Evaluation of tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 levels in patients diagnosed with multi-system inflammatory syndrome in children

Abstract

Investigations are still ongoing about the pathophysiology of multi-system inflammatory syndrome in children, which can progress with serious morbidity and mortality after COVID-19 infection. In this study, we aimed to investigate whether fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels play a role in the diagnosis of the disease and on cardiac involvement. Twenty-three patients (11 girls, 12 boys) and 26 healthy controls (10 girls, 16 boys) were included in the study. The mean age of the patient and control group was 8.45 ± 2.43 and 10.73 ± 4.27 years, respectively. There was no difference between the fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels of the patient and control groups. When the patients with myocardial involvement in the patient group were compared with the patients without myocardial involvement in terms of fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels, no difference was found between these groups. The correlation of fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels with other laboratory parameters was investigated in the patient group. Fibroblast growth factor-2 was moderately inversely correlated with white blood cell count (r = -0.541, p = 0.008), absolute neutrophil count (r = −0.502, p = 0.015) and C-reactive protein (r = −0.528, p = 0.010). Fibroblast growth factor-2 was strongly inversely correlated with erythrocyte sedimentation rate (r = −0.694, p =<0.001). Our data show that fibroblast growth factor-2 and tumour necrosis factor alpha stimulated gene-6 do not provide sufficient information about diagnosis and cardiac involvement in multi-system inflammatory syndrome in children.

During the COVID-19 pandemic, a hyper-inflammatory condition began to appear in children. The Royal College of Pediatrics and Child Health referred to this condition as paediatric multi-system inflammatory syndrome temporally associated with COVID-19. ¹ Afterwards, more cases occurred all around the world, and the disease was referred multi-system inflammatory syndrome in children by the U.S. Centers for Disease Control and Prevention and the World Health Organization. ^2,3

Multi-system inflammatory syndrome in children can be severe and cause fatal complications. ⁴ This disease should be considered in patients with the evidence of SARS-CoV-2 infection who develop a multi-system disease with fever, gastrointestinal symptoms, skin signs, hypotension and/or shock, and cardiac dysfunction with elevated biomarkers of cardiac injury.

Previous studies have shown that transient coronary artery dilatation due to a cytokine storm may develop in systemic-onset juvenile idiopathic arthritis, and this may be due to the same mechanism in patients with multi-system inflammatory syndrome in children. ^5,6 Another theory about the cardiac injury is that virus may directly cause myocarditis. The virus can directly cause myocardial damage by entering cardiomyocytes by way of the angiotensin-converting enzyme 2 receptor. The virus also has the ability to activate CD8+ T lymphocyte migration to cardiomyocytes. ⁶

Expansion of T cell clonotypes and the presence of autoantibodies directed against several self-antigens have been reported in multi-system inflammatory syndrome in children. ^7,8

Tumor necrosis factor alpha-stimulated gene-6 is a secreted product of tumour necrosis factor alpha-treated cells. ⁹ Tumor necrosis factor alpha-stimulated gene-6 is expressed in response to proinflammatory mediators. ^9,10 Tumor necrosis factor alpha-stimulated gene-6 inhibits neutrophil migration to inflammatory sites. Tumor necrosis factor alpha-stimulated gene-6 plays a pivotal role in fighting against inflammation and remodelling extracellular matrix. In previous studies, it has been shown that tumour necrosis factor alpha-induced gene-6 levels are high in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis, and it is highly elevated in the synovial fluid following injury. ^11–13 Tumor necrosis factor alpha-stimulated gene-6 is a strong anti-inflammatory marker. ^14,15 Some growth factors such as transforming growth factor β, epidermal growth factor, and fibroblast growth factor upregulate the synthesis of tumour necrosis factor alpha-stimulated gene-6 in certain cell types. ^16,17

Fibroblast growth factor-2 is a crucial member of the fibroblast growth factor family. It is associated with endothelial cell migration, proliferation, and injury repairment. Fibroblast growth factor-2 is a major heparin-binding angiogenic inducer. ¹⁸ A study showed that fibroblast growth factor-2 stimulates migration and proliferation of endothelial cells and inhibits inflammation. ¹⁹ Fibroblast growth factor-2 provides protection against acute lung injury by recruiting neutrophils in influenza A virus infection. ^20,21

There are no disease-specific biomarkers for the diagnosis of multi-system inflammatory syndrome in children. In this study, we planned to investigate whether tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 levels can be helpful in diagnosis in patients diagnosed with multi-system inflammatory syndrome in children, the pathophysiology of which is still unclear.

Materials and method

Two groups, the patient group and the control group, were included in the study. The patient group consisted of patients diagnosed with multi-system inflammatory syndrome in children according to U.S. Centers for Disease Control and Prevention diagnostic criteria in the paediatric cardiology outpatient clinic between 2020 and 2022. Patients with a history of drug use due to any chronic disease were not included in the study. The control group included healthy children admitted to the paediatric outpatient clinic without chronic disease, active infection, or chronic drug use. Written informed consent was obtained from the parents of all the patients and the control group. Ethics committee approval was obtained from the ethics committee of Cumhuriyet University (2022-05/04).

Sample collection

Fasting blood samples were collected from all patients into serum tube and K3-EDTA tubes (Becton Dickinson, UK). Patient samples were taken on admission. The serum sample tubes were allowed to clot before centrifugation. After centrifugation, the serum was aliquoted and frozen at −80°C.

Biochemical analyses

The quantitative sandwich ELISA technique was used for the detection of serum fibroblast growth factor-2 (Wuhan USCN Business Co., Ltd, China) and tumour necrosis factor alpha-stimulated gene-6 (Wuhan Cloud Clone Corp., Ltd, China). Complete blood count, D-dimer, fibrinogen, N-terminal pro-brain natriuretic peptide, troponin I, creatinine kinase, creatinine kinase-MB, C-reactive protein, and erythrocyte sedimentation rate values were evaluated.

Statistical analyzes

Data were analysed using SPSS 22.0 computer software. Normality of distribution of numerical variables was evaluated. Data are expressed as ranges, means, medians and standard deviation. Patients were further divided into two subgroups according to the presence or absence of cardiac involvement at the time of admission. Numerical data were compared between the groups using Mann–Whitney U-test (not normally disturbed subjects) and sample t-test (normally disturbed subjects). Descriptive statistics were used to report demographical and clinical data from patients and healthy controls. We used Spearman’s rank difference correlation to examine the correlation between tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 and acute phase reactants, complete blood count parameters, and cardiac markers. A p value less than or equal to 0.05 was considered statistically significant.

Results

Twenty-three patients (11 girls, 12 boys) diagnosed with multi-system inflammatory syndrome in children and 26 healthy controls (10 girls, 16 boys) were included in the study. The mean age of the patient group was 8.45 ± 2.43 years, and the mean age of the control group was 10.73 ± 4.27 years. The most common symptom of the patient group was fever, and it was present in all patients. The mean time from fever onset to diagnosis was 4.14 ± 1.61 days. Cardiovascular system involvement was the most common with 69.5% (n = 16) of the patients, while gastrointestinal system involvement was second with 65.2% (n = 15). Other system symptoms and findings included skin involvement 56.5% (n = 13), conjunctivitis 47.8% (n = 11), oropharyngeal involvement 34.7% (n = 8), cervical lymphadenopathy 4.3% (n = 1), respiratory tract involvement 13% (n = 3), neurological involvement 8.6% (n = 2), and skeletal system involvement 4.3% (n = 1). Among the cardiovascular findings, there were 69.5% (n = 16) of patients with valve regurgitation. The most common valve involvement was observed in the mitral valve with a rate of 65.2% (n = 15). This was followed by tricuspid valve (21.7%), aortic valve (8.6%), and pulmonary valve involvement (8.6%), in order of frequency. Myocarditis was detected in 17.3% (n = 4), pericarditis in 17.3% (n = 4), perimyocarditis 8.6% (n = 2), and myopericarditis 4.3% (n = 1) of patients. Transthoracic echocardiography revealed 17.3% (4) of patients with left ventricular systolic dysfunction (ejection fraction <55). Coronary artery aneurysm, dilatation, and increased coronary artery wall echogenicity were not observed in any patient.

The laboratory characteristics of the patient group and the healthy control group are shown in Table 1. When the two groups were compared, the absolute neutrophil count and absolute neutrophil count/absolute lymphocyte count ratio in the patient group were found to be statistically significantly higher than the control group, while the levels of absolute lymphocyte count, haemoglobin, haematocrit, red blood cell, and platelet were found to be statistically significantly lower. When the two groups were compared in terms of tumour necrosis factor alpha-induced gene-6 levels; the median tumour necrosis factor alpha-stimulated gene-6 level of the patient group was 3549.70 pg/ml (11.30–4366), and the median tumour necrosis factor alpha-stimulated gene-6 level of the control group was 3504.15 pg/ml (2370.70–4366). The difference between the two groups was not statistically significant (p = 0.818). When the two groups were compared in terms of fibroblast growth factor-2 levels; the median fibroblast growth factor-2 level of the patient group was 181.60 pg/ml (5–882.60), and the median fibroblast growth factor-2 level of the control group was 183.05 pg/ml (50.90–709.80). The difference between the two groups was not statistically significant (p = 0.659). The median of tumour necrosis factor alpha stimulated gene-6 level was 3810.10 pg/ml (11.30–4054.10) in patients with myocardial involvement (n = 7), and 3096 pg/ml (24.70–4366) in patients without myocardial involvement (n = 16) (p = 0.462). The median of fibroblast growth factor-2 level was 201.40 pg/ml (5–252) in patients with myocardial involvement (n = 7) and 168 pg/ml (53.80–882.60) in patients without myocardial involvement (n = 16) (p = 0.688).

Table 1. Laboratory findings of patient group and control group.

ALC: Absolute lymphocyte count, ANC: Absolute neutrophil count, CK: Creatinine kinase, CK-MB: Creatinine kinase-MB, CRP: C-reactive protein, ESR: Erythrocyte sedimentation rate, FGF-2: Fibroblast growth factor-2, MIS-C: Multi-system inflammatory syndrome in children, Nt-Pro-BNP: N-terminal pro-brain natriuretic peptide, RBC: Red blood cell, TSG-6: Tumor necrosis factor alpha stimulated gene-6, WBC: White blood cell.

Student’s t-test was used in independent groups for numerical variables matching normal distribution. Mann-Whitney U test was used for numerical variables that do not confirm the normal distribution. p < 0.05 was considered statistically significant.

The correlation of tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 levels with other laboratory parameters was investigated in patient group (Table 2). Fibroblast growth factor-2 was moderately inversely correlated with white blood cell count (r = −0.541, p = 0.008), absolute neutrophil count (r = −0.502, p = 0.015), and C-reactive protein (r = −0.528, p = 0.010). Fibroblast growth factor-2 was strongly inversely correlated with erythrocyte sedimentation rate (r = −0.694, p = <0.001). There was no statistically significant correlation between tumour necrosis factor alpha stimulated gene-6 and other parameters.

Table 2. Correlation of TSG-6 and FGF-2 levels of patient group with other laboratory parameters.

ALC: Absolute lymphocyte count, ANC: Absolute neutrophil count, CK: Creatinine kinase, CK-MB: Creatinine kinase-MB, CRP: C-reactive protein, ESR: Erythrocyte sedimentation rate, FGF-2: Fibroblast growth factor-2, MIS-C: Multi-system inflammatory syndrome in children, Nt-Pro-BNP: N-terminal pro-brain natriuretic peptide, TSG-6: Tumor necrosis factor alpha-stimulated gene-6, WBC: White blood cell.

Spearman correlation test was used. p < 0.05 was considered statistically significant.

Discussion

The COVID-19 pandemic poses a difficulty to public health and remains a mystery across the world. ²² Due to associated comorbidities, multi-system inflammatory syndrome in children has showed up as a threat to paediatric populations diagnosed with COVID-19 shortly after the pandemic. The result of an exacerbated immune system response or a maladaptive response, multi-system inflammatory syndrome in children is characterised by hyper-inflammation and cytokine storm. ^23,24 Some studies have found an increase in the proinflammatory and anti-inflammatory system in the acute phase in multi-system inflammatory syndrome in children, similar to the mixed antagonist response syndrome seen in sepsis in children. ²⁵ This life-threatening response requires urgent management.

Multi-system inflammatory syndrome in children has been reported in hundreds of children worldwide. ^26,27 The disease remains up to date with many question marks. Although the pathophysiology of COVID-19 or multi-system inflammatory syndrome in children has not been understood in detail, some studies have explained conditions of exacerbated immunological response, cytokine storm, hyper-inflammation, reactive epithelial change, vascular damage, and coagulopathy. ^28,29 Diorio et al. ³⁰ have found evidence of microangiopathy in multi-system inflammatory syndrome in children, and a recent autopsy study of a child who died from multi-system inflammatory syndrome in children revealed abundant viral particles across several tissues. ³¹ However, most of the explanation about pathogenesis remains unknown and warrants further research.

The fibroblast growth factor-2 family has biological functions on various cell types, with fibroblast growth factor-2 being a potent angiogenic factor in vivo. The fibroblast growth factor-2 family exerts its effects by signalling to target cells via cell surface tyrosine kinase receptors. ^32–34 Tumor necrosis factor alpha-stimulated gene-6 is an inflammation-associated secreted protein that has diverse tissue-protective and anti-inflammatory properties. Although tumour necrosis factor alpha-stimulated gene-6 exhibits anti-inflammatory and tissue-protective properties, it has also been implied to play a role in some lung disease pathologies. It is well known that tumour necrosis factor alpha-stimulated gene-6 expression is upregulated during inflammation. ³⁵ Tumor necrosis factor alpha-stimulated gene-6 is increased during inflammation: including cardiovascular disease, lung disease, autoimmune diseases and several different cancers. ³⁶ Tumor necrosis factor alpha-stimulated gene-6 has also been shown to have potent anti-inflammatory properties in acute lung injury, peritonitis, and rheumatoid arthritis. ³⁷

The aim of this study was to investigate the potential diagnostic value of tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 in multi-system inflammatory syndrome in children. We wanted to investigate a new biomarker because i) there are variable case definitions for multi-system inflammatory syndrome in children, ii) the clinical features of multi-system inflammatory syndrome in children can also be seen in other types of infections/malignancies and other rheumatic diseases, iii) myocardial dysfunction, which is an important reason of morbidity and mortality in multi-system inflammatory syndrome in children, can occur insidiously. We found that fibroblast growth factor-2 level was not different between patients and healthy controls. We did not find any study investigating fibroblast growth factor-2 levels in patients with multi-system inflammatory syndrome in children. However, we found that different results were obtained in studies conducted in adult COVID-19 patients. In a study conducted in 59 COVID-19 patients, the authors found that fibroblast growth factor-2 level was statistically significantly lower in the patient group compared to the healthy group. ³⁸ In another study, fibroblast growth factor-2 level was found to be higher in patient group than healthy control, but no statistically significant difference was found. ³⁹ The different results in these two studies may be due to the time of blood collection and differences severity of disease. While COVID-19 mostly affects the respiratory system, multisystem inflammatory syndrome in children is a multisystemic process, our different results may be explained with this. Since we took blood samples at the time of diagnosis, when the proinflammatory process was more dominant, we may have measured the fibroblast growth factor-2 level before it wasn't elevated yet. Because we know that increased transforming growth factor β-1 and fibroblast growth factor-2 levels have been associated with pulmonary fibrosis in the late period. When we compared multi-system inflammatory syndrome in children, which has sepsis, septic shock, and Kawasaki disease in its differential diagnosis, with studies evaluating fibroblast growth factor-2 conducted in these diseases; similar to our study, Faiotto et al. ⁴⁰ found that circulating fibroblast growth factor-2 levels were similar in patients with sepsis/septic shock patients and healthy controls. The reason for this similarity can be explained by the predominance of the proinflammatory process in both disease groups in the acute period. We support this view by the statistically significant inverse correlation of fibroblast growth factor-2 with white blood cell count, absolute neutrophil count, C-reactive protein, and erythrocyte sedimentation rate. Although coronary involvement is less expected in multi-system inflammatory syndrome in children, also known as Kawasaki-like syndrome, there was no patient with coronary artery involvement in our study. The effect of fibroblast growth factor-2 on localised stenosis, extracellular matrix increases and occlusion and recanalisation in Kawasaki disease has been shown in autopsy reports. ⁴¹ According to these results, if multi-system inflammatory syndrome in children patients with coronary involvement are examined, the fibroblast growth factor-2 level may be found to be higher in late period. In the adult myocardium, fibroblast growth factor-2 is involved in hypertrophy and angiogenesis, as well as the response to ischaemia–reperfusion injury. ⁴² In our study, we found that the median level of fibroblast growth factor-2 in patients with myocardial involvement was higher than in patients without myocardial involvement, although it was not statistically significant. We suggest that the reason of fibroblast growth factor-2 elevation may be for compensatory purposes.

In addition, we found that there was no difference between patients and healthy controls in terms of tumour necrosis factor alpha stimulated gene-6 levels. Qu et al. ⁴³ showed that increased plasma tumour necrosis factor alpha-stimulated gene-6 concentration is a new marker for the diagnosis of non-cardioembolic acute ischaemic attack, a disease that triggers an inflammatory cascade. In the study of Xu et al., they found that the plasma level of tumour necrosis factor alpha-stimulated gene-6 in dilate cardiomyopathy patients with heart failure increased significantly, and the plasma levels of tumour necrosis factor alpha-stimulated gene-6 could be used as a new predictor for cardiac function, myocardial fibrosis, and prognosis. N-terminal pro-brain natriuretic peptide and tumour necrosis factor alpha-stimulated gene-6 levels were statistically significantly positively correlated. It has been suggested that increased N-terminal pro-brain natriuretic peptide and tumour necrosis factor alpha-stimulated gene-6 levels can be used as a marker of poor prognosis in these patients. ⁴⁴ In our study, although the tumour necrosis factor alpha-stimulated gene-6 level was high in the patient group and myocardial involvement group, there was no significant difference, and no correlation was found with N-terminal pro-brain natriuretic peptide. Our myocardial involvement group patients had a picture of acute heart failure. If tumour necrosis factor alpha-stimulated gene-6 levels are evaluated in patients who develop chronic heart failure in the late period, maybe different results can be obtained.

In our study, the absolute neutrophil count and absolute neutrophil count/ absolute lymphocyte count ratio in the patient group were found to be statistically significantly higher than the control group, while the levels of absolute lymphocyte count, haemoglobin, haematocrit, red blood cell, platelet were found to be statistically significantly lower. Zhao et al. ⁴⁵ found that patients with severe multi-system inflammatory syndrome in children patients showed elevated white blood cell count, absolute neutrophil count levels, decreased absolute lymphocyte count levels, similar platelet levels compared to patients with non-severe multi-system inflammatory syndrome in children. This lymphopenia and decreased absolute neutrophil count/absolute lymphocyte count ratio observed in patients may be the result of a cytokine storm.

The most important limitation of our study is the small sample size. This small size may reduce statistical power and affect heterogeneity. Measurement of tumour necrosis factor alpha-stimulated gene-6 and fibroblast growth factor-2 levels only at the time of diagnosis may also be misleading. Different results could have been obtained with repeated measurements in long-term follow-up.

In conclusion, it has been shown that fibroblast growth factor-2 and tumour necrosis factor alpha-stimulated gene-6 levels do not provide guidance in the diagnosis of multi-system inflammatory syndrome in children. Furthermore, based on the results of this study, these results may be modified by conducting studies with larger sample sizes and longer follow-up periods.