kit, 20μg protein were added to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were separated by electrophoresis and were then transferred to Immobilon-FL PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated with anti-IL-5 antibody (Santa Cruz Biotechnology) and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology) antibody at 4° C overnight, followed by incubation with secondary antibody at room temperature for 1 hour. The proteins were visualized and quantified using a 2-color infrared imaging system (Odyssey; LI-COR Biosciences). Protein expression levels were normalized to the GAPDH control.

Total mARN was collected after coronary tissue and cells were lysed using the TRIzol reagent. Then, complementary DNA (cDNA) was synthesized from 2μg of total mRNA using oligo (dT) primers and a reverse transcription kit according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplifications were performed using a LightCycler 480 SYBR Green Master Mix (all from Roche). The primer sequences are listed in Table 1. The relative mRNA expression levels were measured and were normalized to GAPDH in the corresponding sample.

All the coronary samples were fixed with 4% neutral paraformaldehyde, and after being embedded in paraffin, the samples were cut into 5 μm slices and mounted onto slides. Immunofluorescence staining was used to detect IL-5 expression in each sample. To determine the source of IL-5, double immunofluorescence staining with the anti-CD4 antibody, anti-CD68 antibody, and anti-IL-5 antibody was performed.

We selected a whole new set of consecutive patients with chest pain who were suspected to have CAD at the People's Hospital of Guangxi Zhuang Autonomous Region from June 2016 to December 2017. All patients underwent coronary angiography. According to the clinical symptoms and the results of the coronary arteriogram and electrocardiogram, the patients were divided into 4 groups: a) Non-CAD (NCAD, n=36) group. Inclusion criteria: chest pain was not accompanied by electrocardiogram changes, coronary stenosis, or coronary spasm when an intracoronary acetylcholine injection was given during coronary angiography; b) SAP group (n=51). Inclusion criteria: typical exertion-induced chest discomfort associated with down-sloping or a horizontal ST-segment depression> 1mm in an exercise test; c) UAP group (n=44). Inclusion criteria: chest pain at rest with definite ischemic electrocardiographic changes: ST-segment changes and/or T-wave inversions, and d) Acute myocardial infarction (AMI, n=29) group. Inclusion criteria: myocardial infarction confirmed by a significant rise in troponin I and creatine kinase MB levels.

The standard of grouping was based on a previous study by Lin et al.19 We excluded patients who had had an AMI within the previous 3 months, or unstable angina pectoris within the previous month.20 We also excluded patients with evidence of significant concomitant diseases, in particular, hemodynamic valvular heart disease, cardiomyopathy, known malignant diseases, and febrile conditions.21 The diagnosis was established the clinicians with extensive clinical experience. Gensini scores were used to assess stenosis in the coronary arteries. The detailed algorithm of Gensini scores has been previously described in by Ye et al.22

The patients themselves or their families provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region.

Blood samples were obtained upon patient arrival at the emergency unit. Blood samples were collected before coronary angiography was performed and before any treatment was provided. The time of onset for all patients was less than 6hours. The samples were centrifuged at 4000×g for 20minutes and then the supernatants were collected and stored at−80° C until the beginning of the experiments. The samples were removed from a−80° C environment and then thawed at room temperature. Plasma IL-5 (R&D Systems), IL-17, and IFN-γ (both from eBioscience) levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.

Male adult C57BL/6 mice were anesthetized with 2% isoflurane, and the spleens were isolated. The splenic single cell suspension was prepared and cultured in RPMI 1640 (Gibco) complete culture medium. The Cluster of Differentiation 4 (CD4+) Th cells were positively selected using CD4 magnetic beads (Miltenyi Biotech) and an auto magnetic activated cell sorting separator.23 After activation by anti-CD3 and anti-CD28 (both from eBioscience), the CD4+Th cells were treated with oxidized low-density lipoprotein (50 μg/mL, Servicebio) or recombinant mouse IL-5 (rIL-5, Genetex). Then the cells were cultured in a humidified CO2 incubator with 5% CO2 at 37°C for 24hours.24,25

The CD4+Th cells cultured above were collected and stained with FITC (fluorescein isothiocyanate) anti-CD4 (FITC-CD4). After treatment with fixation/permeabilization concentrate, the cells were stained with phycoerythrin (PE)-labeled anti-IFN-γ (PE-IFN-γ) and PE-labeled anti-IL-17 (PE-IL-17). The isotype controls were included for compensation and to confirm antibody specificity. Th1 cells were defined as CD4+/IFN-γ+, and Th17 cells were defined as CD4+/IL-17+. The cell stimulation cocktails and all of the flow cytometry antibodies were purchased from eBioscience and used according to the manufacturer's instructions.

Plasma cytokine concentrations and clinical characteristics are expressed as medians (lower quartile to upper quartile) and were compared with the Mann-Whitney U test. The correlations between IL-17, IFN-γ, clinical characteristics and IL-5 were analyzed using the Spearman correlation analysis. Simple linear regression analysis and subsequent binary linear regression analysis were used to determine whether IL-5 was an independent biomarker of CAD. The data from cell culture are expressed as mean±standard deviation and were compared with Student t tests. All data were analyzed with SPSS 23.0 software, and P <.05 was considered statistically significant.

Among the patients who provided coronary tissue, levels of cardiac troponin I (cTnI) and C-reactive protein (CRP) were significantly higher in the CAD groups than in the deceased donor group. No differences were found between the groups in other clinical characteristics, including age, sex, smoking, drinking, systolic blood pressure (SBP), fasting glucose (Glu), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and diastolic blood pressure (DBP). Clinical data are listed in Table 2.

IL-5 was significantly lower in the coronary plaque of CAD patients than in that of deceased donors (Figure 1A-B). Both CD4+Th cells and macrophages were the source of IL-5, of which the macrophages were the main source (Figure 1C-D).

Figure 1.

Expression and source of IL-5 in human coronary tissue. A The IL-5 levels in the coronary plaque of CAD patients and deceased donors were measured by western blotting. B. The IL-5 mRNA levels in these 2 groups were measured by RT-qPCR. C, D. The source of IL-5 was analyzed by double immunofluorescence staining in coronary tissue (400×). CAD, coronary artery disease; CD, cluster of differentiation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. * P < .05.

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Levels of Glu, TC, SBP, LDL, CRP and cTnI were significantly lower in the NCAD group than in the other 3 groups. Other clinical characteristics, including age, sex, smoking and drinking, TG, HDL, and DBP, showed no obvious differences. None of the clinical characteristics was obviously different between SAP, UAP and AMI groups except SBP and cTnI. SBP and cTnI levels were highest in the AMI group, higher than in the UAP group, and were lowest in the SAP. Clinical data are listed in Table 3.

The results of ELISA showed that IL-5 levels were gradually decreased in the NCAD, SAP, and UAP groups, whereas no differences were found between the UAP and AMI groups (Figure 2A). Both IL-17 and IFN-γ levels gradually increased in the NCAD, SAP, UAP and AMI groups (Figure 2B-C). Spearman correlation analysis showed that IL-5 levels were negatively correlated with IL-17 levels and IFN-γ levels in CAD patients (Figure 2D-E). In addition, IL-17 levels were positively correlated with IFN-γ levels (Figure 2F).

Figure 2.

Cytokines in CAD patients. Plasma IL-5 (A), IL-17 (B), and IFN-γ (C) concentrations in the NCAD and CAD groups. Correlations between IL-5 (D), IL-17 (E) and IFN-γ levels (F). Correlations between Gensini Score and plasma IL-5 (G), IL-17 (H) and IFN-γ (I) levels. AMI, acute myocardial infarction; CAD, coronary artery disease; IFN-γ, interferon-γ; IL, interleukin; NCAD, non-CAD; SAP, stable angina pectoris; UAP, unstable angina pectoris.

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The correlation between IL-5, IL-17, IFN-γ levels and Gensini scores was detected to further determine whether the 3 cytokines are associated with the severity of CAD. The results showed that the Gensini score was positively correlated with IL-17 and IFN-γ levels but was negatively correlated with IL-5 (Figure 2G-I).

The results of the simple linear regression analysis showed that IL-5, IL-17, IFN-γ, cTnI, TC, LDL, and Glu exhibited a trend toward a correlation with the prevalence of CAD (P <.05). Then, the 3 cytokines and 4 clinical characteristics were analyzed by binary linear regression analysis, and the results showed that IL-5 (β=−0.273, 95%CI,−0.455 to−0.091; P=.003), IL-17 (β=0.228, 95%CI, 0.068 to 0.388; P=.006) and IFN-γ (β=0.232, 95%CI, 0.081 to 0.382; P=.003) were associated with the presence of CAD (as shown in Table 4).

The results of flow cytometry analysis showed that oxidized low-density lipoprotein treatment significantly increased both Th1 and Th17 percentages, while regulatory T cell (Treg) numbers decreased (Figure 3A-D). These effects could be reversed by rIL-5. The mRNA of the characteristic Th1 and Th17 cytokines, such as IFN-γ, TBX21, IL-18, RORγT, IL-17, and IL-23, showed a similar trend as the Th1 and Th17 cells, respectively (Figure 3E).

Figure 3.

Effect of IL-5 on Th1, Th17 and Treg differentiation in vitro. (A). Th1 and Th17 cells were identified among CD4+Th cells based on their expression of CD4+IFN-γ+ and CD4+IL-17+, respectively. The Th1 (B), Th17 (C) and Treg (D) levels in control, OX-LDL and OX-LDL+rIL-5 groups. (E). The IFN-γ, TBX21, IL-18, RORγT, IL-17, IL-23 and IL-35 mRNA levels in the 3 groups were measured by RT-qPCR. CD, cluster of differentiation; IFN-γ, interferon-γ; IL, interleukin; OX-LDL, oxidized low-density lipoprotein; PE, phycoerythrin; rIL-5, recombinant mouse IL-5; RoRγ, retinoic acid receptor-related orphan receptor gamma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TBX, T-box transcription factor; Th, T helper cells; Treg, regulatory T cell. aP < .05 vs control group. bP < .05 vs OX-LDL group.

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First, an animal experimental model of ischemic heart disease or infarction is needed to show the deleterious effect of IL-5KO or the beneficial effect of IL-5 stimulation in vivo. Second, our sample size is insufficient, and more patients are needed to validate our results. Third, the study participants enrolled from the hospital may have caused selection bias because inpatients are generally considered to have a more severe disease state. Finally, there was a lack of follow-up visits for CAD patients.