Association of HMGB1, S100A12 and IL-17A Expression with IVIG-Resistant Kawasaki Disease and Treatment Options

Background: Kawasaki disease (KD) is a medium vessel vasculitis of unknown aetiology that predominantly affecting coronary arteries. The damage-associated molecular pattern molecules (DAMPs) such as HMGB1, S100A12 and IL-17A have been reported to predict poor response to IVIG. Here, we explored the the role of HMGB1, S100A12 and IL-17A in the detection of intravenous immunoglobulin (IVIG)-resistant in KD patients, and to investigate the value of different adjunctive therapy. Method: 126 KD patients and as well as age- and sex-matched 16 febrile control subjects were enrolled in our study. The fresh peripheral blood were collected from KD patients and febrile controls, analyzed the demographic or clinical data and various laboratory parameters. We also measured changes in serum levels of IL-17A and mRNA expression levels of HMGB1 and S100A12 were tested in IVIG-resistant KD patients. Further we explored the association between coronary arteries lesions and different treatment options about IVIG retreatment, methylprednisolone and iniximab for IVIG-resistant KD patients. Result: Regarding laboratory parameters, KD individuals were found to have lower levels of lymphocyte(L)%, prealbumin, CD4 + , CD8 + and higher levels of WBC, neutrophil (N)%, CRP, ESR, NT-proBNP, ALT, CD4 + /CD8 + (P<0.05 or P<0.01). For KD group, the 53 IVIG-resistant patients had signicantly higher levels of S100A12, HMGB1, serum IL-17A, N%, CRP, NT-proBNP, TBIL, ALT, AST and lower levels of L%, PLT (P<0.05 or P<0.01) in comparison to the IVIG-responsive patients. For patients with IVIG-resistant, IVIG retreatment, methylprednisolone or iniximab were used. Methylprednisolone showed better in improving clinical symptoms and CRP than the IVIG retreatment and iniximab (P> 0.05). Conclusion: IVIG-resistant was associated with overreaction of inammation.The levels of HMGB1,S100A12 and IL-17A suggested to be reliable predictors for IVIG-resistant in KD. In addition, the adjunctive therapy of methylprednisolone and iniximab showed more effective in relieving clinical symptoms than IVIG retreatment.


Introduction
Kawasaki disease (KD)is an acute febrile illness that causes systemic vasculitis in young children [1]. KD is now the most common cause of acquired heart disease in developing countries [2]which can lead to serious coronary artery lesion [3,4]. Timely a single infusion of 2 g/kg intravenous immunoglobulin (IVIG) along with aspirin [5] can declined the incidence of coronary artery lesions (CALs) effectively [1,6].
However, 10-20% of KD patients develop recurrent or persistent fever after rst dose IVIG treatment are classi ed as IVIG resistant [7,8]. These IVIG resistant patients have a higher risk of CALs compared with responders. Therefore, identifying some biomarkers of patients who are likely to be IVIG-resistant would guide the choice of treatment option. So as to prevent CALs by inhibiting the in ammation reaction in the early stage.
The DAMPs such as S100 calcium-binding protein A12 (S100A12) and high-mobility group protein B1 (HMGB1) have been reported to be a sensitive indicator for disease activity and in ammation in many Page 3/19 in ammatory disorders. Studies have found HMGB1 and S100A12 are closely associated with the development of CAL in KD [5,9,10]. These markers are released from stressed cells and binding with receptor for advanced glycation end products (RAGE) or Toll-like receptors (TLRs) to activate the NF-κB pathway to produce endothelial damage and lead to CALs development in KD patients [11]. In addition, Elevated Th17 cells and decreased T regulatory cells have also been demonstrated in the acute stage of KD. Plasma levels of Th17 and Interleukin-17A (IL-17A) are also highly expressed in KD. IL-17A, tumor necrosis factora (TNF-α) and IL-6 secreted by Th17 cells have proin ammatory properties, which can collectively mobilize, recruit and activate neutrophils, thus mediating in ammation of the tissues [12][13][14].
IVIG resistance is a high risk factor for coronary involvement [15]. Here we aim to identify the clinical features and laboratory factors that are predictive of IVIG resistant KD. And early pre-judgment of nonresponse to IVIG has an important guiding role in managing KD [16]. Higher level of C-reactive protein (CRP), neutrophils(N), AST, NT-proBNP and lower level of platelet(PLT) counts have been known as the risk factors for IVIG non-response KD [17][18][19][20][21][22]. Thus, IVIG-resistant patients are associated with severe in ammatory reactions, adjunctive treatment are needed to interfere the overwhelming in ammatory process [23]. To identify predictive biomarkers which related to IVIG resistance in the early stage is meaningful. In this study, we measured some laboratory characteristics as potential risk factors for IVIG resistance KD. And we found HMGB1, S100A12 and IL-17A in children KD are at high risk for IVIG resistance.
Recently, although several adjunctive therapies such as IVIG retreatment, corticosteroids, in iximab and Others are available, comparative robust data on which to IVIG-resistant treatment decisions are scarce [24]. IVIG appears to have a wide range of anti-in ammatory effect, and possible mechanisms of action include regulation of cytokine production, increased regulation of T-cell activity, neutralization of toxins and so on [1,25]. Methylprednisolone attributes to suppression of persistent vascular in ammation. In iximab (TNF-α blockade) binds speci cally to human TNF-α and is indicated for the treatment of immune-modulated in ammatory disorders. Thus, We summarized the experience with three different IVIG retreatment options to guide the choice of Optimal therapeutic agents to clinicians for the children with IVIG resistance.

Patients
Written informed consents were obtained from parents or guardians of all study participants. 126 KD patients who were diagnosed according to the criteria established by the American Heart Association(AHA) in Wuhan Children's Hospital from 2015 to 2019 [1]. The diagnosis of classic KD is according to the presence of fever at least 5 days and the presence of ≥4 of the 5 following principal clinical criteria: 1. strawberry tongue, ssure and erythema lips 2. bilateral nonpurulent conjunctivitis; 3. maculopapular orerythema multiforme-like rashes; 4. redness and swelling of the hands and feet or periungual membrane desquamation; 5. unilateral cervical lymphadenopathy. We took the peripheral blood samples from all patients when they were admitted to hospital in acute stage. Another 16 patients with an acute febrile infectious disease were selected as a normal control group. Moreover, KD group was further divided into IVIG-responsive group and IVIG-resistant group.
All the KD patients were treated with an initial IVIG infusion at 2g/kg [26]. We de ned IVIG-responsive as defervescence within 36 to 48 hours after the initial IVIG infusion and no recurrence(temperature >38℃) , IVIG-resistant patients were persistent fever (T>38℃) beyond 36 to 48 h after standard therapy [5,26]. In our study, there were 53 KD patients turn into IVIG-resistant after initial IVIG and Acetylsalicylic Acid (ASA) therapy. Among them, 23 IVIG-resistance patients were continued with second round IVIG at 2g/kg; 26 IVIG-resistant patients received methylprednisolone (20mg/kg/d for 3 consecutive days); and 4 patients were treated with in iximab (5 mg/kg intravenously over 2 hours). Laboratory indicators at admission(KD1), after innitial IVIG(KD2),and at different treatment(KD3) laboratory variables were recorded. All of them were followed up regularly in the outpatient department.

Quantitative RT-PCR
The expression levels of mRNA for S100A12 and HMGB1 in whole blood were measured in 73 IVIGresponsive and 53 IVIG-resistant KD patients using real-time PCR. Total RNA from whole blood of KD patients was isolated by using RNA-Trizol (Takara, Dalian, China), and reverse transcription (RNA→cDNA) was carried out following the the manufacturer's instruction of kit (PrimeScript™ RT Master Mix kit, Code: RR036A). After the template cDNA was synthesized, the ampli cation steps and the reaction conditions were followed with the instruction of kit ( SYBR® Premix Ex Taq™ kit (Catalogue No: RR420A). The procedure were repeated for three times and the data were analyzed using the 2 −ΔΔCt method [27]. Sequences of primers for real-time PCR are included in Table 1.

Serum IL-17A Levels
We measured the level of serum IL-17A in patients by the ELISA in according to the manufacturer's instruction [27]. The sensitivity of the human IL-17A ELISA kit (Elabscience, Catalogue No: E-EL-H0105c) was 18.75 pg/mL, and the intra-and inter-assay precision was below 10%.

Statistical Analysis
Data are expressed as the mean ± SD for a percentage for categorical variables. We analyzed demographic, quantitative data or mRNA expression levels with Student's t-test or one-way ANOVA. SPSS version 12.0 software was used for statistical analyses. The gures in this study were generated by using GraphPad Prism 5.0 software.

Results
Laboratory characteristics of the KD group and control group All laboratory data used in this study were obtained at admission before the initiation of therapy. The variance of laboratory parameters between the control group and KD group were analyzed. The levels of L%, PA and CD8 + , CD4 + T cell were lower in KD individuals. Higher levels of WBC, N%, CRP, ESR, NT-proBNP, ALT and CD4 + /CD8 + in KD group. these differences were statistically signi cant (P<0.05 or P<0.01). There were no other statistically signi cant differences in indicators of PCT, TBIL, ALB and AST between the KD patients and control group (Table 2, Figure 1).

Demographic data
The demographic and clinical characteristics of the study participants are shown in Table 3. The acute infections among the normal control group were upper or lower respiratory tract infections or gastroenteritis. Of all the participants, 32 patients (25.40%) had CAL formation, and 53 patients (42.06%) were IVIG-resistant.
Detecting HMGB1 and S100A12 expression Comparison with KD group, the relative expression levels of HMGB1 and S100A12 mRNA were higher than NC group (P<0.05, Figure3A). Subsequentely, compared with IVIG-responsive group, the mRNA levels of HMGB1 and S100A12 in IVIG-resistant group were markedly elevated (P<0.05, Figure3B). Moreover, the Real-time PCR melt curve indicated that the primers of S100A12 and HMGB1 were speci c (Figure3C-D).

Laboratory characteristics in IVIG-responsive and IVIG-resistant group
In the KD group, the laboratory data of the IVIG-responsive patients and the IVIG-resistant patients were obtained before IVIG-treatment. The variance of laboratory parameters between the IVIG-responsive and the IVIG-resistant were analyzed. In terms of laboratory data of KD group, patients who were IVIGresistant had higher levels of N%, CRP, NT-proBNP, TBIL, ALT and AST prior to IVIG therapy when compared with those who were IVIG-responsive. Meanwhile, compared with IVIG-responsive patients, the IVIG-resistant patients had lower levels both of L% and PLT. PCT, ESR, ALB, CD4 + , CD8 + and CD4 + /CD8 + expression levels remained higher than normal value. However, there had no signi cant difference when compared with IVIG-responsive patients and the IVIG-resistant patients (Table 4, Figure 2).

Improvement of clinical symptoms in the IVIG-resistant after different treatments
The recovery of clinical symptoms in the acute phase of the three groups of IVIG resistance after discharge were shown in Table 5. The study compared the effects of three adjunctive therapies from seven aspects: the pyrexia before starting treatment, average time of hospital stay, antipyretic time, mucosal congestion subsiding time, lymph node swelling subsiding time, redness and swelling of the hands and feet subsiding time and CALs formation before starting treatment. Among 53 IVIG resistance patients, 16 of them had CAL formation before treatment. The antipyretic time and mucosal congestion/redness and swelling of the hands and feet subsiding time of methylprednisolone group and in iximab group were all shorter than IVIG retreament group (P<0.05 or P<0.01). There had no difference between three treatment options in lymph node swelling subsiding time(P>0.05). All the follow up information was collected within 2 weeks and 2 months after discharge respectively.

Various laboratory characteristics in the IVIG-resistant after different treatments
Patients who were IVIG-resistant had no statistical difference in the following laboratory data before KD1 and KD2 when different drugs treatment during hospitalization. In KD3, compared with IVIG retreatment and in iximab group, methylprednisolone group showed more signi cant lowering CRP (P<0.05). The other laboratory characteristics after three adjunctive therapies, there had no signi cantly difference(P>0.05, Table 6). After discharge, we found that methylprednisolone group and in iximab group were more effective than IVIG retreatment group in alleviating in ammatory indexes (P<0.05 or P<0.01, Table 5). All the follow up information was collected within 2 weeks and 2 months after discharge respectively.

Discussion
Kawasaki disease (KD) is an acute febrile illness associated with vasculitis that affects infants and young children. It has become the main cause of acquired heart disease during childhood. And the coronary artery lesion signi cantly impair the quality of life [28][29][30]. The coronary wall in ammation, endothelial dysfunction and impaired vascular remodeling contribute to the development of coronary artery abnormalities (CAAs) and thrombosis [31]. Developing the early judgment of the factors of IVIGresistant and CALs is crucial. Although several predictive biomarkers and genes have been described, follow-up studies are inadequate. Especially consistent reported for the risk factors of IVIG-resistant and developing CALs. The precise mechanisms of IVIG-resistant and anti-in ammation in KD patients still remain unclear [13]. Our study aimed to identify the predictor for IVIG resistance, we investigated laboratory data collected before the initial IVIG treatment. IVIG resistant patients have higher levels of N%, CRP, NT-proBNP, TBIL, ALT, AST and lower levels of L%, PLT. Several previous studies reported that CRP, ESR, ALT, γ-GT and NT-proBNP could be used for predicting resistance to IVIG therapy and patients who are at high risk for CALs [4,[31][32][33][34]. To predict the ability of response to IVIG before initial therapy, would allow clinicians to identify those potential IVIG resistant patients and give them more aggressive treatments [35]. Our results demonstrated that the higher levels of WBC, N%, CRP, ESR, NT-proBNP, ALT, CD4 + /CD8 + ratio and lower levels of L%, PA, CD8 + , CD4 + can be considered as predictor for IVIG-resistant.
Our data suggest that resistant to initial IVIG treatment and are a high risk for CALs may due to severe coronary in ammation.
The DAMPs such as HMGB1 and S100A12 have proven to be sensitive markers for disease activity and in ammation in numerous in ammatory disorders [36][37][38]. KD triggers the release of HMGB1 and S100A12, which activate toll-like receptors (TLRs) and receptor for advanced glycation endproducts (RAGE) in the affected area, leading to an exaggerated in ammatory response and cell death [39]. Here, we found signi cant increased levels of HMGB1 and S100A12 in total KD patients, especially in IVIGresistance group. These results indicating that HMGB1 and S100A12 maybe can help to assess the severity of vasculitis in KD. Th17 cells are a newly discovered T-helper cell subset associated with the proin ammatory stage of autoimmunity. Their differentiation requires both TGF-b combined with either IL-10 or IL-21 and results in the speci c expression transcription factor retinoic acid-related orphan receptor ct(RORct), and the production of in ammatory cytokines including IL-6 and IL-17A[40]. Th17-and Tregrelated Cytokines participats in many autoimmune diseases. Among them, IL-17A is obviously associated with KD and to provoke proin ammatory responses [13,41]. In addition, studies reported that genetic aberrations in certain intracellular signaling pathways involving immune effector functions can increased susceptibility to KD and development of CALs [31]. In our study, we found the level of IL-17A increased in KD group before the initial treatment. Consistent with this, IL-17A remains at a high level with IVIGresistant patients. In addition, KD group had lower percentage of CD4 + T cell, CD8 + T cells and higher CD4 + /CD8 + ratio when compared to the NC group. Clinical risk factors predict patient resistance to IVIG and treatment regimens to reduce the risk of developing CALs which remain controversy. These ndings strongly suggest that compared with the IVIG responsive group, the immune disorder may be more signi cant and serious in the IVIG resistant group.
Although IVIG is a powerful anti-in ammatory and immune regulating drug, there are still some children can't relieve the clinical symptoms after initial IVIG treatment effectively. IVIG resistance as one of the most important issue to be solved urgently for KD patients. Many experts recommend retreatment with a second dose of IVIG for IVIG-resistant. But in our study, IVIG retreatment did not show a particular advantage in alleviating clinical symptoms and in ammatory indicators. Therefore, how to choose proper therapies for IVIG-resistant KD should based on well understanding of the mechanism of that resistance. Results from our laboratory suggests that the striking anti-in ammatory effects of IVIG were weakened by addition of excessive activate in ammatory response. For IVIG-resistant, we show the IVIG retreatment, methylprednisolone, and in iximab which have different advantages in suppression of the in ammation. Compared with in iximab and IVIG retreatment group, the methylprednisolone showed more advantages in reducing CRP in the short-term follow-up (P < 0.05). But there's no difference between the three treatments in alleviate the levels of WBC, N %, ESR, ALB, ALT, AST and PLT (P > 0.05). Although whether to the use of corticosteroids in KD is still controversial, in our study methylprednisolone group showed better in improving clinical symptoms than the IVIG retreatment and in iximab group during hospitalization. After discharge, we found that methylprednisolone group and in iximab group were more effective than IVIG retreatment group in alleviating in ammatory indexes at follow-up. The main Complications of in iximab is that it can increase the risk of latent infection or opportunistic infection. Fortunately, there's no infusion reactions or complications were happened in our study. We suggests that in iximab is effective and well tolerated for IVIG-resistant KD. It can also possible to prevent CALs effectively by inhibiting overactive in ammatory response.
In our study, several details deserve further attention. First, the relatively small sample size of this study might prevent some of the detected data from being statistically signi cant. Second, our study results need to be validated across different hospitals, different regions, different races. Lastly, IVIG-resistant was associated with higher levels of HMGB1and S100A12, but the exact signaling pathway remains unknown. Therefore, further work to explore the mechanism of IVIG-resistant is necessary. Further studies on the mechanism of IVIG-resistant may help identify new methods to predict and treat IVIG-resistant patients effectively in KD.

Conclusions
Accumulating evidence suggests that higher levels of WBC, N%, CRP, ESR, NT-proBNP, ALT, CD4 + /CD8 + ratio and lower levels of L%, PA, CD8 + , CD4 + T cells showed useful for predicting in KD patients with IVIGresistant. And high level of HMGB1, S100A12 and serum IL-17A before the initial treatment could also predict IVIG-resistant. In addition, methylprednisolone showed better outcome in improving clinical symptoms than the IVIG retreatment and in iximab.

Abbreviations KD Kawasaki disease IVIG Intravenous immunoglobulin
CALs coronary artery lesions DAMPs damage-associated molecular pattern molecules HMGB1 high-mobility group protein B1 S100A12 S100 calcium-binding protein A12 IL-17A Interleukin-17A Declarations Ethics approval and consent to participate Written informed consents were obtained from parents or guardians of all study participants. The experimental protocol was approved by the Ethics Committee of Wuhan Children's Hospital.

Consent for publication
All the authors agreed to publish.

Availability of data and material
The data used and analyzed in the present study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interest. Tables Table 1 The sequences of all primers for rt-PCR.      Laboratory characteristics changes in IVIG-resistant patients during KD1, KD2 and KD3 points of Kawasaki disease. KD1: before the treatment of initial IVIG (A); KD2: after the treatment of initial IVIG (B); KD3: after the second dose of different treatment options, including IVIG retreatment, methylprednisolone and in iximab group (C). *P< 0.05 compared with IVIG-retreatment group. Figure 1 Laboratory characteristics between with KD group and NC group. NC group: normal control group; KD group: Kawasaki disease group. *P< 0.05, **P< 0.01

Figure 3
Page 19/19 (A): The relative expression levels of HMGB1 and S100A12 mRNA between NC group and KD group. (B) The relative expression levels of HMGB1 and S100A12 mRNA between IVIG-responsive group and IVIGresistant group. The melt curve of S100A12(C) and HMGB1(D).Data are shown as the mean ± SD. # : P< 0.05