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Abstract

Heart failure (HF) has been major health concern affecting 1%–2% of world adult population. Role of various cytokines in chronic heart failure (CHF) have been demonstrated in different populations; however, association of an important cytokine, interleukin-1β (IL-β), is poorly documented. Furthermore, polymorphism in promoter region is shown to be linked with cytokines levels. In this study, we explored plasma levels of IL-1β in healthy controls (HCs) and different clinical categories of CHF and association of common IL-1β promoter variants with susceptibility to development of HF. In all, 354 CHF patients admitted to Department of Cardiology at the first affiliated hospital of Soochow University were enrolled in this study. These patients were further clinically sub-categorized into New York Heart Association (NYHA)-I to IV based on NYHA criteria. A total of 77 HCs were included in the current investigation. Plasma levels of IL-1β were quantified by enzyme-linked immunosorbent assay (ELISA) and common promoter gene polymorphisms in IL-1β gene were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). CHF patients displayed higher plasma IL-1β compared to HCs. Interestingly, plasma levels of IL-1β were associated with severity of HF patients: NYHA-IV had highest levels, and least quantity was noticed in NYHA-I cases. Prevalence of heterozygous and homozygous mutant for C-511T polymorphisms were significantly higher in CHF patients when compared to HCs. Importantly, these observations remained valid for NYHA-III and IV sub-groups in comparison to controls. Elevated plasma levels of IL-1β were observed in 511 mutants (CT and TT) than wild type (CC), indicating important function variants determining plasma levels of cytokine in both controls and patients. In conclusion, IL-1β (C-511T) variants are associated with elevated plasma IL-1β and predisposed to severe chronic HF in Chinese.

Keywords

Chinese heart failure interleukin polymorphism

Introduction

Heart failure (HF) is an emerging public health concern and is complicated clinical syndrome affecting around 1%–2% of adult population worldwide.¹ The exact etiology of HF is not known; however, multiple factors have been attributed. HF is defined as inability of heart to supply adequate blood and oxygen to peripheral tissue system. The major clinical features include shortness of breath, dyspnea or fatigue, tachycardia, and weakened exercise tolerance. The HF subjects have been classified based on severity of clinical manifestations, and the most established functional stratification criteria was adopted by New York Heart Association (NYHA) as classes I, II, III, and IV based on clinical symptoms and physical activity of the patient.¹

In the last couple of years, there was a significant rise in morbidity and mortality in Chinese population due to cardiovascular ailments. The information from ‘Report on Cardiovascular Disease in China, 2011’ suggested that 230 million people were affected from cardiovascular diseases of which about 4.2 million (0.018%) subjects suffered from HF.²

Various cytokines and inflammatory mediators play vital role in poor prognosis of HF. Particularly, plasma cytokines and chemokines are associated with deterioration of functional classes of HF as graded by NYHA and cardiac performance. For example, increased levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and IL-18 as well as chemokines such as MCP-1 and CXCL-8/IL-8 have been associated with severity of HF.³

Among different inflammatory mediators, IL-1 is an apical pro-inflammatory cytokine that induces many other inflammatory mediators in hosts. Earlier data suggested an essential role of IL-1 signaling in HF by dampening cardiac function and inducing cardiomyocyte apoptosis.⁴ Among different members of IL-1 family, importance of IL1-β in mediating pathogenesis has been realized in the context of HF. An association of serum level IL-1β with acute decompensation of HF contributing to development of vascular damage and atherosclerosis indicates a pathogenic role of the cytokine in acute HF.⁵ Although role of IL-1β has been investigated in case of acute HF, no serious attempt is taken to examine the potential role of this cytokine in the context of chronic heart failure (CHF).

Differential levels of IL-1β have been reported among human and attributed to various gene polymorphisms.^6,7 Mutations at promoter region hamper or facilitate binding of different transcription factors and affect transcription rate and ultimately net protein production. In addition, variants in coding region possibly code deformed protein and degrade out from the blood stream. Various single-nucleotide polymorphisms (SNPs) have been reported in IL-1β gene; however, SNPs those affect plasma levels of cytokines are poorly defined. Four SNPs (C-3737T, G-1464C, C-511T, and T-31C) have been investigated widely in literature and believed to have functional relevance as those located in the promoter region of IL-1β gene.^6,7

In this study, we performed a hospital-based case-control study to examine role of plasma IL-β with severity of CHF in a Chinese population. In addition, possible association of common promoter IL-1β gene polymorphisms with susceptibility/resistance against development of CHF was explored.

Materials and methods

Subjects

Confirmed CHF patients as per NYHA’s criteria after clinically accessed by trained clinicians to all patients admitted to Department of Cardiology at the first affiliated hospital of Soochow University and those patients were subjected to electrocardiogram, chest X-ray, echo Doppler study before enrolled in this study. A total of 354 CHF patients were included in the current investigations comprising four distinct clinical categories (1 NYHA-I (n = 92), (2) NYHA-II (n = 96), (3) NYHA-III (n = 86), and (4) NYHA-IV (n = 80). All patients were treated with standard treatment protocol as instructed by American Heart Association⁸ and European Society of Heart study.⁹ Pool of patients with other disorders/clinical complications such as autoimmune diseases, acute coronary syndromes, liver dysfunctions, chronic infections, and malignancy were excluded from this study. In all, 77 healthy matched subjects with no history of any heart-related anomalies, hailing from similar geographical area, were enrolled as controls. In addition, all controls had no history of autoimmune diseases and hypertension. The study and protocol were approved by Institutional Human Ethical Committee of Soochow University, and written informed consent was obtained from all enrolled subjects. About 5 mL of intravenous blood samples were collected from each participant with ethylenediaminetetraacetic acid (EDTA), mixed thoroughly, and centrifuged at 400g for 20 min for isolation of plasma. Isolated plasmas were stored at −80 C till use for quantification of cytokines. Leftover blood samples were mixed equal amount of 8M urea and stored at −80 C till isolation of genomic DNA.

Isolation of blood genomic DNA

Stored blood samples were thawed and whole genomic DNA was isolated in 200 µL of samples by SIGMA DNA mini blood isolation kit according to manufacturer’s instructions.

IL-1β gene polymorphisms genotyping

The four SNPs (C-3737T, G-1464C, C-511T, and T-31C) in IL-1β promoter region were genotyped by polymerase chain reaction followed by restriction fragment length polymorphism. Primers sequences, amplicon size, restriction enzymes, and fragment size after digestion are mentioned in Supplementary Table 1. About 20% of controls and patients were randomly picked, sequenced, and 100% concordance was noticed between these two genotyping techniques.

Table 1.

Distribution of IL-1β (C-511T) polymorphism in healthy controls and CHF patients.

Genotype/allele	HC (n = 77)	CHF (n = 354)	P value	OR (95% CI)
CC	46 (60)	110 (31)	Ref	1
CT	27 (35)	188 (53)	<0.0001	2.91 (1.71–5.01)
TT	4 (5)	56 (16)	0.0002	5.85 (2.04–15.76)
C	119 (77)	408 (58)	Ref	1
T	35 (23)	300 (42)	<0.0001	2.5 (1.68–3.72)

HC: healthy controls; CHF: chronic heart failure; OR: odds ratio; CI: confidence interval.

Quantification of plasma IL-1β levels

Plasma levels of IL-1β in healthy controls (HCs) and patients were quantified by enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by the manufacturer (R&D Systems).

Statistical analysis

All statistical analyses were conducted in GraphPad prism 6.03. Mean plasma levels of IL-1β in HC and CHF patients were compared by student’s t-test. Furthermore, difference of mean IL-1β levels in different clinical categories was accessed by analysis of variance (ANOVA) followed by Tukey’s post-test. Distribution of IL-1β promoter polymorphisms in HCs and CHF patients or different clinical groups of CHF was analyzed by Fisher’s exact test. A P value less than 0.05 was taken as significant for plasma levels of IL-1β comparison. However, for genotype comparison, a probability value <0.001 (0.05/4) was considered as significant after Bonferroni correction as in this study four SNPs were considered for genetic association study.

Results

Baseline characteristics

In the present hospital-based case-control study, 354 CHF patients were enrolled. Out of them, 238 were male and 116 were female. Furthermore, 77 HCs (gender ratio 7.3 male:2.7 female) were enrolled having mean age of 52.73 years. The mean age of patients was 54.42 years and comparable with those of controls. Based on NYHA criteria, CHF patients were further categorized into NYHA-I, NYHA-II, NYHA-III, and NYHA-IV having mean ± SD left ventricular ejection fraction (LVEF) of 60.82 ± 10.38, 47.93 ± 7.89, 36.26 ± 5.7, and 27.95 ± 8.44, respectively.

Plasma levels of IL-1β in patients and controls

Plasma levels of IL-1β were quantified in both HCs (n = 77) and HF patients (n = 332) by ELISA. The results are shown in Figure 1(a). Plasma level of IL-1β was significantly elevated in patients with CHF as compared to HC, and the difference was statistically significant (P < 0.0001).

Figure 1.

Plasma levels of IL-1β in healthy controls and chronic heart failure patients. (a) Plasma levels of IL-1β were quantified by ELISA in 77 controls and 332 chronic heart failure patients. The mean plasma levels were compared by student’s t-test. Elevated levels of IL-1β were observed in CHF compared to HC (P < 0.0001). Furthermore, CHF patients were sub-categorized into NYHA-I, II, III, and IV and plasma levels of IL-1β were compared among them (b) by ANOVA followed by Tukey’s post test. Severe clinical categories (NYHA-III and IV) had higher levels of IL-1β than mild clinical condition (NYHA-I and II). A P value <0.05 was considered as significant (*: P<0.05; ****: P<0.0001).

Association of plasma IL-1β in different clinical categories of CHF patients

Based on biochemical, clinical, and physical characteristics, CHF patients were further sub-categorized into four major sub-groups (NYHA-I, II, III, and IV), and plasma levels of IL-1β were compared among them. As shown in Figure 1(b), a progressive increase in IL-1β levels was observed among four different classes of CHF patients with maximum titer in NYHA-IV. Furthermore, we investigated possible correlation between plasma IL-1β with LVEF, and no significant correlation of these variables was observed (data not shown).

IL-1β (C-511T) variants are associated with susceptibility to CHF

We genotyped four common SNPs in promoter region of IL-1β gene (C-3737T, G-1464C, C-511T, and T-31C) and analyzed their possible association with development of CHF (Table 1). The prevalence of heterozygous and homozygous mutant of IL-1β C-511T polymorphism was significantly higher in CHF patients when compared to HCs (CT: P < 0.0001, odds ratio (OR) = 2.91, 95% confidence interval (CI) = 1.71–5.01; TT: P = 0.0002, OR = 5.85, 95% CI = 2.04–15.76). Furthermore, the minor allele was also more frequent in CHF cases than HCs (P < 0.0001, OR = 2.5, 95% CI = 1.68–3.72; Table 1). Distribution of other SNPs (C-3737T, G-1464C, and T-31C) was comparable among HCs and CHF patients (data not shown).

IL1β (C-511T) polymorphism is correlated with plasma levels of IL-1β

Polymorphisms at promoter region of gene possibly affect binding of transcription factors and altered expression of mRNA. As the investigated SNPs are located in the promoter region of IL-1 β gene, we hypothesized that variants might be associated with plasma levels of IL-1β. We analyzed the association between four promoter variants with levels of IL-1β and noticed a significant link between IL-1β (C-511T) variant with plasma quantity of IL-1β: mutant displayed higher levels compared to wild type (Figure 2). No significant association was observed between other genetic polymorphisms (C-3737T, G-1464C, and T-31C) and plasma levels of IL-1β (data not shown).

Figure 2.

IL-1β (C-511T) polymorphism is associated with plasma levels IL-1β. Plasma levels of IL-1β in patients and HCs were quantified by ELISA and compared in different genotypes for IL-1β (C-511T) polymorphism. Subjects with TT genotype had higher levels of plasma IL-1β compared to CC and CT. Mean plasma levels of IL-1β in different C-511T genotypes were compared by ANOVA and Tukey’s post test. A P value <0.05 was taken as significant (*: P<0.0001).

IL-1β (C-511T) variants are associated with clinical severity of CHF patients

We observed a significant association of IL-1β (C-511T) variants with susceptibility to development of CHF and altered plasma levels in different clinical categories of CHF patients. Thus, we tested possible association of IL-1β (C-511T) polymorphism with different clinical manifestation of HF. As shown in Table 2, NYHA-III and IV clinical group patients displayed higher prevalence of both heterozygous (NYHA-III: P < 0.0001, OR = 12.35, 95% CI = 5.18–28.46; NYHA-IV: P < 0.0001, OR = 7.09, 95% CI = 3.15–15.72) and homozygous mutants (NYHA-III: P < 0.0001, OR = 28.75, 95% CI = 7.92–87.96; NYHA-IV: P < 0.0001, OR = 17.25, 95% CI = 5.19–51.51) compared to HCs. Similarly, minor allele of IL-1β (C-511T) polymorphism was also highly frequent in severe clinical categories (NYHA-III and IV) than HCs (NYHA-III: P < 0.0001, OR = 4.50, 95% CI = 2.75–7.26; NYHA-IV: P < 0.0001, OR = 3.95, 95% CI = 2.45–6.44).

Table 2.

Prevalence of IL-1β (C-511T) polymorphism in various clinical categories of heart failure patients and healthy controls.

Genotype/allele	HC (n = 77)	NYHA-I (n = 92)	NYHA-II (n = 96)	NYHA-III (n = 86)	NYHA-IV (n = 80)	HC vs NYHA-I (P value; OR; 95% CI)	HC vs NYHA-II (P value; OR; 95% CI)	HC vs NYHA-III (P value; OR; 95% CI)	HC vs NYHA-IV (P value; OR; 95% CI)
CC	46 (60)	42 (46)	48 (50)	8 (9)	12 (15)	Ref; 1	Ref; 1	Ref; 1	Ref; 1
CT	27 (35)	42 (46)	38 (40)	58 (67)	50 (62.5)	0.10; 1.70; 0.90–3.28	0.41; 1.34; 0.71–2.58	<0.0001; 12.35; 5.18–28.46	<0.0001; 7.09; 3.15–15.72
TT	4 (5)	8 (8)	10 (10)	20 (24)	18 (22.5)	0.35; 2.19; 0.68–6.87	0.25; 2.39; 0.71–7.27	<0.0001; 28.75; 7.92–87.96	<0.0001; 17.25; 5.19–51.51
C	119 (77)	126 (68)	134 (70)	74 (43)	74 (46)	Ref. 1	Ref. 1	Ref. 1	Ref. 1
T	35 (23)	58 (32)	58 (30)	98 (57)	86 (54)	0.08; 1.56; 0.96–2.54	0.14; 1.47; 0.90–2.38	<0.0001; 4.50; 2.75–7.26	<0.0001; 3.95; 2.45–6.44

HC: healthy controls; NYHA: New York Heart Association; OR: odds ratio; CI: confidence interval.

Discussion

In this study, we quantified plasma levels of IL-1β and genotyped four common SNPs in promoter of IL-1β gene in a large Chinese cohort. Higher levels of IL-1β were observed in CHF patients compared to HCs and cytokines levels correlated with disease severity. In addition, genotyping of common IL-1β promoter polymorphisms revealed a significant association of -511 variants with predisposition to CHF. Interestingly, plasma levels of IL-1β were also associated with IL-1β (-511) gene polymorphism suggesting as a functional variant controlling levels of IL-1β in plasma. To the best of our knowledge, this is the first study considering both genetic association and measurement of plasma levels in Chinese population.

There are substantial amount of information depicting the role of several pro-inflammatory cytokines in promoting pathogenesis of CHF. Among these inflammatory molecules, the inerleukin-1 family of receptors and ligands constitute the prominent category and is associated with severity of heart diseases. The potential use of IL-1 receptor blockade to improve clinical outcome of HF has been well reported.³ IL-1β constitutes one of the members of IL-1 family that has been used as a therapeutic target for many inflammatory diseases. Owing to the importance of this cytokine in different diseases, we were interested to examine its biological role in the context of CHF in a Chinese cohort.

In this investigation, we found significantly higher levels of serum IL-1β in CHF subjects as compared to HCs. Our data were corroborated with earlier findings suggesting an elevated level of this cytokine in CHF subjects as compared to HCs as observed in a hospital-based study in Bucharest.¹⁰ To correlate the titer of IL-1β with disease severity, we examined the cytokine level in various NYHA classes of CHF and found a gradual elevation. This indicates a relationship of high levels of cytokine with direct deterioration of NYHA functional classes. Although there is no human study available to associate the titer of IL-1β with severity of CHF, earlier experimental studies indicate an enhanced intra-cardiac IL-1β levels correlating directly with increased acute Coxackeivirus B3-induced myocarditis in susceptible strains of mice and disease severity.^11,12 This investigation substantiates the role of this pro-inflammatory cytokine as a potential biomarker to examine severity of CHF.

Reports on association between IL-1β polymorphisms and predisposition to CHF or various clinical categories of CHF are very rare. In this study, we observed a significant association of IL-1β (C-511T) variants with susceptibility to development of CHF. On sub- clinical categorization, our data suggested role of (C-511T) mutant with development of NYHA-III and IV. This is the first report to investigate association of IL-1β gene polymorphism with different clinical categories. However, the mechanism how (C-511T) variants are associated with CHF or clinical severity is not known. A recent study reported association of IL-1β variant (+3954) with susceptibility to coronary slow flow phenomenon (CSFP) in Turkish patients. However, it failed to demonstrate role of IL-1β (-511) and IL1 receptor antagonist (IL-1Ra) variable number tandem repeats (VNTR) with CSFP.¹³

Furthermore, genotype–phenotype linked between IL-1β polymorphism and plasma levels were explored. IL-1β (C-511T) variants were associated with high plasma levels of IL-1β than wild type. The exact reason of such observation is not known. It is possible that changes in nucleotide from ‘C’ to ‘T’ could enhance binding of certain transcription factors and that may lead to over transcription of IL-1β mRNA. However, such association was not noticed for other polymorphism of IL-1β promoter variants. The observation of this study is in line with an earlier report; IL-1β (-511) variants are associated with higher transcript when compared to wild type.⁷

In conclusion, plasma levels of IL-1β are elevated in CHF patients and associated with disease severity. IL-1β (C-511T) genetic variant is correlated with higher levels of plasma cytokine and associated with predisposition to severe CHF in Chinese population. Although we have included larger sample of patients for the current investigation, further studies are required in other population with enrolment of decent number of samples to validate our conclusions.

Supplemental Material

Supplementary_Table – Supplemental material for Association of IL-1β polymorphisms and plasma levels with chronic heart failure: A case-control study in Chinese patients

Supplemental material, Supplementary_Table for Association of IL-1β polymorphisms and plasma levels with chronic heart failure: A case-control study in Chinese patients by Yuan Ji, Jiyong Ge and Xun Li in European Journal of Inflammation

Footnotes

Acknowledgements

We would like to thank all patients and healthy controls for their voluntary participation.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental material

Supplemental material for this article is available online.

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