Association Between IL-23 and Coronary Arterial Lesions in Children with Kawasaki Disease

The pathogenesis of coronary artery lesions (CALs) in KD patients has been thought of as an unknown stimulus that triggers an inammatory cascade with activation of the immune system. However, interleukin-23 (IL-23) is supposed as a key cytokine in inammatory and autoimmunity diseases. The role of IL-23 in the pathogenesis of CALs has not been fully elucidated. This study explored the relationship between the serum IL-23 levels and CALs in patients with KD. We collected blood specimens from 90 children with KD before intravenous immunoglobulin (IVIG) therapy. Levels of IL-23, IL-6, IL-17A, IL-10, MCP-1 and VEGF were measured in 190 cases, including 4 groups: KD with CALs (n = 46), KD without CALs (n = 44), febrile control group, (FC, n = 40) and normal control group, (NC, n = 60). Clinical parameters were tested in all subjects. IL-23 was signicantly elevated in the KD group compared with the febrile and normal control groups, especially increased in the KD patients with CALs. Serum levels of IL-23 in KD patients were positively associated with WBC, CRP, IL-6, IL-17A, IL-10, MCP-1, and VEGF in children with KD. In conclusion, IL-23 may be involved in the pro-inammatory process and the pathogenesis of CALs in KD patients.


Introduction
Kawasaki disease (KD) is a self-limited autoimmune systemic vasculitis disease predominantly in children under 5 years [1]. Its most serious complication is coronary arteritis lesions (CALs) or aneurysms that may cause ischemia, myocardial infarction, and sudden cardiac death [2]. Although the pathogenesis of CALs in KD patients has not been fully elucidated, an unknown stimulus triggers an in ammatory cascade with activation of the immune system is thought to be a cause of the disease [3].
Interleukin-23(IL-23) is a recently discovered cytokine of the IL-12 family and a heterodimeric protein composed of an IL-23-speci c p19 subunit and a p40 subunit shared with IL-12. IL-23 is mainly produced by activated macrophages and dendritic cells [7][8]. IL-23 is supposed as a key cytokine in in ammatory and autoimmunity diseases such as atherosclerosis, rheumatoid arthritis (RA), and asthma [9][10], which promotes Th17 cell differentiation and activation to produce in ammatory factors( IL-6, IL-17, TNF-α, and GM-CSF). Moreover, IL-23 can produce IL-1, TNF-α, and IL-23 itself through activating in ammatory macrophages [11][12]. However, whether IL-23 involve in the pathogenesis of KD is still unclear. Thus, we examined serum IL-23 levels and the correlation with other in ammatory cytokines (IL-6, IL-17A, IL-10, MCP-1, and VEGF) to explore whether IL-23 has a role in developing CALs in KD patients.  [13]. 60 healthy children (32 males and 28 females, 2.19±2.22 years old) as the normal control group(NC) and 40 children with an acute febrile infectious disease (20 males and 20 females, 2.84±1.63 years old) as the febrile control group(FC).

Materials And
KD patients were divided into 2 groups based on the presence or absence of CALs: KD with CALs (n=46) and KD without CALs (n=44). CALs were de ned by having an actual internal diameter of 3 mm or more in a child under 5 years; 4 mm or more in a child 5 years or older, or if the internal diameter of the segment was at least 1.5 times greater than that of an adjacent segment according to examination by echocardiography [14]. Echocardiography was obtained before administering intravenous immunoglobulin (IVIG) or within 2 weeks of the onset. We collected blood specimens from KD patients before IVIG therapy within 1 week after symptom onset. Serum was stored at -80℃ for future analysis.

Statistical analysis
All data are expressed as number and percentage (n, %) or mean ± standard deviation (SD). One-way ANOVA and 2-tailed unpaired t-test were used to analyze the statistical signi cance of concentrations of cytokines. Correlation between two groups was tested by Pearson's correlation analysis. A two-tailed pvalue of <0.05 was considered signi cant. SPSS version 18 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

Results
3.1. Serum IL-23 levels were higher in KD patients than that in normal children and acute febrile infectious patients.
The serum samples analyzed in this study are the same samples analyzed in the previous report [15]. It shows that no statistically signi cant differences in age and gender among three groups: the KD patients group, the normal control group(NC), and the febrile control group(FC) [15]. The detected serum IL-23 levels ranged from 0.4 to 2507.3 pg/ml. As shown in Fig. 1 3.3. The relationship between IL-23 and clinic characteristics and in ammatory factors.
Our previous report has showed that levels of HB and RBC markedly decreased, and levels of WBC, PLT, CRP, ESR and PCT were signi cantly elevated in the KD group than those in NC and FC groups. Moreover, IL-6, IL-17A, IL-10, MCP-1 and VEGF were markedly increased in the KD group compared with the NC and FC groups (p<0.05) [15]. In this study, we further compare the relationship between IL- 23 (Table 1).  IL-23 is produced mainly by activated dendritic cells and macrophages, which is supposed as a key cytokine in in ammatory and autoimmunity diseases such as atherosclerosis, asthma, and rheumatoid arthritis (RA) [7][8][9][10]. However, few studies have examined the role of IL-23 in the pathogenesis of KD. Our research shows that serum IL-23 levels in KD patients were higher than those in normal children and febrile infectious patients, indicating that IL- 23  IL-10 is an immunomodulatory cytokine with anti-in ammatory and immunosuppressive properties, which inhibits the production of in ammatory cytokines(such as IL-1β, IL-6, and TNF-α) and is implicated in the pathogenesis of in ammatory and autoimmune diseases [21][22][23]. IL-10 has been reported to be involved in the pathogenesis of KD. The adeno-associated virus (AAV) -mediated induction of IL-10 prevents vascular in ammation, brosis, and lethality in a murine model of KD [24][25]. Some research shows that IL-23 can enhance IL-10 production, consistent with our results that IL-23 levels were positively correlated with IL-10 levels [26][27]. Therefore, we speculate that IL-23 may have an effect on inducing the expression of IL-10 in the acute phase of KD.

Discussion
This research found that serum IL-23 levels were positively associated with WBC, CRP, MCP-1, and VEGF levels in KD patients. MCP-1 is a new member of the CC-chemokine family that activates and attracts the monocytes, which has been con rmed to be positively associated with the development of KD [28][29][30].
There is almost no research on the relationship between IL-23 and MCP-1. Our study showed that serum IL-23 levels positively correlated with MCP-1 in KD patients so that IL-23 may promote the expression of MCP-1 and may promote vascular in ammation during the acute phase of KD can be speculated.
In ammation predictors of in ammation include WBC and CRP. High levels of CRP serve as an independent risk factor for predicting giant aneurysms in KD [31]. Furthermore, VEGF, a potent proangiogenic protein, can increase vascular permeability and endothelial cell proliferation, which is considered a predictor of CAL in acute KD [32][33][34][35]. Moreover, IL-23 could promote VEGF production in mammary cancer and human colorectal carcinoma [36][37]. Our study showed that serum IL-23 levels were positively correlated with CRP, WBC, and VEGF. Therefore, it can be speculated that IL-23 may promote the production of VEGF and the formation of the CAL by promoting the pro-in ammatory process in KD patients.
The main limitation of our study is the relatively small number of recruited patients and lack of comparison between before and after treatment with IVIG of KD patients. Absence of more proin ammatory cytokines(TNF-a, IL-1β, INF-γ, and TGF-β) measurement at the same time.
In conclusion, our study demonstrated that serum IL-23 levels increased in the KD patients, notably KD patients with CALs. Moreover, serum IL-17A, IL-10, IL-6, MCP-1, and VEGF levels were positively correlated with IL-23 in the acute phase of KD. The results of this study indicate that IL-23 might play an proin ammatory role in the response of vasculitis in KD. Additional research is needed to further clarify the mechanism of IL-23 in the pathogenesis of KD vasculitis.

Declarations
Acknowledgments This work was supported by National Natural Science Foundation of Chinaunder Grant: No.81500273.
Compliance with ethical standards Con ict of interest All authors have no actual or potential con icts of interest with other people or organizations with 3 years of initiating the work presented here.
Ethical approval The study protocol was approved by the Ethics Committee of Children's Hospital of Chongqing Medicine University, and written informed consent forms were obtained from the parents of all subjects. Figure 1 Comparisons of serum IL-23 levels among KD, FC and NC groups.

Figure 2
Comparisons of serum IL-23 levels between the KD with CALs group and KD without CALs group.