Turk J Biochem 2019; 44(6): 797–802

Research Article

Tuğba Kandemir Gülmez, Can Acipayam*, Metin Kilinç and Nurten Seringeç Akkeçeci The role of -9 and interleukin-17 in myocarditis with different etiologies Farklı etyolojilerle oluşan miyokarditlerde interlökin-9 ve interlökin-17’nin rolü https://doi.org/10.1515/tjb-2018-0491 Results: It was found that IL-17 levels were statistically Received December 2, 2018; accepted February 28, 2019; previously significant in all acute rheumatic fever, Myocarditis ­published online March 30, 2019 and Kawasaki patients compared to the control group Abstract (p = 0.001) and that cut-off was 4.30 pg/mL. This value was determined to be 71% sensitive and 67% specific for Objective: Myocarditis is an inflammatory disease of the IL-17 (AUC = 0.761). cardiac muscle. Prognosis is most often good but, in some Conclusions: Both of the mean and median levels of patients, the disease can be fulminant. Our aim with this IL-17 were significantly higher in pediatric patients with study was to determine interleukin-9 (IL-9) and interleukin-17 myocarditis than in healthy children. Our study made us (IL-17) levels in myocarditis cases with different etiologies. think that complications of myocarditis and associated Materials and methods: Thirty one patients with myocar- morbidity can be prevented by IL17 inhibitors. The high ditis and 30 healthy controls of similar age and gender levels of IL17 found in our study may be a reference for without a history of chronic disease were included in the future study. study. All 31 patients were clinically myocarditis. In some of Keywords: Acute rheumatic fever; Myocarditis; Kawasaki; these patients, the cause of myocarditis is acute rheumatic IL-9; IL-17. fever or Kawasaki disease. Serum samples of the patients were taken during diagnosis in order to analyze serum IL-9 and IL-17 levels and sedimentation rate, CRP, ASO, pro- Öz BNP, CK-MB, and Troponin-I tests were performed. Amaç: Miyokardit, kalp kasını tutan inflamatuar bir has- talıktır. Prognoz genel olarak iyidir, ancak bazı hastalarda *Corresponding author: Can Acipayam, MD, Department of hastalık fulminant seyredebilir. Bu çalışma ile amacımız, Pediatric, Division of Pediatric Hematology and Oncology, Faculty of farklı etiyolojili miyokardit olgularında IL-9 ve IL-17 düzey- Medicine, Sutcu Imam University, Avşar Kampüsü, Kahramanmaras lerini belirlemektir. 46100, Turkey, Phone: +90-3443003765, Fax: +90-3443003409, Gereç ve yöntem: Çalışmaya 31 miyokarditli hasta ve e-mail: [email protected]. https://orcid.org/0000-0002- kronik hastalık öyküsü olmayan benzer yaş ve cinsiyet te 6379-224X Tuğba Kandemir Gülmez: Department of Pediatrics, Faculty 30 sağlıklı kontrol dahil edildi. 31 hastanın tümü klinik of Medicine, Sutcu Imam University, Kahramanmaras, Turkey, olarak miyokardit idi. Bu hastaların bazılarında, miyokar- e-mail: [email protected] ditin nedeni akut romatizmal ateş veya Kawasaki hastalı- Metin Kilinç: Department of Biochemistry, Faculty of Medicine, ğıdır. Serum IL-9 ve IL-17 düzeylerini analiz etmek için tanı Sutcu Imam University, Kahramanmaras, Turkey, sırasında serum örnekleri alındı ​​ve sedimantasyon hızı, e-mail: [email protected] Nurten Seringeç Akkeçeci: Department of Physiology, Faculty CRP, ASO, pro-BNP, CK-MB ve Troponin-I testleri yapıldı. of Medicine, Sutcu Imam University, Kahramanmaras, Turkey, Bulgular: Kontrol grubu ile karşılaştırıldığında Akut e-mail: [email protected] romatizmal ateş, Kawasaki ve diğer nedenlerle oluşan tüm 798 Tuğba Kandemir Gülmez et al.: The role of interleukin-9 and interleukin-17 in myocarditis miyokardit hastalarında IL-17 düzeylerinin ­istatistiksel invocation, activation and migration of neutrophils. Th-17 olarak anlamlı fark olduğu bulundu (p = 0.001) ve cut-of cells are among the main triggers of tissue inflammation değeri 4.30 pg/mL idi. Bu değerin IL-17 için %71 duyarlı ve and they have been found to be related to the pathogene- %67 spesifik olduğu tespit edildi (AUC = 0.761). sis of the most of experimental autoimmune diseases and Sonuçlar: Miyokarditli pediatrik hastalarda IL-17’nin orta- various inflammatory events in humans. ­IL-17-mediated lama ve ortanca düzeyleri sağlıklı çocuklardan anlamlı inflammation is not protective, has serious immuno- derecede yüksekti. Çalışmamız, miyokardit kompli- pathological consequences and carries a risk of activation kasyonlarının ve myokardite bağlı morbiditenin IL-17 of auto­immunity [11]. IL-9 is a that has a pleio- inhibitörleri tarafından önlenebileceğini düşündürdü. tropic effect on different cell types and that is produced Çalışmamızda bulunan yüksek IL-17 düzeyleri gelecekteki by T cells. Biological functions of IL-9; stimulating prolif- çalışmalar için referans olabilir. eration of activated T cells, increasing the production of immunoglobulin in B cells, stimulating the proliferation Anahtar Kelimeler: Akut romatizmal ateş; Miyokardit; and differentiation of mast cells and stimulating hemat- Kawasaki; IL-9; IL-17. opoietic stem cells [12]. During the literature review, it was determined that there are several studies on IL-9 and IL-17. However, there Introduction is no study conducted with myocarditis patients. This makes our study first in investigating the relation with Myocarditis is characterized with inflammation and IL-17 and IL-9 in myocarditis patients. necrosis of myocardium and is developed independently from coronary artery disease [1]. Among acquired cardiac diseases, it has very high morbidity and mortality in child- hood and young adults, especially in the neonatal period Methods [2]. Therefore, in our study, we received myocarditis cases with various causes such as acute rheumatic fever (ARF), Myocarditis cases who applied to Kahramanmaras Kawasaki disease which affect the heart throughout life. Sutcu Imam University, Faculty of Medicine Research We have investigated interleukin-9 (IL-9) and interleu- and Application Hospital Pediatrics Department kin-17 (IL-17) levels, which have previously been involved between February and August 2017 and who developed in inflammatory diseases by thinking that there is an auto- myocarditis due to various etiologies were included in inflammatory process in myocarditis cases with different the study. etiologies. The Ethics Committee approval was obtained for Coronary thrombus, myocardial infarction, and cor- this study. The content of the study was explained to respondingly sudden death can develop in patients the relatives of the patients included in the study in diagnosed with Kawasaki disease who do not receive detail and their written consents were taken. In this treatment. The risk of coronary artery disease significantly study, there were 31 patients in the patient group and reduces with early diagnosis and treatment [3]. 30 individuals in the control group. Patients who were Cardiac involvement is seen in nearly half of the cases aged between 0 and 18 years and who were recently in ARF, the most common and most important cause of diagnosed with myocarditis, ARF and Kawasaki disease acquired cardiac diseases in childhood [4, 5]. In acute were included in the study. All of our patients have myo- rheumatic fever, cardiac involvement varies from mild carditis but six patients had myocarditis due to ARF and and temporary cardiac involvement to exudative pancar- seven patients had myocarditis due to Kawasaki disease. ditis showing a mortal course [6]. Patients were evaluated with echocardiography (ECO) The pathological basis of myocarditis is associated who were diagnosed clinically compatible with myo- with viral cytopathic effect [7], whereas, it is associated carditis. The diagnosis of myocarditis was confirmed by with infectious agents and superantigen theory in Kawa- ECO. The control group consisted of 30 healthy children saki disease [8] and with cross-reactivity of antibodies who were in the similar age group and gender with the produced against streptococci in ARF [9, 10]. These find- patient group, who did not have a history of the previ- ings show that these diseases have an immunological ous known cardiac disease, who did not have chronic basis. disease history and who did not get any infection diag- Th-17 cells produce IL-17. IL-17 is involved in the nosis. The age and gender were similar in the patient inflammatory process. IL-17 is the key cytokine in the and control groups. Tuğba Kandemir Gülmez et al.: The role of interleukin-9 and interleukin-17 in myocarditis 799

Blood samples of the patients included in the study disease (89.33 IU/mL) and with myocarditis (254.33 IU/mL) were taken for sedimentation rate, CRP, ASO, Pro-BNP, (p < 0.05). There was no statistically significant difference CK-MB and Troponin-I during the diagnosis and tested on found between subgroups in terms of sedimentation rate, the same day. 4–5 cc venous blood samples were collected pro-BNP, CKMB, and Troponin-I. from the patients during the diagnosis in order to analyze There was no statistically significant difference deter- IL-9 and IL-17 levels in biochemistry tubes. Samples stored mined between the control group and the patient group at −80°C were dissolved at room temperature. Based on in terms of serum IL-9 level (p = 0.077) (Table 1). There the recommendations of the company, samples were was no statistically significant difference found among analyzed using ‘Enzyme-Linked ­Immunosorbent Assay’ the patient subgroups (ARF-Kawasaki, ARF-Myocarditis (ELISA) method with Human IL-17A Platinum ELISA and and Kawasaki-Myocarditis) in terms of IL-9 measurement Human IL-9 Platinum ELISA kits in the ELISA reader (p-values: 0.568, 0.952, 0.368, respectively). (Thermo Scientific Multiscan FC Elisa Reader). The results There was a statistically significant difference noticed obtained were given in pg/mL. between the control group and the patient group in terms The findings obtained from the experiments were of serum IL-17 level (p = 0.001) (Table 1). It was found that analyzed using “SPSS 16.0 for Windows” statistical IL-17 levels were statistically significantly higher in all package software. The conformity of the data to a normal patients with myocarditis. There was no statistically sig- distribution was tested using Kolmogorov-Smirnov nificant difference found between ARF-Kawasaki, ARF- Test and the homogeneity of variances was tested Myocarditis and Kawasaki-Myocarditis subgroups in using “Homogeneity of Variance Test-Levene Statistic”. terms of IL-17 measurement (p-values: 0.668, 0.155, 0.205, “Independent Sample T Test” was used for the inde- respectively) (Figures 1 and 2). pendent samples in the analysis of the data that were Roc Analysis was performed for the IL-17 level which not conforming to a normal distribution and that were was found to be statistically high at a significant level not homogeneous variances. “Mann-Whitney U Test” and cut-off was found as 4.30 pg/mL. This value was and “Kruskal-Wallis Variance Analysis” were used for determined as 71% sensitive and 67% specific for IL-17 the analysis of the data that were non-conforming to a (AUC = 0.761). normal distribution and that were non-homogeneous data. Mann-Whitney U-test was used for the double com- parisons and statistical significance was evaluated by Table 1: IL-9 and IL-17 measurements in patient and control groups. performing Bonferroni correction. “Chi-square test” was used for the analysis of the qualitative data. For statis- Groups/parameters IL-9 (pg/mL) IL-17 (pg/mL) tical significance, the probability value (p-value) was Control 4.412 ± 0.730 4.272 ± 0.743 selected as 0.05. Test results that had a p-value lower Patient 4.049 ± 0.841 4.805 ± 0.832a than 0.05 were considered significant. All values were ARF 4.053 ± 0.720 5.353 ± 1.357 given as “mean ± standard deviation”. Kawasaki 4.255 ± 4.433 5.002 ± 0.699 Myocarditis 3.978 ± 0.995 4.527 ± 0.459

aUsing “Kruskal-Wallis Variance Analysis”, p < 0.05, significant.

Results IL-9 and IL-17 measurements in patient and control groups 6 There were 31 patients in the patient group and 30 indi- viduals in the control group. Of the patients, 23 were 5 male and 8 were female and the mean age was found as 4 8.7 years (1 year–17 years).

Biochemical parameters were assessed in patients 3 IL-9 with ARF, myocarditis and Kawasaki disease. The mean ogram/L IL-17 pik CRP value was 69.71 mg/L in patients with acute rheumatic 2 fever and 16.94 mg/L in myocarditis group, and there was 1 a significant difference found between them (p = 0.005).

ASO levels of patient subgroups were compared and ASO 0 Control Patient levels of patients with ARF (949.86 IU/mL) were found to be significantly higher than those in patients with Kawasaki Figure 1: IL-9 and IL-17 measurements in patient and control groups. 800 Tuğba Kandemir Gülmez et al.: The role of interleukin-9 and interleukin-17 in myocarditis

IL-9 and IL-17 measurements among patient sub-groups the fact that IL-17 levels were statistically significantly high 6 in patients with ARF, myocarditis, and Kawasaki diseases

5 that involve autoimmunity in etiology supports this study in terms of the relation between autoimmunity and IL-17. 4 There is no study on IL-17 in myocarditis; however, there are studies supporting the fact that IL-17 level is high 3 IL-9 in acute coronary syndromes among cardiac diseases. IL-17 IL-17 levels were found to be higher in patients with the acute 2 coronary syndrome [17]. In another study conducted on the acute coronary syndrome, it has been thought that 1 IL-17 and other pro-inflammatory are involved in

0 the pathogenesis of acute coronary syndrome [18]. There ARA Kawasaki Miyokardit are also studies investigating IL-17 levels in atherosclero- Figure 2: IL-9 and IL-17 measurements among patient subgroups. sis cases. In mice in which experimental atherosclerosis model has been developed by creating Apolipoprotein E deficiency, it has been found that IL-17A is functionally Discussion blocked, resulting in decreased infiltration of inflammatory cells into atheroma plaques and development of vascular Inflammatory factors are involved in the etiopathogenesis inflammation [19]. All these studies support the fact that of myocarditis [13]. For this reason, which are inflammatory IL-17 leads to inflammation in cardiac diseases. In a study markers, IL-9 and IL-17, were evaluated in our study. It has conducted on patients with the acute coronary syndrome, been reported that IL-9, one of the involved in there was a positive relation determined between IL-17A inflammatory response, is responsible for airway hyper- levels and it has been reported that IL-17 leads to platelet sensitivity and increased mucus secretion generally in aggregation by using the ERK2 signaling pathway [20]. asthmatic patients and experimental models in In a study conducted on psoriasis patients, it has the studies. In a previous study conducted on rats, it has been found that Th17 increases in skin lesions of patients been shown that increased IL-9 in transgenic mouse lungs and that anti-TNF agent used in the treatment of psoriasis leads to hyperplasia, increased airway inflamma- disease have an effect by decreasing Th17 cells [11]. In our tion and bronchial hyperreaction [14]. In another study, study, the fact that IL-17 was determined to be high suggests it has been reported that the expression of IL-9 increases that the use anti-TNF agents may give positive results in the the mucus secretion by affecting the sub-groups of mucin treatment processes of these diseases in terms of prognosis. both in vivo and in vitro [15]. In our study, serum IL-9 There has been a significant improvement in plaques with level was found to be higher in the patient group than in treatment, the first anti-IL-17A agent that is the control group; however, it was not statistically signifi- rapidly used in recent years in the treatment of moderate cant. Our study is an original study in terms of IL-9 assess- and severe plaque psoriasis [21]. On the contrary, there is no ment in cardiac patients. In future studies, significant study conducted on secukinumab treatment in childhood. findings in terms of IL-9 may be obtained by performing The fact that a significant response is obtained with anti- repeated measurements in larger patient groups and at dif- IL-17A treatment in psoriasis disease suggested that treat- ferent stages of the diseases in cardiac diseases. ments such as anti IL-17 may be beneficial in patients with A high level of IL-17 in patients with myocarditis was myocarditis; that the disease may be taken under control the most important finding in our study. In this study, the with early treatment before the development of sequelae, mean serum IL-17 level was determined to be higher in the in this study. In two different studies conducted, it has been patient group compared to the control group and the dif- found that cerebrospinal fluid and serum of patients with ference was statistically significant (p = 0.001). It has been multiple sclerosis contains a high level of IL-17 [22–25]. In reported in previous studies that IL-17 has an active role this study, it was suggested that measurement of IL-17 levels in autoimmune diseases, in particular; and that high IL-17 from pleural effusion fluid in patients with cardiac involve- levels in rheumatologic diseases are associated with poor ment progressing with pericardial effusion may contribute prognosis. In a study conducted on patients with rheuma- to the diagnosis and prognosis. toid arthritis, it has been shown that TNF, IL-1 and IL-17 Studies conducted on IL-17 are usually designed to cytokine expressions are high in patients and that this high investigate asthma and its pathophysiology. In these level is a determinant for joint damage [16]. 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