Introduction

Kawasaki disease (KD) is an acute, self-limiting systemic vasculitis primarily affecting children under five years of age.¹ Its defining pathological hallmark is inflammation of medium-sized blood vessels, with a marked predilection for the coronary arteries. The most severe complication of KD is the development of coronary artery lesions (CALs), which can progress to coronary artery aneurysms, significantly elevating the risk of myocardial infarction, ischemic heart disease, and arterial rupture.² Despite extensive research, the precise pathogenesis of KD remains poorly understood.³ However, emerging evidence highlights immune system dysfunction as a central mechanism that leads to the overproduction of inflammatory cytokines, and result in vasculitis and the formation of CALs.⁴ Studies have consistently demonstrated elevated serum pro-inflammatory cytokine levels, such as interleukin-17 (IL-17),⁵ IL-1β,⁶ IL-6,^7,8 and tumor necrosis factor- α (TNF-α), in patients with KD. Conversely, anti-inflammatory cytokine IL-35 levels are significantly reduced⁹ These imbalances in pro- and anti-inflammatory mediators are closely associated with vasculitis and the development of CALs observed in KD.

Thymosin β4 (Tβ4) is a 43-amino acid lymphopoietic peptide widely distributed across various tissues, with prominent expression in the myocardium and vascular smooth muscle.¹⁰ It serves as a key regulator of actin dynamics, mediating a wide range of biological functions, including actin regulation,¹¹ inhibition of apoptosis,¹² modulation of inflammatory responses,¹³ and promotion of angiogenesis.¹⁴ The anti-inflammatory properties of Tβ4 have been extensively demonstrated in preclinical studies. For instance, in mouse models of ethanol- and lipopolysaccharide-induced liver injury, as well as neonatal models of fetal alcohol spectrum disorder, Tβ4 can attenuate the inflammation response by suppressing the production of pro-inflammatory cytokines such as TNF-α and IL-1β.^13,15 Additionally, Tβ4 plays a pivotal role in angiogenesis. In a mouse model of hindlimb ischemia, Tβ4 enhanced blood vessel formation by upregulating vascular endothelial growth factor, thereby increasing the proliferation and migratory capacity of endothelial progenitor cells.¹⁴ Recent evidence further underscores the essential role of Tβ4 in all three stages of cardiac vessel development—vasculogenesis, angiogenesis, and arteriogenesis in animal models. Remarkably, Tβ4 facilitates coronary vascularization during both childhood and adulthood by supporting cardiomyocyte survival and exerting protective effects on the heart.¹⁰

Despite the well-documented roles of Tβ4 in inflammation and angiogenesis, whether its involvement in the pathogenesis of KD remains unexplored. This study aimed to address this gap by evaluating serum Tβ4 levels in children with acute KD and exploring their correlation with clinical parameters and CALs.

Methods

General Characteristics of Participants

This study included children diagnosed with KD at the Children’s Hospital of Chongqing Medical University between April 2023 and October 2024. Diagnosis was made in accordance with the American Heart Association’s diagnostic and treatment guidelines.¹⁶ An age-matched healthy control (HC) group consisting of children without any medical conditions, was also recruited. All participants underwent screening to exclude underlying inflammatory, immunological, metabolic, hematological, or cardiac disorders. Ethical approval for the study was obtained from the Ethics Committee of the Children’s Hospital of Chongqing Medical University (Approval Number: ID245-2024). Written informed consent was obtained from the parents or legal guardians of all participants.

Echocardiographic evaluations were conducted on patients with KD before the initiation of treatment with intravenous immunoglobulin (IVIG) and anticoagulants. Based on coronary artery z-scores, patients with KD were categorized into two groups: those with z-scores ≥2.0 were classified as the KD-CALs group, while those with z-scores <2.0 were designated as the KD-NCALs.¹⁷ All patients with KD received standard treatment consisting of intravenous immunoglobulin (2 g/kg as a single infusion) and oral aspirin (initially 30–50 mg/kg/day in divided doses during the acute phase, followed by 3–5 mg/kg/day as a single daily dose during the subacute phase), according to the AHA guidelines.¹⁶

Blood Sample Collection and Processing

Blood samples were collected from patients with KD during the acute phase of the disease, prior to the administration of IVIG or anticoagulant therapy, as well as from healthy children in the HC group. The samples were centrifuged at 3000 rpm for 10 minutes to separate the serum, which was subsequently stored at −80°C for further analysis.

Serum Tβ4, Cytokine Levels, and Laboratory Variables

Serum Tβ4 levels were measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Cusabio, China). A comprehensive panel of laboratory parameters was also evaluated, including white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb), platelet (PLT) count, neutrophil percentage (N%), lymphocyte percentage (L%), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), and creatine kinase isoenzymes (CK-MB). Coagulation markers such as prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were also assessed. Additionally, liver function tests were performed, measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Serum cytokine levels were quantified for IL-2, IL-12p70, interferon-alpha (IFN-α), IL-8, IL-4, IL-5, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, and IL-6.

Statistical Analysis

Statistical analyses were conducted using IBM SPSS Statistics for Windows (version 25.0; IBM SPSS Corp., Armonk, NY, USA). Normally distributed data were compared using Student’s t-test, while non-normally distributed data were analyzed with the Mann–Whitney U-test. Categorical variables were evaluated using the chi-square test. The relationship between serum Tβ4 levels and clinical parameters was examined through Spearman’s rank correlation analysis. Results are reported as mean ± standard deviation (SD) for normally distributed data, median (P25, P75) for non-normally distributed data, or counts and percentages (n, %) for categorical variables. A P-value of <0.05 was considered statistically significant.

Results

Participant Demographics

The study included 90 children diagnosed with KD, comprising 54 males and 36 females, mean age of 2.50 ± 1.59 years. These patients were further classified into two subgroups: 43 in the KD-CALs group and 47 in the KD-NCALs group. Additionally, 55 children (32 males and 23 females, mean age of 2.96 ± 1.77 years) in the HC group were included. There were no statistically significant differences in age or sex between the KD and HC groups (P >0.05).

Serum Tβ4 Levels in All Participants

As shown in Figure 1, the serum Tβ4 levels in the KD group (0.903 mg/mL [0.575, 1.155], n = 90) were significantly lower compared to the HC group (1.190 mg/mL [0.840, 1.441], n = 55) (P <0.05).

Figure 1 Serum Tβ4 levels in the KD group and the HC group. **P<0.01.

Laboratory Variables and Serum Tβ4 Levels in the KD-CAL and KD-NCAL Groups

As shown in Table 1, there were no significant differences in WBC, RBC, Hb, PLT, N%, L%, CRP, ESR, PCT, AST, ALT, CK-MB, PT, APTT, TT or IL-2, IL-12p70, IFN-α, IL-8, IL-4, IL-5, TNF-α, IFN-γ, IL-17A, IL-1β, IL-6 between the KD-CALs and KD-NCALs groups (P >0.05). However, compared to the KD-NCALs group, the KD-CALs group showed significantly higher serum IL-10 levels and significantly lower Tβ4 levels (P < 0.05).

Table 1 Laboratory Variables and Serum Tβ4 Levels in the KD-CALs and KD-NCALs Groups

Relationship Between Serum Tβ4 Levels and IVIG Treatment in Patients with KD

As shown in Figure 2, serum Tβ4 levels increased significantly following IVIG treatment. The median Tβ4 level before IVIG treatment was 0.783 ng/mL (0.531, 1.142), while the level after treatment was 1.572 ng/mL (0.717, 2.277) (P < 0.05).

Figure 2 Serum Tβ4 levels increase after IVIG treatment. **P<0.01.

Correlations Between Tβ4 Levels and Laboratory Variables in Patients with KD

Serum Tβ4 levels were positively correlated with RBC and Hb levels while they showed negative correlations with PCT, TT, IL-2, IL-12p70, IFN-α, IL-4, IL-10, TNF-α, and IL-1β (P <0.05; Table 2). No significant correlations were observed between serum Tβ4 levels and other variables, including WBC, PLT, N%, L%, CRP, PT, APTT, ESR, AST, ALT, CK-MB, IL-8, IL-5, IFN-γ, IL-17A, or IL-6 in the KD group (P >0.05).

Table 2 Correlations Between Tβ4 Levels and Laboratory Variables in Patients with KD

Correlations Between Tβ4 Levels and Laboratory Variables in the KD-CALs and KD-NCALs Groups

There were no significant correlations between serum Tβ4 levels with WBC, RBC, Hb, PLT, N%, L%, PCT, ESR, CRP, AST, ALT, CK-MB, APTT, PT, IL-2, IL-12p70, IFN-α, IL-8, IL-5, IL-10, IFN-γ, IL-17A, or IL-6 (P >0.05), negative correlations were observed with TT, IL-4, TNF-α, and IL-1β in the KD-CALs group (P <0.05). However, There was a positive correlation between serum Tβ4 levels and RBC and negatively correlated with PCT, IL-2, IL-12p70, IFN-α, TNF-α, and IL-1β in the KD-NCALs group (P <0.05; Table 3).

Table 3 Correlations Between Tβ4 Levels and Laboratory Variables in the KD-CALs and KD-NCALs Groups

Discussion

KD is an acute systemic vasculitis, CALs being its most severe complication. While previous research has suggested that acute inflammation, coagulation abnormalities, and endothelial dysfunction contribute to the pathogenesis of KD, the precise mechanisms remain unclear.^18,19 Tβ4 is known for its anti-inflammatory properties¹¹ and ability to promote angiogenesis¹⁴ in cardiovascular disease, but whether it is involved in the pathogenesis of KD remains unclear. This study aimed to evaluate serum Tβ4 levels in patients with KD and their relationship with CALs. Key findings include: (1) Serum Tβ4 levels in the KD group were significantly lower than those in the HC group; (2) Tβ4 levels were further reduced in the KD-CALs group compared to the KD-NCALs group; (3) serum Tβ4 levels increased significantly post-IVIG treatment in children with KD; (4) There were positively correlated between Tβ4 levels and RBC and Hb levels, while negatively correlating with TT, PCT, IL-2, IL-12p70, IFN-α, IL-4, TNF-α, and IL-1β in the KD group. Notably, negative correlations between Tβ4 levels and TT, IL-4, TNF-α, and IL-1β were specifically observed in the KD-CALs group.

Tβ4 is an endogenous peptide with protective and regenerative properties, demonstrated in models of cellular and organ injury.^13,15 It is increasingly recognized as a potential biomarker in a range of conditions, including cardiovascular disease,¹⁰ hepatic disorders,²⁰ infectious,²¹ and autoimmune diseases.²² Tβ4 is a key regulator of actin dynamics, promoting angiogenesis,¹⁴ inhibiting apoptosis,¹² and modulating inflammation.¹³ While its role has been explored in cardiovascular diseases,²³ rheumatoid arthritis,²⁴ sepsis,²⁵ and liver disorders,¹⁵ but its involvement in acute inflammatory diseases like KD remains less documented. We found that serum Tβ4 levels were significantly lower in the KD group compared to healthy controls, with even lower levels observed in patients with CALs. These findings suggest that Tβ4 may be involved in the inflammatory pathogenesis of KD and the progression of CALs. Tβ4 is known to be actively secreted by endothelial and immune cells under physiological and reparative conditions and plays an important role in promoting endothelial cell migration, angiogenesis, and tissue repair.^26,27 IVIG treatment in KD has been shown to reduce systemic inflammation and coronary artery damage by downregulating proinflammatory cytokines such as IL-6, TNF-α, and MMP-9,^28,29 while also decreasing markers of cellular injury and necrosis.³⁰ Consistent with these effects, we observed a significant increase in serum Tβ4 levels following IVIG administration. Given the reduction in tissue damage and inflammation, this increase is unlikely to be due to passive leakage due to cell death. Instead, it may reflect an upregulation of active Tβ4 production in response to vascular repair and immune modulation. However, due to the observational nature of the study and the partial overlap in Tβ4 levels across groups, further mechanistic investigations are needed to elucidate the underlying pathways and to determine whether Tβ4 could serve as a meaningful indicator of disease activity or vascular involvement in KD.

IL-2, IL-12p70, IFN-α, TNF-α, and IL-1β are classic pro-inflammatory cytokines that play distinct roles in the activation and progression of inflammation through various mechanisms. IL-2³¹ and IL-12p70³² promote the differentiation of Th1 cells, thereby enhancing immune responses and exacerbating inflammation. Additionally, Tβ4 exhibited protective effects by reducing TNF-α and IL-1β levels in a colitis mouse model,³³ However, no studies have directly linked Tβ4 with IL-2, IFN-α or IL-12p70. Our study is the first to show that Tβ4 levels are negatively correlated with IL-2, IL-12p70, IFN-α, TNF-α, and IL-1β levels in patients with KD. These associations suggest a potential interaction between Tβ4 and the cytokine milieu during the acute phase of KD. Lower Tβ4 levels were correlated with elevated concentrations of both pro- and anti-inflammatory cytokines, indicating that Tβ4 may be involved in complex immunomodulatory pathways. Whether Tβ4 is regulated by inflammatory cytokines, contributes to their expression, or both, remains to be elucidated through further mechanistic studies.

In contrast, IL-10 and IL-4, classical anti-inflammatory cytokines, are significantly elevated during the acute phase of KD.³⁴ IL-4 promotes Th2 immune responses while suppressing Th1-mediated inflammation, potentially mitigating disease progression.³⁵ However, the precise role of IL-4 in KD remains unclear. Previous studies suggest that Tβ4 may protect against elevated IL-10 levels,³¹ but no direct evidence has linked Tβ4 with IL-4. Our study is the first to demonstrate that Tβ4 levels are negatively correlated with both IL-10 and IL-4 in patients with KD. These findings may suggest a compensatory mechanism: IL-4 and IL-10 may be upregulated following Tβ4 downregulation to mitigate inflammation. Their expression may not directly reflect Tβ4 levels but instead be mediated by broader immunoregulatory pathways that underlie Tβ4’s protective effect on the endothelium and the promotion of tissue remodeling. Notably, IL-4 can induce endothelial activation and monocyte recruitment via MCP-1,³⁶ whereas IL-10 is linked to IVIG resistance and coronary complications,³⁷ underscoring their context-dependent pathogenic potential. Furthermore, Wang et al indicated that serum IL-4 serves as an independent predictor of coronary artery disease.³⁸ Our research further revealed that Tβ4 levels are negatively correlated with IL-4 in patients with KD-CALs, suggesting that Tβ4 may play a crucial regulatory role in the pathogenesis of KD-CALs.

Furthermore, Tβ4 levels were negatively correlated with TNF-α and IL-1β in the KD group. Both of these pro-inflammatory cytokines play a crucial role in the pathogenesis of coronary artery damage in KD. TNF-α inhibitors are already being used to prevent coronary aneurysms,³⁹ and IL-1β has been shown to be closely linked to inflammation and vascular damage in KD cell models.⁴⁰ Tβ4 may be associated with reduced levels of TNF-α and IL-1β, potentially contributing to its observed protective role, although the underlying mechanisms remain to be clarified.

Prolonged TT is commonly indicative of coagulation abnormalities, reflecting impairments in fibrin generation and stabilization.⁴¹ Previous studies have demonstrated that Tβ4 enhances the stability of the fibrin network and supports coagulation by facilitating the attachment of factor XIIIa (transglutaminase) to fibrin and collagen.⁴² Coagulation dysfunction has been closely associated with the development of CALs⁴² in patients with KD. Our study revealed that there were negative correlation between Tβ4 levels and TT, suggesting that reduced Tβ4 levels may prolong TT, compromise fibrin formation and stability, and promote a hypercoagulable state. This, in turn, may contribute to coronary artery thrombogenesis and further result in myocardial ischemia in patients with KD.

Conclusions

Our study demonstrated that serum Tβ4 levels were significantly lower in patients with KD, particularly in those with CALs. Tβ4 levels were negatively correlated with several pro-inflammatory cytokines (such as IL-2, IL-12p70, IFN-α, TNF-α, and IL-1β) and anti-inflammatory cytokines (such as IL-10 and IL-4). While these associations suggest that Tβ4 may be involved in the inflammatory milieu of KD and the development of CALs, the specific mechanism remains unclear. Moreover, although serum Tβ4 levels increased following IVIG treatment, the underlying mechanism remains unclear. Due to the observed overlap of Tβ4 levels between groups, its potential diagnostic or protective value should be further investigated in larger, mechanistic studies. Furthermore, this study has several limitations. First, the sample size was relatively small and recruited from a single center, which may limit the generalizability of the results. Second, the observational design of the study precludes conclusions about causality between Tβ4 levels and cytokine expression. Third, we did not perform mechanistic experiments to directly explore the functional roles of Tβ4 in Kawasaki disease. Further studies incorporating in vitro and in vivo functional analyses as well as larger, multicenter cohorts are warranted to validate and expand upon our findings.