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Pathological and clinical effects of interleukin-6 on human myocarditis

Open ArchivePublished:April 02, 2021DOI:https://doi.org/10.1016/j.jjcc.2021.03.003

      Highlights

      • Serum interleukin-6 (IL-6) level was increased in patients with myocarditis.
      • IL-6 was expressed in inflammatory cells of cardiac tissue with myocarditis.
      • Higher serum IL-6 level was correlated with severe clinical course in myocarditis.
      • Increased serum IL-6 level reflects myocardial injury and cardiac dysfunction.

      Abstract

      Background

      Numerous basic studies have shown a relationship between interleukin-6 (IL-6) and the development or severity of myocarditis. However, there has been no study in which the effect of IL-6 levels in patients with myocarditis was evaluated.

      Methods

      We enrolled control patients (n = 12) and consecutive patients with acute myocarditis (n = 13), including lymphocytic, eosinophilic, and giant cell myocarditis, and investigated the pathological and clinical effects of IL-6 on human myocarditis.

      Results

      The serum IL-6 level in patients with myocarditis (16.7 [9.9, 103.8] pg/mL) was significantly higher than that in the control patients (1.4 [1.0, 1.9] pg/mL) (P<0.001). Immunohistochemical analysis showed that IL-6 was expressed in infiltrating inflammatory cells of endomyocardial biopsy samples from all patients with myocarditis. Moreover, the log-transformed value of serum IL-6 level showed significant positive correlations with serum creatine kinase (CK) level, CK-MB level, peak CK level, peak CK-MB level and C-reactive protein level (all P ≤ 0.005) and a negative correlation with the left ventricular (LV) ejection fraction (p = 0.014). We divided the patients with myocarditis into a low IL-6 group (9.9 [4.5, 14.2] pg/dL, n = 7) and a high IL-6 group (108.9 [51.1, 130.9] pg/dL, n = 6). The degree of infiltration of IL-6-expressing inflammatory cells in myocardial samples obtained from patients in the high IL-6 group was significantly more severe than that in samples obtained from patients in the low IL-6 group. Furthermore, patients in the high IL-6 group significantly more frequently received catecholamine therapy (P = 0.005), venoarterial extracorporeal membrane oxygenation (P = 0.029), and artificial respirator support (P = 0.021) in the acute phase of myocarditis.

      Conclusion

      The results suggest that there is a strong impact of IL-6 on cardiac injury and dysfunction in patients with myocarditis.

      Key words

      Introduction

      Myocarditis is a relatively rare but potentially life-threatening inflammatory disease that can develop even in healthy people. Viral infection is the primary cause of myocarditis, but it has various other aetiologies including bacterial or protozoal infections, toxins, drug reactions, and autoimmune diseases [
      • Kindermann I.
      • Barth C.
      • Mahfoud F.
      • Ukena C.
      • Lenski M.
      • Yilmaz A.
      • et al.
      Update on myocarditis.
      ].
      The natural course of myocarditis and the interactions and temporal changes in cytokines were shown in previous studies using a murine model of myocarditis [
      • Rose N.R.
      Critical cytokine pathways to cardiac inflammation.
      ,
      • Yue-Chun L.
      • Teng Z.
      • Na-Dan Z.
      • Li-Sha G.
      • Qin L.
      • Xue-Qiang G.
      • et al.
      Comparison of effects of ivabradine versus carvedilol in murine model with the coxsackievirus B3-induced viral myocarditis.
      ]. A recent study showed the time courses of innate and adaptive immune responses in myocardial tissues obtained from patients with acute myocarditis [
      • ichi Kawada J
      • Takeuchi S.
      • Imai H.
      • Okumura T.
      • Horiba K.
      • Suzuki T.
      • et al.
      Immune cell infiltration landscapes in pediatric acute myocarditis analyzed by CIBERSORT.
      ]; however, the pathophysiology of myocarditis in human patients has not been fully elucidated. Additionally, although the efficacy of immunosuppressant treatment for giant cell myocarditis has been demonstrated [
      • Cooper L.T.
      Giant cell and granulomatous myocarditis.
      ,
      • Cooper L.T.
      • Hare J.M.
      • Tazelaar H.D.
      • Edwards W.D.
      • Starling R.C.
      • Deng M.C.
      • et al.
      Usefulness of immunosuppression for giant cell myocarditis.
      ], a fundamental treatment for myocarditis has still not been established.
      Tocilizumab, an anti-human interleukin-6 (IL-6) receptor monoclonal antibody, has been developed as a therapeutic for inflammatory diseases including rheumatoid arthritis and juvenile idiopathic arthritis. Tocilizumab is expected to become a new therapeutic option for treating cytokine release syndrome in severe cases of coronavirus diseas (COVID-19) [
      • Zhang C.
      • Wu Z.
      • Li J.W.
      • Zhao H.
      • Wang G.Q.
      Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality.
      ,
      • Luo P.
      • Liu Y.
      • Qiu L.
      • Liu X.
      • Liu D.
      • Li J.
      Tocilizumab treatment in COVID-19: a single center experience.
      ]. The drug has also been reported to be effective in a mouse model of myocarditis [
      • Savvatis K.
      • Müller I.
      • Fröhlich M.
      • Pappritz K.
      • Zietsch C.
      • Hamdani N.
      • et al.
      Interleukin-6 receptor inhibition modulates the immune reaction and restores titin phosphorylation in experimental myocarditis.
      ]. IL-6 has been shown to contribute to the development of various cardiac diseases via the progression of myocardial damage in human subjects [
      • Kanda T.
      • Takahashi T.
      Interleukin-6 and cardiovascular diseases.
      ], but the effect of IL-6 on myocarditis has not been determined. In this study, we investigated the pathological and clinical effects of IL-6 in patients with acute myocarditis.

      Materials and methods

      Study population

      This study was a retrospective study that included patients with acute myocarditis who received treatment in the cardiac care unit (CCU) of Okayama University Hospital during the period from July 2010 to April 2017. We diagnosed myocarditis as active or borderline myocarditis as defined by Dallas pathological criteria [
      • Aretz Thomas
      • Myocarditis H.
      The Dallas criteria.
      ]. Patients with biopsy samples showing inflammatory infiltrates and associated myocytes with or without necrosis and/or degeneration of adjacent myocytes that were not typical of ischaemic damage were defined as patients having myocarditis. According to these criteria, we enrolled 13 consecutive patients with acute myocarditis (mean age: 47±21 years old, female: n = 4 [31%]) in this study. For the control group, we enrolled 12 patients with primary arrhythmia syndromes without any cardiomyopathy or inflammatory disease (mean age: 50±10 years old, female: n = 5 [42%]) whose age and sex matched those of patients in the myocarditis group. The study was approved by the institutional ethics committee of Okayama University (application number: 1908–010). We also used data for serum IL-6 levels in 48 healthy volunteers (mean age: 43±8 years old, female: n = 21 [44%]) whose age and sex matched those of patients in the myocarditis group that were provided by the National Cerebral and Cardiovascular Center as validation data (application number: M30–060–5). All patients in this study provided written informed consent. Informed consent was obtained from a parent and/or legal guardian for subjects who were less than 18 years of age. This study was conducted according to the principles expressed in the Declaration of Helsinki.

      Myocardial tissue samples and pathological evaluation

      Myocardial tissue samples were collected by endomyocardial biopsy (EMB) from all patients with myocarditis when the patients were admitted to our hospital. We obtained myocardial samples from 8 of the 12 patients in the control groups by EMB, which was performed to rule out the possibility of cardiomyopathy including cardiac sarcoidosis. By using a disposable bioptome (Argon Medical Devices, TX, USA), five biopsy specimens from the right side of the interventricular septum were obtained from each patient with the long sheath technique and the jugular vein approach. Tissues were fixed with 10% formalin, embedded in paraffin, cut into 5-µm-thick sections using a microtome, and placed on adhesive glass slides. The slides were air-dried at room temperature for 2 h.
      Haematoxylin and eosin (H&E) staining was performed using standard protocols. We used H&E staining to evaluate infiltration, type of inflammatory cells and injury to adjacent cardiomyocytes. In this study, we diagnosed patients as having giant cell myocarditis when their cardiac tissue samples showed multinucleated giant cells in inflammatory lesions without sarcoid granuloma [
      JCS Joint Working Group
      Guidelines for diagnosis and treatment of myocarditis (JCS 2009): digest version.
      ,
      • Banba K.
      • Kusano K.F.
      • Nakamura K.
      • Morita H.
      • Ogawa A.
      • Ohtsuka F.
      • et al.
      Relationship between arrhythmogenesis and disease activity in cardiac sarcoidosis.
      ]. Eosinophilic myocarditis was diagnosed when we found eosinophilic infiltrates, eosinophil degranulation, disappearance and fusion of cardiomyocytes, and interstitial edema and fibrosis [
      JCS Joint Working Group
      Guidelines for diagnosis and treatment of myocarditis (JCS 2009): digest version.
      ,
      • Enko K.
      • Tada T.
      • Ohgo K.O.
      • Nagase S.
      • Nakamura K.
      • Ohta K.
      • et al.
      Fulminant eosinophilic myocarditis associated with visceral larva migrans caused by Toxocara canis infection.
      ]. Myocarditis mainly based on lymphocyte infiltration that did not satisfy either of the two above-mentioned diagnostic criteria was diagnosed as lymphocytic myocarditis in this study.

      Immunohistochemical staining

      IL-6 expression was evaluated by performing immunohistochemical staining with an anti-IL-6 antibody. Slides of heart sections embedded in paraffin were deparaffinized, rehydrated, and then subjected to antigen retrieval by boiling (30 min) in 10 mM citrate buffer at pH of 6.0. Immunoenzymatic staining was performed using an antihuman IL-6 antibody (NCL-l-IL6; Leica Biosystems, Newcastle Upon Tyne, UK) and a Leica BOND-III automated immunostainer (Leica Microsystems, Wetzlar, Germany). The antibody was applied at a 1:50 dilution for 60 min at room temperature. Negative control sections were incubated without a primary antibody. The sections were evaluated independently by three authors (N.A., Ka.N., and Ta.T.) without any information about the clinical course and outcome of the patients.
      The degree of in-situ IL-6 expression was scored on the basis of the number of infiltrating inflammatory cells that were positive for anti-IL-6 staining in 5 randomly selected separate fields (× 40) from 3 sections per patient. The grades were based on the average numbers of anti-IL-6-positive inflammatory cells per field and according to the following criteria: grade 0 (n = 0), grade 1 (n = 1–9), grade 2 (n = 10–25), and grade 3 (n>25) (Fig. 1).
      Fig 1
      Fig. 1Representative findings of each grade of anti-IL-6 staining in cardiac tissue. The grade was scored according to the average number of inflammatory cells that were positive for anti-IL-6 staining in 5 different fields (× 40) as follows: grade 0 (n = 0) (A), grade 1 (n = 1–9) (B), grade 2 (n = 10–25) (C), and grade 3 (n>25) (D). Scale bars = 50 µm. IL-6: interleukin-6.

      Measurement of IL-6 levels and other laboratory data

      Serum IL-6 levels in patients at the time of admission were measured by using the sandwich enzyme immunoassay kit D6050 (R&D Systems, Minneapolis, MN, USA) for the control group and myocarditis group and the kit HS600B (R&D Systems) for the validation group. Other laboratory parameters, including cardiac enzymes (creatine kinase [CK] and creatine kinase-MB [CK-MB]) and brain natriuretic peptide (BNP), were measured using venous blood samples collected at the time of patients’ admission by standard laboratory techniques with an automatic analyser. Serum CK and CK-MB levels and plasma BNP level were measured repeatedly during hospitalization to estimate the severity of myocarditis.

      Collection and evaluation of clinical data

      Information on age, sex, blood pressure, heart rate, New York Heart Association (NYHA) functional classification, medical history, smoking history, laboratory data, LV function as evaluated by transthoracic echocardiography at the time of admission, and cause of myocarditis was obtained from medical records for the patients. LV function was also evaluated just before discharge. Additionally, treatment and the presence of life-threatening arrhythmia during hospitalization and clinical events (in-hospital death and cardiac death within 3 years after diagnosis) were examined.

      Statistical analysis

      Standard statistical methods were used in this study. Normally distributed continuous variables are expressed as means ± standard deviation, and comparisons were performed by using the unpaired t-test for two groups and one-way analysis of variance for three groups followed by Bonferroni post hoc correction. Non-normally distributed continuous variables are expressed as medians (interquartile range). Logarithmic transformation was also used for non-normally distributed continuous variables to transform them to normally distributed continuous variables when they were used for analysis. Categorical variables are presented as absolute values and frequencies and were compared using Fisher's exact test. Differences with P<0.05 were considered significant. We compared serum IL-6 levels between controls and patients with myocarditis to evaluate the increase in IL-6 levels in myocarditis. We also divided patients with myocarditis into two groups according to the median serum IL-6 levels (a low IL-6 group and a high IL-6 group) to evaluate the effect of IL-6 on the clinical findings and the course of the disease. Pearson's correlation coefficient was calculated to evaluate the correlation between two normally distributed continuous variables. We performed all analyses in this study using EZR version 1.41 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) [
      • Kanda Y.
      Investigation of the freely available easy-to-use software “EZR” for medical statistics.
      ].

      Data availability

      The data generated or analysed during this study are available from the corresponding author upon reasonable request.

      Results

      Serum IL-6 levels in patients with myocarditis

      We evaluated the serum IL-6 levels in control patients (n = 12) (patients with primary arrhythmia) and patients with myocarditis (n = 13). The median IL-6 levels in the control group and myocarditis group were 1.4 (1.0, 1.9) pg/mL and 16.7 (9.9, 103.8) pg/mL, respectively. After performing logarithmic transformation, the serum IL-6 level in patients in the myocarditis group was significantly higher than that in patients in the control group (P<0.001). The median serum IL-6 level in the validation group (healthy volunteers) was 0.8 (0.6, 1.4) pg/mL. There was no significant difference between the log-transformed serum IL-6 levels in the validation group and control group (P = 0.360). On the other hand, the log-transformed serum IL-6 level in the myocarditis group was significantly higher than that in the validation group (P<0.001) (Fig. 2).
      Fig 2
      Fig. 2Comparison of serum interleukin-6 (IL-6) levels in the validation group, control group, and myocarditis group. Box plots show the median, interquartile range, and minimum/maximum values in the groups.
      Based on the median serum IL-6 level, patients with myocarditis were divided into a low IL-6 group (9.9 [4.5, 14.2] pg/dL, n = 7) and a high IL-6 group (108.9 [51.1, 130.9] pg/dL, n = 6).

      Histological evaluation of cardiac tissue

      H&E staining in the control group (n = 8) showed no infiltration or injury of myocardial cells in any of the patients (Fig. 3A, B). According to the diagnostic criteria described in the Methods section, 8 (62%) of the 13 patients with myocarditis were diagnosed with lymphocytic myocarditis, three (23%) were diagnosed with eosinophilic myocarditis, and two (15%) were diagnosed with giant cell myocarditis. Fig. 3E, F, I, J, M, and N show representative images of H&E staining of cardiac tissues obtained from patients with myocarditis. In subsequent immunohistochemistry using an anti-IL 6 antibody, none of the eight patients in the control group showed specific staining (Grade score = 0) (Fig. 3C and D), while all of the patients in the myocarditis group showed IL-6 expression in infiltrating inflammatory cells (Grade score ≥ 1) including eosinophils (Fig. 3G and H), lymphocytes (Fig. 3K and L), and monocytes/macrophages (Fig. 3O and P).
      Fig 3
      Fig. 3Haematoxylin and eosin (H&E) staining and immunohistochemical staining with anti-interleukin-6 (IL-6) antibody of cardiac samples. Cardiac tissue from patients with myocarditis shows considerable infiltration of inflammatory cells accompanied by destruction of cardiomyocytes (E, F I, J M, and N). Cardiac tissue from a control patient does not show infiltration of inflammatory cells or destruction of myocardial cells (A, B). IL-6 expression can be seen in various infiltrating inflammatory cells, including eosinophils (G, H), lymphocytes (K, L), and monocytes/macrophages (O, P), in cardiac samples from a patient with myocarditis. However, a sample from a control patient does not show any specific staining by anti-IL-6 antibody (C, D). Scale bars = 50 µm.
      Furthermore, the proportion of patients whose myocardial samples showed grade 3 of anti-IL-6 staining was higher in the high IL-6 group (n = 5 [83%]) than in the low IL-6 group (n = 1 [14%]) (Table 1).
      Table 1Grading scores of anti-IL-6 staining in patients with myocarditis.
      Low IL-6 group (n = 7)High IL-6 group (n = 6)P value
      Grading score
      1,%4 (57)0 (0)0.070
      2,%2 (29)1 (17)1.000
      3,%1 (14)5 (83)0.029
      Values are expressed as the absolute number of cases (relative percentage). IL-6: interleukin-6.

      Serum IL-6 levels and clinical data

      The clinical data for the low and high IL-6 groups are shown in Table 2. There was no significant difference between the two groups in basic patient characteristics at admission including age, sex, blood pressure, heart rate, prevalence of hypertension and diabetes mellitus, smoking history, and medical treatment with an immunosuppressant. On the other hand, the percentage of patients with NYHA functional classification IV at admission was significantly higher in the high IL-6 group than in the low IL-6 group (P = 0.029). The log-transformed serum C-reactive protein (CRP) level, CK level and CK-MB level at admission and the log-transformed peak CK level and peak CK-MB level during the hospital stay were significantly higher (P = 0.006, P = 0.010, P = 0.002, P = 0.002 and P = 0.001, respectively) and the LV ejection fraction (LVEF) at admission was significantly lower (P = 0.016) in the high IL-6 group than in the low IL-6 group (Fig. 4A-E, 4 G). LVEF evaluated just before discharge was not significantly different between the low IL-6 group and the high IL-6 group. In addition, there was no significant difference between the log-transformed plasma BNP level at admission (P = 0.642, Fig. 4F) and that at discharge (P = 0.836, not shown in the figure) of patients in the two groups. The proportions of patients’ aetiological type and pathological subtype of myocarditis were not significantly different between the two groups (Table 2). One patient in the low IL-6 group who had been treated with prednisolone at 10 mg for polymyositis was diagnosed as having autoimmune myocarditis. One patient in the low IL-6 group was diagnosed as having autoimmune myocarditis due to systemic lupus erythematosus, and that patient started receiving oral prednisolone therapy during hospitalization.
      Table 2Basic characteristics and examination findings of patients with acute myocarditis at admission in the low and high IL-6 groups.
      Low IL-6 group (n = 7)High IL-6 group (n = 6)P value
      Age, years54 ± 1638 ± 230.174
      Female sex, n (%)2 (29)2 (33)1.000
      Systolic BP, mmHg116 ± 27103 ± 200.355
      Diastolic BP, mmHg69 ± 1856 ± 140.203
      Heart rate, beats/min83 ± 2293 ± 430.592
      NYHA classification
      II, n (%)3 (43)0 (0)0.192
      III, n (%)3 (43)1 (17)0.559
      IV, n (%)1 (14)5 (83)0.029
      Medical history
      Hypertension, n (%)1 (14)1 (17)1.000
      Diabetes mellitus, n (%)1 (17)0 (0)1.000
      Smoking, n (%)2 (29)3 (50)0.592
      Medication of immunosuppressant
      Prednisolone1 (17)0 (0)1.000
      Laboratory data
      wbc, × 103/µl7.7 [5.8, 9.7]9.1 [6.4, 12.9]0.456
      hemoglobin, g/dl13.1  ±  1.614.0  ±  2.50.459
      CRP, mg/dL1.41 [0.47, 2.79]7.82 [4.41, 13.01]0.006
      CK, IU/L250 [116, 299]987 [639, 1398]0.010
      CK-MB, IU/L10 [
      • Savvatis K.
      • Müller I.
      • Fröhlich M.
      • Pappritz K.
      • Zietsch C.
      • Hamdani N.
      • et al.
      Interleukin-6 receptor inhibition modulates the immune reaction and restores titin phosphorylation in experimental myocarditis.
      ,
      • Banba K.
      • Kusano K.F.
      • Nakamura K.
      • Morita H.
      • Ogawa A.
      • Ohtsuka F.
      • et al.
      Relationship between arrhythmogenesis and disease activity in cardiac sarcoidosis.
      ]
      48 [34, 63]0.002
      Peak of CK, IU/L289 [116, 361]1343 [956, 7213]0.002
      Peak of CK-MB, IU/L10 [
      • Savvatis K.
      • Müller I.
      • Fröhlich M.
      • Pappritz K.
      • Zietsch C.
      • Hamdani N.
      • et al.
      Interleukin-6 receptor inhibition modulates the immune reaction and restores titin phosphorylation in experimental myocarditis.
      ,
      • Kanda Y.
      Investigation of the freely available easy-to-use software “EZR” for medical statistics.
      ]
      100 [55, 131]0.001
      eGFR, mL/min/1.73 m282 ± 2162 ± 140.087
      IL-6, pg/mL9.9 [4.5, 14.2]108.9 [51.1, 130.9]0.001
      BNP, pg/dL317.6 [200.4, 654.2]626.5 [490.7, 810.5]0.642
      bnp (at discharge), pg/dl94.3 [40.0, 179.3]72.1 [35.1, 106.6]0.836
      hemoglobin a1c,%5.9  ±  0.55.7  ±  0.30.391
      TTE findings
      LVDd, mm50 ± 846 ± 60.327
      LVDs, mm39 ± 1040 ± 60.816
      LVEF,%47 ± 1527 ± 90.016
      LVDd (at discharge), mm50 ± 850 ± 51.000
      LVDs (at discharge), mm35 ± 935 ± 60.941
      LVEF (at discharge),%53 ± 1557 ± 100.666
      Aetiological type
      Viral infection,%2 (29)2 (33)1.000
      Autoimmune disease,%2 (29)0 (0)0.462
      Unknown,%3 (43)4 (67)0.192
      Pathological type
      LM, n (%)5 (71)3 (50)0.592
      EM, n (%)1 (14)2 (33)0.559
      GCM, n (%)1 (14)1 (17)1.000
      Values are expressed as the mean ± standard deviation, median and interquartile range, or absolute number of cases (relative percentage) as appropriate. IL-6: interleukin-6, BP: blood pressure, NYHA: New York Heart Association, WBC: white blood cell, CRP: C-reactive protein, CK: creatine kinase, BNP: brain natriuretic peptide, EGFR: estimated glomerular filtration rate, TTE: transthoracic echocardiogram, LVDd: left ventricular end-diastolic diameter, LVDs: left ventricular end-systolic diameter, LVEF: left ventricular ejection fraction LM: lymphocytic myocarditis, EM: eosinophilic myocarditis, GCM: giant cell myocarditis.
      Fig 4
      Fig. 4Comparison of serum CRP levels (A), CK levels (B), CK-MB levels (C), peak CK levels (D) and peak CK-MB levels (E), plasma BNP levels (F), and LVEF (G) in the low and high IL-6 groups. The CRP, CK, CK-MB, and BNP data were log-transformed to become normally distributed continuous variables. Box plots show the median, interquartile range, and minimum/maximum values in the groups. CRP: C-reactive protein, CK: creatine kinase, CK-MB: creatine kinase-MB, BNP: brain natriuretic peptide, LVEF: left ventricular ejection fraction, IL-6: interleukin-6.

      Relationships between serum IL-6 levels and cardiac biomarkers

      Fig. 5 shows the correlations of IL-6 with CRP, cardiac biomarkers, and LVEF. Pearson's correlation coefficients showed significant positive correlations of log-transformed serum levels of IL-6 with log-transformed serum CRP (R2=0.735, P<0.001), CK (R2=0.684, P<0.001), CK-MB (R2=0.663, P<0.001), peak CK (R2=0.522, P = 0.005) and peak CK-MB (R2=0.548, P = 0.004) levels and a negative correlation with LVEF (R2=0.435, P = 0.014). There was no significant correlation between IL-6 levels and BNP levels at admission (R2=0.128, P = 0.231).
      Fig 5
      Fig. 5Correlations of serum IL-6 levels with serum levels of CRP (A), CK (B), CK-MB (C), peak CK (D) and peak CK-MB (E), plasma BNP levels (F), and LVEF (G). The CRP, CK, CK-MB, and BNP data were log-transformed to become normally distributed continuous variables. R indicates Pearson's correlation coefficient, and R2 is the square of R. IL-6: interleukin-6, CRP: C-reactive protein, CK: creatine kinase, CK-MB: creatine kinase-MB, BNP: brain natriuretic peptide, LVEF: left ventricular ejection fraction.

      Serum IL-6 levels and clinical outcomes

      All of the patients were admitted to the cardiac care unit (CCU) for symptoms of heart failure and/or haemodynamic instability without any other comorbidities, including infectious diseases such as pneumonia or sepsis. Treatment during hospitalization and clinical events in the low and high IL-6 groups are shown in Table 3. Patients in the high IL-6 group significantly more frequently received catecholamine therapy (n = 6 [100%] vs n = 1 [14%], P = 0.005), an intra-aortic balloon pump (n = 6 [100%] vs n = 1 [14%], P = 0.005), venoarterial extracorporeal membrane oxygenation (ECMO) (n = 5 [83%] vs n = 1 [14%], P = 0.029), and artificial ventilation support (n = 4 [67%] vs n = 0 [0%], P = 0.021) during hospitalization than did patients in the low IL-6 group. On the other hand, there was no statistically significant difference between the two groups in the presence of life-threatening arrhythmia or mortality rate during hospitalization or in the cardiac mortality rate within 3 years after diagnosis of myocarditis. One patient in the low IL-6 group died of heart failure during hospitalization. One patient in the low IL-6 group and one patient in the high IL-6 group died due to ventricular tachycardia or ventricular fibrillation after discharge and within 3 years after diagnosis.
      Table 3Treatment and clinical events of patients in the low and high IL-6 groups.
      Low IL-6 group (n = 7)High IL-6 group (n = 6)P value
      Treatment
      mPSL pulse therapy, n (%)3 (43)3(50)1.000
      γ-globulin, n (%)1 (14)4 (67)0.103
      Antiarrhythmic drug, n (%)1 (17)2 (33)0.559
      Catecholamine, n (%)1 (14)6 (100)0.005
      IABP, n (%)1 (14)6 (100)0.005
      ECMO, n (%)1 (14)5 (83)0.029
      Artificial respirator, n (%)0 (0)4 (67)0.021
      Pacemaker, n (%)1 (14)3 (50)0.266
      ICD, n (%)0 (0)1 (17)0.462
      Life-threatening arrhythmia
      Bradycardia (SSS, AVB), n (%)3 (43)4 (67)0.592
      VT/ VF, n (%)1 (14)2 (33)0.559
      Clinical prognosis
      In-hospital death, n (%)1 (14)0 (0)1.000
      CV death within 3 years, n (%)2 (33)1 (17)1.000
      Values are expressed as the absolute number of cases (relative percentage). IL-6: interleukin-6, mPSL: methylprednisolone, γ: gamma, IABP: intra-aortic balloon pump, ECMO: extra-corporeal membrane oxygenation, ICD: implantable cardioverter-defibrillator, SSS: sick sinus syndrome, AVB: atrioventricular block, VT: ventricular tachycardia, VF: ventricular fibrillation, CV: cardiovascular.

      Discussion

      This study showed (1) IL-6 expression in various types of infiltrating inflammatory cells, (2) a significant correlation between serum IL-6 level and severity of the infiltration of inflammatory cells expressing IL-6 in myocardial tissue, and (3) a significant impact of serum IL-6 as a biomarker of cardiac damage and severity in patients with acute myocarditis. To the best of our knowledge, this is the first study to show the effect of IL-6 on myocarditis in patients with pathological and clinical evaluations.

      Serum IL-6 reflects cardiac injury and severity in myocarditis

      IL-6 is a pleiotropic cytokine [
      • Jones S.A.
      Directing Transition from innate to acquired immunity: defining a role for IL-6.
      ] and is secreted by a variety of cells including inflammatory cells. IL-6 is involved in B cell and T cell differentiation, activation of macrophages and natural killer cells, production of acute phase proteins including CRP [
      • Nishikawa T.
      • Hagihara K.
      • Serada S.
      • Isobe T.
      • Matsumura A.
      • Song J.
      • et al.
      Transcriptional Complex Formation of c-Fos, STAT3, and Hepatocyte NF-1α Is Essential for Cytokine-Driven C-Reactive Protein Gene Expression.
      ], and an antiviral response [
      • Kanda T.
      • Takahashi T.
      Interleukin-6 and cardiovascular diseases.
      ]. The results of several studies using an experimental model of infectious myocarditis have suggested that IL-6 has a beneficial effect in early inflammatory stages, while continuous IL-6 expression accelerates myocardial inflammation and injury in the process of viral infection [
      • Kindermann I.
      • Barth C.
      • Mahfoud F.
      • Ukena C.
      • Lenski M.
      • Yilmaz A.
      • et al.
      Update on myocarditis.
      ,
      • Tanaka T.
      • Kanda T.
      • McManus B.M.
      • Kanai H.
      • Akiyama H.
      • Sekiguchi K.
      • et al.
      Overexpression of interleukin-6 aggravates viral myocarditis: impaired increase in tumor necrosis factor-α.
      ,
      • Cihakova D.
      • Rose N.R.
      Chapter 4 pathogenesis of myocarditis and dilated cardiomyopathy.
      ]. Additionally, IL-6 has recently been the focus of a number of studies for the major role it plays in cytokine release syndrome caused by viral infection, including infection with severe acute respiratory syndrome coronavirus 2 [
      • Zhang C.
      • Wu Z.
      • Li J.W.
      • Zhao H.
      • Wang G.Q.
      Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality.
      ,
      • Luo P.
      • Liu Y.
      • Qiu L.
      • Liu X.
      • Liu D.
      • Li J.
      Tocilizumab treatment in COVID-19: a single center experience.
      ].
      Our study of patients with acute myocarditis showed the expression of IL-6 among infiltrating inflammatory cells in myocardial tissue. Furthermore, a higher serum IL-6 level was correlated with severe infiltration of IL-6-expressing inflammatory cells in myocardial tissue and with higher levels of serum CRP and cardiac enzymes. These results suggested that IL-6 released from inflammatory cells causes cardiac damage by inducing and maintaining acute inflammation and that serum IL-6 level is affected by IL-6-producing inflammatory cells in myocardial tissues in patients with acute myocarditis.
      Furthermore, in this study, patients with myocarditis who had high serum IL-6 levels showed severely decreased cardiac dysfunction and poor physical performance and they required catecholamines, mechanical support, and ventilator support as treatment in the acute phase. These results indicate that IL-6 is not only involved in the mechanism of myocarditis but is also an important marker reflecting severity of the disease. According to previous studies, the serum level of IL-6 in patients with biopsy-proven myocarditis can range from 11.9 to 272.4 pg/ml [
      • Matsumori A.
      • Yamada T.
      • Suzuki H.
      • Matoba Y.
      • Sasayama S.
      Increased circulating cytokines in patients with myocarditis and cardiomyopathy.
      ,
      • Kishimoto C.
      • Shioji K.
      • Kinoshita M.
      • Iwase T.
      • Tamaki S.
      • Fujii M.
      • et al.
      Treatment of acute inflammatory cardiomyopathy with intravenous immunoglobulin ameliorates left ventricular function associated with suppression of inflammatory cytokines and decreased oxidative stress.
      ,
      • Kato S.
      • Morimoto S.I.
      • Hiramitsu S.
      • Uemura A.
      • Ohtsuki M.
      • Kato Y.
      • et al.
      Successful high-dose intravenous immunoglobulin therapy for a patient with fulminant myocarditis.
      ,
      • Zeng J.H.
      • Liu Y.X.
      • Yuan J.
      • Wang F.X.
      • Wu W.B.
      • Li J.X.
      • et al.
      First case of COVID-19 complicated with fulminant myocarditis: a case report and insights.
      ], similar to the results of our study. Furthermore, two case studies showed that serum IL-6 levels peaked when myocarditis patients were in haemodynamically severe conditions requiring the induction of ECMO [
      • Kishimoto C.
      • Shioji K.
      • Kinoshita M.
      • Iwase T.
      • Tamaki S.
      • Fujii M.
      • et al.
      Treatment of acute inflammatory cardiomyopathy with intravenous immunoglobulin ameliorates left ventricular function associated with suppression of inflammatory cytokines and decreased oxidative stress.
      ,
      • Kumar K.
      • Guirgis M.
      • Zieroth S.
      • Lo E.
      • Menkis A.H.
      • Arora R.C.
      • et al.
      Influenza myocarditis and myositis: case presentation and review of the literature.
      ]. It is possible that the subjects in the high IL-6 group were admitted to our hospital during the severe phase of myocarditis because our institution is an advanced care hospital and receives patients with severe conditions.
      On the other hand, plasma BNP level measured at the time of admission was not significantly different between the low and high IL-6 groups. However, considering that a high IL-6 level suggests a severe stage of NYHA and LV dysfunction, this lack of difference might be due to the small size of the study population and the large variation in serum BNP levels in myocarditis.
      Furthermore, there was no significant difference between the frequencies of cardiac death in the low and high IL-6 groups in this study. The reason for this lack of difference also might be the small size of the study population and the small number of patients who died in the hospital because of advanced intensive care. Additionally, we could not check physical performance and serum IL-6 levels just before discharge. Therefore, the status and prognosis of patients with myocarditis in the subacute to chronic stages may not have been fully evaluated. These problems are major limitations of this study.

      Clinical implications of IL-6 as a therapeutic target of myocarditis

      A specific therapy for myocarditis has not yet been established. A previous study showed the effectiveness of immunosuppressants for myocarditis, but there was still a persistent risk of sudden cardiac death by life-threatening arrhythmia [
      • Kandolin R.
      • Lehtonen J.
      • Salmenkivi K.
      • Räisänen-Sokolowski A.
      • Lommi J.
      • Kupari M.
      Diagnosis, treatment, and outcome of giant-cell myocarditis in the era of combined immunosuppression.
      ].
      In this study, we found that IL-6 was an important factor that greatly affected the severity of myocarditis pathologically and clinically. A previous study also showed that overexpression of IL-6 promoted myocardial injury in mice with viral myocarditis by interrupting the cytokine network and viral clearance [
      • Tanaka T.
      • Kanda T.
      • McManus B.M.
      • Kanai H.
      • Akiyama H.
      • Sekiguchi K.
      • et al.
      Overexpression of interleukin-6 aggravates viral myocarditis: impaired increase in tumor necrosis factor-α.
      ]. Furthermore, it was shown that treatment with an anti-IL-6 receptor antibody (tocilizumab) improved cardiac dysfunction and LV remodeling in an experimental murine model of Coxsackie virus B3-induced myocarditis [
      • Savvatis K.
      • Müller I.
      • Fröhlich M.
      • Pappritz K.
      • Zietsch C.
      • Hamdani N.
      • et al.
      Interleukin-6 receptor inhibition modulates the immune reaction and restores titin phosphorylation in experimental myocarditis.
      ]. Based on the results of this previous basic research in animal models and our study in patients with myocarditis, IL-6 could become a therapeutic target for myocarditis. Interestingly, in this study, it was found that various types of inflammatory cells infiltrating cardiac tissue expressed IL-6. This finding suggests that, unlike other immunosuppressants such as steroids, anti-IL-6 receptor antibody treatment would provide beneficial effects for pathologically different types of myocarditis by blocking cytokine storms accelerated by IL-6 released from inflammatory cells. However, the function of IL-6 in vivo is complex and pleiotropic [
      • Fontes J.A.
      • Rose N.R.
      • Čiháková D.
      The varying faces of IL-6: from cardiac protection to cardiac failure.
      ]. IL-6 signaling is protective during acute inflammation, while it can also be pathogenic in chronic disease [
      • Diao H.
      • Kohanawa M.
      Endogenous interleukin-6 plays a crucial protective role in streptococcal toxic shock syndrome via suppression of tumor necrosis factor alpha production.
      ,
      • Kallen K.J.
      The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases.
      ,
      • Kaplanski G.
      • Marin V.
      • Montero-Julian F.
      • Mantovani A.
      • Farnarier C.
      IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation.
      ] Additionally, one study showed that IL-6-deficient mice with infectious myocarditis developed increased pathological cardiac myocyte injury accompanied by activation of T cells and infiltration of macrophages and monocytes into myocardial tissue [
      • Poffenberger M.C.
      • Straka N.
      • El Warry N.
      • Fang D.
      • Shanina I.
      • Horwitz M.S.
      Lack of IL-6 during coxsackievirus infection heightens the early immune response resulting in increased severity of chronic autoimmune myocarditis.
      ]. When considering IL-6 as a therapeutic target for myocarditis, it is necessary to understand its complex behavior and control it at an appropriate phase or timing. Further research on IL-6 in patients with myocarditis is required to solve this complex problem.
      In addition, we only focused on data for IL-6 in this study without considering data for other cytokines such as IL-1β, IL-10 and tumor necrosis factor-α. These inflammatory or anti-inflammatory cytokines interact with each other and this interaction is very important for the damage and remodeling of cardiac tissue in myocarditis. Recent studies have shown that the proinflammatory cytokine IL-1 plays an important role in the development of myocarditis and that IL-1 could be a therapeutic target of myocarditis [
      • Abbate A.
      • Toldo S.
      • Marchetti C.
      • Kron J.
      • Van Tassell B.W.
      • Dinarello C.A.
      Interleukin-1 and the inflammasome as therapeutic targets in cardiovascular disease.
      ,

      Onoue K., Saito Y. New treatment for myocardial diseases. J Cardiol 2020. doi:10.1016/j.jjcc.2020.10.016.

      ]. Therefore, the lack of data for other cytokines is a limitation of this study.

      Study limitations

      There were other limitations in our study. First, the sample size was relatively small. Specifically, dividing the subjects with myocarditis into two groups produced subgroups with only 7 and 6 patients, respectively. Although we validated the reproducibility of the data by comparing the serum IL-6 levels in the control group and validation group, for which the data were obtained from different institutions, and also confirmed that the serum IL-6 level in patients with myocarditis was significantly higher than the levels in those two groups, the small sample size is one of the major limitations of this study. Second, we could not accurately assess the amount of serum IL-6 derived from myocardial inflammatory cells because IL-6 is produced not only by the inflammatory cells in myocardial tissue but also by other types of cells in the body. Since myocarditis caused by infection or autoimmune disease is accompanied by strong systemic inflammation, therefore there is a possibility that the serum IL-6 level in patients with acute myocarditis in this study reflects systemic inflammation. Third, we could not evaluate the changes in serum IL-6 levels over time in each subject. All blood samples were obtained at the same time, namely, at admission; however, it is possible that the peak level of serum IL-6 during the acute phase rather than the level at admission was significantly correlated with the prognosis of patients with myocarditis. Finally, although serum cardiac trorponin I and troponin T levels are specific and very important biomarkers of cardiac damage, we evaluated cardiac injury only by serum CK and CK-MB. We could not use data for serum cardiac troponin in this study because patients in this study received evaluation of serum cardiac troponin by various type of methods including measurement of cardiac troponin I, troponin T and high-sensitive troponin T. This is a limitation due to the fact that our study was a retrospective study.
      A prospective and multicentre study is needed to confirm the impact of IL-6 in patients with myocarditis.

      Conclusion

      In patients with acute myocarditis, IL-6 is an important marker that clinically and immunopathologically reflects myocardial injury and severity. IL-6 might be useful as a clinically and pathomechanistically relevant prognostic marker for patients with myocarditis.

      Funding

      This research was supported in part by the Japan Agency for Medical Research and Development (AMED) under Grant Number 20ek0109476h0001 (Kazufumi Nakamura) and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 20K22765 (Naofumi Amioka).

      Disclosures

      The authors declare that there are no conflicts of interest.

      Acknowledgments

      The authors would like to thank K. Akazawa for technical assistance with the experiments in this study.

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      1. Onoue K., Saito Y. New treatment for myocardial diseases. J Cardiol 2020. doi:10.1016/j.jjcc.2020.10.016.