Continued Elevation of Plasma IL-4 and IL-17 Predicts the Progression from VMC to DCM

Objective To investigate plasma cytokines (interferon gamma, interleukin-4, and interleukin-17) in patients with viral myocarditis (VMC) and evaluate their predictive value in the progression from VMC to dilated cardiomyopathy (DCM). Methods A prospective, multicenter, observational study included 536 patients with newly diagnosed VMC admitted in cardiology departments of 24 tertiary super specialised university-affiliated hospitals in the China registry from January 2012 to June 2016. Demographics and clinical characteristics at baseline and after three months were collected, including laboratory blood tests, ECG, echocardiography, and drug treatment in each participating site. The plasma anti-viral antibodies (Abs), anti-heart autoimmune Abs, and cytokines were detected by ELISA. Results Of the 536 patients, 534 were included for analysis after two patients died in less than a month. The plasma levels of IFN-γ, IL-4, and IL-17 were continually higher in patients with incident DCM than in those without incident DCM at baseline, from the 1st month and the 3rd month; all had a P value of <0.0001. There was a positive correlation between IL-4 and LVEDd (r = 0.30, P < 0.0001) and between IL-17 and LVEDd (r = 0.11, P = 0.02). When all these covariates have entered the model simultaneously, elevated IL-4 and IL-17 were still significantly associated with DCM incidence. The RR (95% CI) of DCM incidence were 1.04 (1.02-1.06) for IL-4 and 5.24 (2.81-9.79) for IL-17. Conclusion The continued elevation of plasma IL-4 and IL-17 in VMC patients were associated with a high incidence of DCM at three months, and these two cytokines were independent predictors for the progression from VMC to DCM.


Introduction
Viral myocarditis (VMC) is a common illness worldwide that can lead to severe complications or death in infants and young adults [1,2], which is responsible for sudden death cases in young adults (8.6%-12%) and 9% to 16% of newly onset dilated cardiomyopathy (DCM) [3,4]. The incidence of myocarditis is approximately 1.5 million cases worldwide per year [5]. Acute VMC, a precursor of DCM leading to heart failure, is a triphasic disease involving an initial viral infection, followed by autoimmune response, and finally remodelling of cardiac structure and function [6].
The pathogenesis of DCM secondary to VMC is closely associated with dysfunction of the autoimmune system. CD4 + Th cell subsets (Th1, Th17, and Th2) are involved in the mechanisms for the onset of VMC and DCM and the progressing from VMC to DCM [6][7][8]. The acute viral infection will activate the Th0 cells, and in so doing, this will initiate a cascade of events as follows: Th0 will differentiate to Th1 cells resulting in the production of IFN-γ, thus fueling myocardial cell injury. In the acute stage, Th0 will also differentiate to Th17 cells resulting in the production of IL-17. IL-17, on the one hand, works on cytotoxic T lymphocytes (CTLs) to lead to myocardial cell injury, and on the other hand, it also works on B lymphocytes to produce several anti-heart autoantibodies (AHAs). In the chronic stage, Th0 differentiates to Th2 cells causing the production of IL-4, which promotes B cells to produce AHAs. AHAs can mediate Ca 2+ influx into the myocardial cells and damage them [6][7][8][9][10][11][12]. The objective of our study was to observe the transforming rate progressing from VMC to DCM in China and to decide if cytokines (IL-17, IL-4, and IFN-γ) could predict this progression.

Study Population.
A total of 536 patients with newly diagnosed clinical VMC admitted in 24 cardiology departments of tertiary super specialised hospitals in China were enrolled in the registry in each participating center from January 2012 to June 2016. There were no specific exclusion criteria. A signed informed consent was obtained.

Study
Design. This is a prospective, multicenter, observational study. Demographics and clinical characteristics at baseline and after three months were collected from all patients, including laboratory blood tests, ECG, echocardiography, and drug treatment at each participating site. The plasma anti-viral antibodies (Abs), anti-heart autoimmune Abs, and cytokines were detected by ELISA. VMC clinical types enrolled for the study were divided by four categories: arrhythmia type, heart failure type, acute severe type, and subclinical type according to the 1999 Chinese viral myocarditis diagnostic standard and the 2013 ESCs [4,13]. The following VMC clinical types were enrolled for the study: (a) Arrhythmia type: 1 to 3 weeks after virus infection, mild precordial discomfort, palpitations, ECG reveals premature beats or tachycardia, atrioventricular block, and ST-T changes. There was increased TnI, no clinical manifestations of heart enlargement and heart failure, and gradual recovery after 1 to 2 months of treatment The definition for VMC progressing to DCM is the appearance or persistence of LVEDd ≥ 5:5 cm and LVEF < 45% in patients given optimal medical treatment and followed up for three months [13].

Statistical Analysis.
Baseline characteristics of the viral myocarditis patient cohort were summarised according to the incidence of dilated cardiomyopathy for three months follow-up. All data are presented as mean ± SD for continuous variables, median (interquartile range) if continuous variables were skewed, and n (%) for categorical variables. Comparisons between groups were performed by the chisquare or Fisher exact test (for categorical variables) and the Student t-test or Mann-Whitney U test (for continuous variables). Linear mixed-effect models and logistic regression models with adjustment for gender, age, and baseline echocardiography covariables were used to identify the change of cytokine status (after natural logarithmic transformation) that pose the highest risk for incident DCM. The C statistics for each risk factor were calculated to estimate the predictive values of incident DCM. A P value of <0.05 was considered statistically significant. All statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC).

Patient Characteristics and Incident DCM.
Included in the study were 536 patients of a VMC cohort, of which two patients died in less than one month. A comparison of baseline clinical and laboratory parameters between patients with and without DCM is presented in Table 1. After three months follow-up, 127 (23.78%) newly onset DCM were recorded among 534 patients of the VMC cohort; out of the 127 patients who reached the primary endpoint, significant types were in 46 patients (36.22%) of the acute severe type and in 62 patients (48.82%) of the heart failure type. In comparison with patients without DCM, those patients with DCM presented at an older age, with lower LVEF, larger LVEDD, worse NYHA class, higher NT-pro-BNP levels, and an increased virus infection rate (all with P < 0:0001). Baseline antibodies (against ANT, β1R, MHC, and CaC) and cytokines (IFN-γ, IL-4, and IL-17) were significantly higher in patients who developed DCM as compared to those who did not develop DCM.

Prediction of Incident DCM in Patients with VMC.
Univariate analysis shows all variables concerning incident DCM after three months (Table 2). Adjusted relative risks of incident DCM after three months, multivariate analysis of age, LVEF, IL-4, and IL-17 predicted the occurrence of 2 Disease Markers   Table 4. Dynamic observation showed that the levels of plasma IFN-γ, IL-4, and IL-17 were continually higher in patients with incident DCM than in those without DCM at baseline, the 1st month, and the 3rd month (all P < 0:0001) as shown in the fit plot in Figure 2. For the relationship between cytokine (IFN-γ, IL-4, and IL-17) levels and echocardiographic parameters (LV), after adjusting for age, gender,

Discussion
The present study portrays the incidence of dilated cardiomyopathy from 534 patients who were diagnosed with having clinical viral myocarditis during a mean follow-up of three months, during which 127 (23.78%) patients reached the primary endpoint (incident DCM); several studies revealed different percentages implicating myocarditis of newly onset dilated cardiomyopathy cases [3,4,14]. Here, we first achieved the acquisition of Chinese data about the transforming rate, which gave us a clear picture from the Chinese registry perspective. Moreover, we further explored the role of cytokines in the progression of myocarditis to develop dilated cardiomyopathy. Although it was found that Th cells and related cytokines exert an important function in the progress of VMC to DCM, the cytokines' clinical significance as predictors of incident DCM was scantly investigated. Through a multivariate model, four variables were found to be associated with the progression to DCM, and these were age, LVEF, IL-4, and IL-17. On further analysis, IL-4 and IL-17 significantly improved the area under the ROC curve (AUC) in predicting the 3-month risk of DCM vs. the basic demographic model (age, sex, and LVEF). Increased levels of IFN-γ in the current study did not present any significant function in predicting progression from VMC to DCM. The dual role of IFN-γ can be in VMC pathogenesis, wherein in one hand, when increased it mediates myocardial cell injury, and on the other hand, it inhibits viral replication.
Furthermore, 15 patients out of 99 with subclinical types of VMC were observed progressing to DCM, which was expected in the advanced stages of the disease. A key point to be emphasised is that in developing DCM, the autoimmune mechanism plays a substantial role and not the severity of the patient's baseline condition. So, patients of this type require attention if IL-4 and IL-17 levels are high even when their symptoms are mild.
The baseline UCG left ventricular dimension could not predict the incident DCM. Then, we assessed the correlation between LV dimension and cytokines, where we found positive associations between IL-4 or IL-17 and UCG LV dimension. Despite the above fact, there was no correlation between IFN-γ and UCG LV dimension. Both dynamic changes of IL-4, IL-17, and UCG LV dimension correlations indicated that continued elevation of plasma IL-4 and IL-17 could predict the progression from VMC to DCM.

Limitations
The present study is based on a multicenter observational registry of VMC patients. The critical limitation of this registry is that it lacks endomyocardial biopsy because of the nature of the test as an invasive clinical tool, and it is challenging for patients to accept in China.

Conclusion
The rate of transformation from VMC to DCM was about 23.78% in China, which was in concordance with other observational studies from other countries. The continued elevation of IL-4 and IL-17 in VMC patients was associated with a high incidence of DCM. Since detection for plasma IL-4 and IL-17 is not that complicated, they may serve as significant predictors for VMC evolving to DCM.

Key Points
Question. What is the role of cytokines (IL-4, IL-17, and IFN-γ) in predicting progression from VMC to DCM? Findings. In this prospective multicenter observational study of 536 patients hospitalized with clinical VMC, elevated levels of plasma IL-4 and IL-17 were associated with a high incidence of DCM at 3 months, and these two cytokines were the independent predictors for the progression from VMC to DCM.
Meaning. Inflammation through inflammatory cytokines is essential for the progression of VMC to DCM. IL-4 and IL-17 are significant predictors of incident DCM.

Data Availability
The deidentified participant data are accessible by contacting the corresponding author Yuhua Liao via liaoyh27@163.com.

Disclosure
An earlier version of this article prepared by J. Yuan was presented as an abstract presentation in the European Heart Journal, 2017.