Circulating Th17 cells are not elevated in patients with chronic heart failure

Abstract

Background. Increasing evidences have been obtained that immune activation and inflammation play critical roles in the pathogenesis of chronic heart failure (CHF). T helper (Th) 17 cells are a newly found pro-inflammatory T cell subtype. We therefore assessed the hypothesis that circulating Th17 cells increased in patients with CHF. Hypothesis. Th17 cells and its cytokine might be elevated in patients with CHF. Methods. A total of 92 patients with CHF and 59 healthy donors were enrolled in the study. The frequencies of circulating Th17 cells were determined by flow cytometry. The interleukin (IL)-17 protein levels in the serum and supernatant of phytohemagglutinin (PHA)-stimulated periphery blood mononuclear cells (PBMCs) were detected using ELISA and the mRNA expression of retinoic acid-related orphan receptor (ROR)γt, which is the key transcription factor of Th17 cells was measured by RT-PCR. Results. There were no significant differences in the frequency of circulating Th17 cells, serum level of IL-17, and expression of RORγt in PBMCs between CHF patients and healthy controls. IL-17 protein level in the supernatants of PHA-stimulated PBMCs was also comparable between CHF patients and health donors. Conclusions. Circulating Th17 cells are not elevated in patients with CHF.

Key words::

• chronic heart failure
• inflammation
• T helper 17 cells

Introduction

Chronic heart failure (CHF) is one of the most severe global epidemics with high prevalence, incidence and mortality, which has become a major public burden for most country (1). It has been revealed that the etiologies of CHF are various, including coronary heart disease, hypertension, dilated cardiomyopathy, etc. (2). The development and progression of CHF are closely related to the imbalance between pro-inflammatory and anti- inflammatory mediators (3). So investigating the cytokines network has become a hot spot in order to explain the pathogenesis of CHF and explore an effective treatment for CHF, and several attempts have been carried out recently (4–7).

Interleukin 17 (IL-17) as a pro-inflammatory cytokine is mainly produced by CD4⁺ T helper 17(Th17) cells, which is a new subgroup of T cells that is distinct from T helper type 1 and 2 lineages (8,9). Th17 cells and its effector cytokine IL-17 have been demonstrated to play an important role in the inflammatory response and enhance the expression of several inflammatory cytokines, including IL-6, IL-23 and TNF-α (9,10). It has been shown that in several inflammatory disorders, such as rheumatoid arthritis (RA) (11) and inflammatory bowel disease (12), Th17 cells’ frequency and serum IL-17 level were increased, which further contributes to the inflammatory responses.

We and others have reported that Th17 cells and IL-17 were linked to the pathogenesis of several cardiovascular diseases, including atherosclerosis (13–16), hypertension (17), viral myocarditis (18), myocardial ischemia/reperfusion injury (19), and dilated cardiomyopathy (20). To assess whether Th17 cells are elevated in patients with CHF, we compared the frequencies of circulating Th17 cells, the levels of IL-17 in serum and the expression of its key transcriptional factor retinoic acid-related orphan receptor γt (RORγt) in periphery blood mononuclear cells (PBMCs) between CHF patients and healthy controls. We also compared the capacity of PBMCs to produce IL-17 in response to phytohemagglutinin (PHA).

Methods

Subjects

A total of 92 CHF patients (58 men and 34 women, 52 = 13 years old) and 59 healthy blood donors (33 men and 26 women, 47 = 9 years old) were enrolled in the investigation. Patients were classified as having non-ischemic heart failure (NIHF) (n = 52, 31 men and 21 women) if they had no history of myocardial infarction and did not have significant coronary artery stenosis upon coronary angiography. Patients were considered to have ischemic heart failure (IHF) (n = 40, 27 men and 13 women) if the coronary angiography presented significant coronary artery disease (50% stenosis in more than one major epicardial coronary artery) or the patients had a history of myocardial infarction or previous revascularization. The diagnostic criteria is based on clinical history, symptom, physical examination, echocardiography, electrocardiography, chest X-ray, and levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), conforming to available guidelines regarding CHF.

Patients in certain condition as follows were excluded: (1) those took anti-inflammatory drugs, for example, non-steroidal anti-inflammatory drugs, steroids, etc., (2) those suffered with collagen disease, disseminated intravascular coagulation, renal failure, advanced liver disease, malignant disease, thromboembolism, other inflammatory disease (such as pneumonia, septicemia), atrial fibrillation, rheumatic heart disease, valvular heart disease, or congenital heart disease, or (3) those had pacemakers (21). Moreover, those patients with higher serum cholesterol than target values after risk stratification (22), who were under medical treatment (statin) in the recent 3 months, were also excluded. The investigation conforms to the principles outlined in the Declaration of Helsinki. The trial was approved by the ethics committee of Tongji Medical College of Huazhong University of Science and Technology and patients and controls provided written informed consent.

Peripheral blood mononuclear cells (PBMCs) isolation

Blood samples were obtained and PBMCs were prepared using Ficoll-Hypaque density gradient centrifugation (Sigma, USA) for flow cytometric analysis, real-time polymerase chain reaction (RT-PCR), or cell culture. Serum was obtained after centrifugation and stored at −80°C.

Flow cytometric analysis of Th17 cells

Isolated PBMCs were suspended at a density of 2 = 10⁶ in complete RPMI 1640 supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin, 2 mM glutamine and 10% heat-inactivated fetal calf serum (Gibco BRL, USA), and stimulated with ionomycin (1 μmol/L, Sigma, USA), phorbol myristate acetate (PMA) (100 ng/ml, Alexis Biochemicals, USA), and monensin (2 μg/ml, eBioscience, USA), for 4 hours at 37°C. Then the cells were harvested and stained with FITC anti-human CD4 (eBioscience, USA) at 4°C for 20 minutes. After the surface staining, the cells were stained with PE-Cy7 anti-human IL-17A (eBioscience, USA) after fixation and permeabilization according to the manufacturer’s instructions (eBioscience, USA). Isotype controls were given to enable correct compensation and confirm antibody specificity. Finally, stained cells were analyzed on a FACScan cytometer (BD FACSCalibur, USA) equipped with Cell Quest software (BD Bioscience PharMingen) (21).

RORγt expression determined by RT-PCR

According to the manufacturer’s instructions, total RNA of freshly isolated PBMCs was extracted by TRIzol lysis buffer (Invitrogen, USA). And then, cDNA was prepared with Revertra Ace kit (Takara, Japan). The expression of RORγt was detected using SYBR Green Master Mix (Takara, Japan) and samples were analyzed by the ABI Prism7900 Sequence Detection System (Applied Biosystems, USA). Primer pairs were as follows: RORγt: F:5′-TGAGAAGGACAGGGAGCCAA-3′, R:5′-CCACA GATTTTGCAAGGGATCA-3′. For each sample, the expression level of target gene was normalized to that of GAPDH housekeeping gene.

Cell culture

Freshly isolated PBMCs were stimulated with PHA (10 μg/ml) at the density of 2 × 10⁶/ml in complete RPMI1640 with 10% heat-inactivated fetal calf serum (Gibco BRL, USA) for 24 hours. Then the supernatant was collected and stored at −80°C for the detection of IL-17 by ELISA.

IL-17 protein detected by enzyme-linked immunosorbent assay (ELISA)

IL-17 levels in the serum or supernatant of phytohemagglutinin (PHA)-stimulated PBMCs were analyzed using Human IL-17 ELISA Kit (Bender Medsystems, USA), following the manufacturer’s instructions. The minimal detectable concentration was 0.5 pg/ml, and intra-assay and inter-assay coefficients of variation were < 10%. All samples were detected in duplicate.

Statistical analysis

Data are presented as mean ± standard deviation (S.D.). Data were analyzed by one-way ANOVA (more than 2 groups) or student’s t test (2 groups). In all cases, p < 0.05 was considered significant.

Results

Circulating Th17 cells frequencies

The basic clinic characteristics of study subjects were shown in Table I. There were no significant differences in age or gender between the four groups. As shown in Figure 1, the frequencies of Th17 (CD4⁺ IL17⁺ /CD4⁺ T cells) in patients with CHF (0.99 ± 0.72), NIHF (0.98 ± 0.70), and IHF (1.02 ± 0.74) were similar with those in healthy controls (0.83 ± 0.70).

Figure 1. Frequencies of Th17 in the peripheral blood of CHF patients and non-CHF controls. (a) Representative FACS pictures from a single person in each group. (b) The percentage of Th17 cells was comparable among CHF, NIHF, IHF and non-CHF controls. n.s. = not significant.

Table I. Clinical characteristics of the study population.

	CHF patients (n = 92)	NIHF patients (n = 52)	IHF patients (n = 40)	Non-CHF controls (n = 59)
Age (year)	53 ± 13	50 ± 12	55 ± 13	47 ± 9
Gender (male/female)	58/34	31/21	27/13	33/26
NYHA (II/III/IV)	12/52/28	7/29/16	5/23/12	–
LVEF (%)	33.2 ± 10.9*	32.4 ± 10.4*	34.1 ± 11.5*	67.6 ± 4.9
LVEDD (cm)	6.5 ± 0.7*	6.6 ± 0.7*	6.4 ± 0.7*	4.6 ± 0.3
Hypertension (%)	46	15	77	12
NT-proBNP (pg/ml)	4401 ± 4200	4476 ± 3942	4338 ± 4633	–
Medication (%)
ACEI/ARBs	85	92	72	5
Digitalis	35	40	30	0
β-blocker	73	75	71	0
Diuretics	70	69	71	0

Data are presented as mean ± standard deviation (S.D.), percentages or number. NIHF, non-ischemic heart failure; IHF, ischemic heart failure; NYHA, New York Heart Association; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic dimension; NT-ProBNP, N-terminal Pro B-type natriuretic peptide; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker. *p < 0.05 versus non-CHF controls.

Expression of RORγt in PBMCs from patients with CHF

As a key transcription factor for the differentiation of Th17 cells, the expression of RORγt in PBMCs from CHF group and control group was investigated. As shown in Figure 2, the mRNA expression of RORγt was slightly increased in the PBMCs of CHF (1.29 ± 1.45), NIHF (1.25 ± 1.37), and IHF (1.34 ± 1.57) patients compared with those of healthy controls (1.14 ± 1.30), but the differences were not statistically significant.

Figure 2. The ratio of RORγt/GAPDH mRNA were comparable among the four groups. n.s. = not significant.

The concentrations of IL-17 in patients with CHF

As shown in Figure 3, IL-17 protein was only detected in 22 cases of 92 CHF group (23.9%) and 18 cases of 59 control group (30.5%), and the levels of IL-17 were similar in the CHF group (2.03 = 1.03 pg/ml) and control group (2.15 ± 2.33 pg/ml).

Figure 3. The serum levels of IL-17 in CHF patients were similar with that in non-CHF controls. n.s. = not significant.

The levels of IL-17 in the culture supernatants of PHA-stimulated PBMCs in patients with CHF

Since IL-17 levels in the serum were not changed in CHF patients, we further compared the levels of IL-17 in the supernatant of PHA-stimulated PBMCs between the two groups. As shown in Figure 4, IL-17 protein in the culture supernatants of PHA-stimulated PBMCs were detected in 10 cases of 14 CHF patients (71.4%) and 11 cases of 16 non-CHF controls (68.8%) and the levels of IL-17 were not increased in the CHF patients (227.7 ± 146.2 pg/ml) compared to healthy controls (205.8 ± 155.2 pg/ml).

Figure 4. The level of IL-17 in the supernatants of PHA stimulated-PBMCs from CHF patients was similar with that from non-CHF controls. n.s. = not significant.

Discussion

In the present study, we first demonstrated that circulating Th17 cells frequencies, serum IL-17 levels, and the expression of RORγt in PBMCs were comparable between CHF patients and non-CHF controls. We further detected the levels of IL-17 in the supernatants of PHA-stimulated PBMCs and the results indicated that IL-17 levels in the supernatant of PHA-stimulated PBMCs were also similar in CHF patients and non-CHF controls. Taken together, our data suggested that circulating Th17 cells are not elevated in CHF patients.

The results from limited reports concerning the Th17 cells in CHF patients are discordant (4,5). One study (4) involving human subjects from Shandong Province in Northern China reported that the frequencies of circulating Th17 cells in patients with CHF were significantly increased compared with the non-CHF controls. And one study (5) enrolling subjects from the same region reported that the frequency of peripheral Th17 cells, serum levels of Th17 related cytokines, and expression of RORγt were higher in patients with dilated cardiomyopathy (DCM) than those of non-CHF controls. Other two studies (6,7), in which samples were obtained from people in Guangxi Province in Southern China, reported that IL-17 levels in both plasma and culture supernatant of non-stimulated PBMCs from DCM patients were not significantly increased compared to non-CHF controls, while IL-17 levels in the culture supernatants of PHA-stimulated PBMCs and the expression of RORγt in PHA- stimulated PBMCs were significantly elevated in patients with DCM compared to non-CHF controls. However, our study, which has the largest sample size including 92 CHF patients and 59 non-CHF controls, found that the frequencies of circulating Th17, IL-17 levels in the serum and culture supernatants of PHA-stimulated PBMCs were not significantly elevated in CHF patients compared to non-CHF controls, and RORγt mRNA expression did not either. The explanation for the discrepancy may be the differences in study subjects, experimental methods, and reagents.

In the present study, we demonstrated that circulating Th17 cells were not altered in patients with CHF compared with those in non-CHF controls. However, we still could not rule out the possibility that Th17 cells contributed to the progress of CHF. Recently, it has reported that IL-17 promoted myofibroblast to produce MMP through OPG/RANKL and IL-17 is essential for the progression of dilated cardiomyopathy in mice with myocarditis (23,24). We therefore could speculate that cardiac-infiltrated Th17 cells may participate the ventricular remodeling after cardiac injury. Although circulating Th17 cells did not change in CHF patients as demonstrated above, it does not mean that there is on change in cardiac-infiltrated Th17 cells in the disorder. For example, it has been shown that patients with RA had similar number of circulating CD4⁺ CD25^high Treg cells but increased number of synovial CD4⁺ CD25^high Treg cells when compared to non-RA controls (25). Based on this thinking, further study on the cardiac-infiltrated Th17 cells is needed.

Acknowledgements

This work was supported in by National Basic Research Program of China (973 Program: 2007CB512005 and 2012CB517805 to X.C.); Grants from National Natural Science Foundation of China (No. 30871067 and 81170303 to X.C.); Program for New Century Excellent Talents in University of China (NCET-09-0380 to X.C.).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.