Necrotic Change of Tunica Media Plays a Key Role in the Development of Coronary Artery Lesions in Kawasaki Disease

Abstract

Background: Alarmins resulting from cell death or oxidative stress are involved in the development of Kawasaki disease (KD) vasculitis. In a previous study, we demonstrated the potential role of interleukin (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated.

Methods and Results: We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) using a coculture assay. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-suppression of tumorigenicity 2 (ST2) antibodies compared with conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Primary necrosis of HCASMCs induced significant release of IL-33. In cocultures of necrotic HCASMCs with HCAECs, the necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in the HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs.

Conclusions: There is potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

Kawasaki disease (KD) is an acute, self-limiting, febrile systemic vasculitis of unknown etiology associated with the development of coronary artery lesions (CALs) in infants and young children.^1–3 Although the etiology of KD has not been fully elucidated, the innate immune activity triggered by certain factors plays a role in the development of KD vasculitis. The innate immune pathogen-associated molecular patterns of microbes activate proinflammatory signals in innate immune and vascular cells through pattern recognition receptors, followed by the production of large amounts of chemokines and cytokines. Moreover, the damage-associated molecular patterns (DAMPs)/alarmins from cell death and oxidative stress are thought to be involved in the pathogenesis of KD and the development of CALs.^4–8 Various alarmins, such as S100 proteins and high mobility group box 1, are elevated in the serum of patients with KD during the acute phase.^4,8 Alarmins exert pleiotropic effects on platelets, monocytes, neutrophils, endothelial cells, and vascular smooth muscle cells (SMCs) through receptor-mediated and receptor-independent mechanisms, followed by proinflammatory cytokine release, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α.⁴

Recently, IL-33, a member of the IL-1 family of cytokines, was reported to be involved in KD pathophysiology during both the acute and long-term phases.⁶ IL-33 acts as an alarmin and alerts the immune system against tissue damage.⁹ We have previously shown that IL-33 has more potential to induce elevated production of proinflammatory cytokines in human coronary artery endothelial cells (HCAECs) than TNF-α, which is one of the major leading cytokines in KD.⁶ In that study, we showed that human coronary artery SMCs (HCASMCs) may be a major source of IL-33 release in KD.⁶ Based on those results, we hypothesized that the edematous dissociation (necrotic change) of the tunica media composed of HCASMCs observed in patients who died 6–8 days after the onset of KD might be involved in the exacerbation of KD vasculitis.¹⁰ This necrosis occurs before significant infiltration by inflammatory cells from the intima and adventitia, indicating that the structural weakening of the arterial wall due to SMC death is a precursor to further inflammatory damage.¹¹ To test this hypothesis, we performed experiments to confirm the relationship between edematous dissociation of the tunica media and worsening KD vasculitis using an in vitro model of coronary arteritis in KD closer to in vivo conditions.

Materials and Methods

Cell Culture Conditions

HCAECs were purchased from Lonza (Walkersville, MD, USA) and maintained in endothelial cell growth medium-2 (EGM-2) BulletKit (Lonza) at 37℃ under humidified 5% CO₂ as previously described.⁶ HCASMCs were cultured in SMC growth medium-2 (SmGM-2) BulletKit (Lonza) under the same conditions.

Primary Necrosis of HCASMCs Induced by Membrane Rupture

The HCASMCs were washed once in pH 7.0 N-(2hydroxyethyl)piperazine-N’-ethanesulfonic acid (HEPES) buffer and subsequently exposed to recombinant human IL-1β or TNF-α (R&D Systems) at a concentration of 10 ng/mL for 4 h to mimic KD conditions. After stimulation, the HCASMCs were washed again in HEPES buffer and then frozen for 30 min at −80℃ and thawed again to cause primary necrosis.¹² Immediately after thawing, SmGM-2 medium was added at a density of 4.0×10⁴ cells/mL for the next steps of the coculture assay.

Coculture Assay of HCAECs and HCASMCs

We used a coculture assay using the Transwell^® cell culture insert system (Corning Inc. NY, USA) to investigate the interactions between HCAECs and HCASMCs.^13–15 The cell culture insert system, with its porous membrane, allows the exchange of soluble factors and facilitates cell-to-cell communication between 2 cell types. HCAECs were seeded into the lower chamber of 24-well plates (4.0×10⁴ cells/well), and incubated at 37℃, 5% CO₂ for 24 h. Subsequently, HCASMCs were seeded into the upper chamber (2.0×10⁴ cells/well) followed by coculture with HCAECs in the lower chamber. This setup mimics the in vivo environment of KD coronary arteritis, where endothelial cells line the inner surface of the coronary artery and necrotic SMCs form the middle layer. Note that after 24 h of coculturing, the supernatant was collected from the lower chamber for subsequent analyses.

Anti-Inflammatory Effects of Anti-IL33/Suppression of Tumorigenicity 2 (ST2) Axis Antibodies on the HCAECs

The anti-inflammatory effects of anti-IL-33 and anti-ST2 antibodies for KD vasculitis were evaluated and compared with those of conventional therapies such as intravenous immunoglobulin (IVIG) or anti-TNF-α antibodies. The doses of the anti-IL-33 and anti-ST2 antibodies were determined according to a previous report.^16–21 The dose of high-dose IgG (representative of IVIG) and anti-TNF-α antibodies were calculated from the ratio of experimental dose to clinical dose of anti-IL-33 antibodies.^1,22 The HCAECs were pretreated with 5 μg/mL of anti-IL-33 antibody (Catalog no. AF3625, R&D Systems), 5 μg/mL of anti-ST2/IL-33R antibody (Catalog no. AF523, R&D Systems), 2 mg/mL of IgG (Venoglobulin IH5%I.V., Japan Blood Products Organization, Tokyo, Japan), or 5 μg/mL of anti-TNF-α antibody (Catalog no. MAB610, R&D Systems) for 30 min before coculture. Note that 5 μg/mL of goat IgG isotype control was used as the control (Catalog no. 02-6202, ThermoFisher Scientific).

Enzyme-Linked Immunosorbent Assay (ELISA)

To evaluate the levels of IL-33 in the supernatant of necrotic HCASMCs and IL-6 in the supernatant of HCAECs, we performed an ELISA (R&D Systems). The lower detection limits for IL-33 and IL-6 were 0.4 pg/mL and 0.7 pg/mL, respectively.

Multiplex Cytokine Analysis

We performed a comprehensive cytokine assay using a Bio-Plex 3D system (Bio-Rad Laboratories Inc., Hercules, CA, USA) and a Bio-Plex Human cytokine48/chemokine40/inflammation37 screening panel (Bio-Rad Laboratories Inc.), as previously described.²³ Briefly, 1×diluted beads (50 μL) were added to each well of a 96-well plate. The plate was washed twice with 100 μL of Bio-Plex buffer and protected from light. Note that 50 μL of the standards, the HCAEC supernatants diluted 10-fold, and blank were added to each well. The plate was sealed and incubated on a shaker (Stat Fax^TM 2200, 220 V Incubator/Shaker, CA, USA) at 850 rpm for 1 h at room temperature (RT). Subsequently, the plate was washed 3 times with 100 μL of buffer with the Bio-Rad Bio-Plex Pro^TM II Wash station (Bio-Rad Laboratories, Inc., USA). The 10×detection antibodies (25 μL) were added to each well and incubated (30 min, RT). The plate was washed with 100 μL of buffer 3 times before adding 50 μL of 1×streptavidin alkaline phosphatase (SA-PE) to each well and incubated (10 min, RT). Subsequently, the beads were resuspended in 125 μL of assay buffer and incubated on a shaker at 850 rpm for 2 min at RT. The plates were read using a Bio-PlexMAGPIX Multiplex Reader. Bio-Plex Manager^TM software (version 6.1) was used to analyze data from the multiplex immunoassays. All cytokines are expressed as pg/mL and extrapolated from standard curves. Inter- and intraplate variabilities were determined with CV <20% and Accuracy [(Obs/Expected)*100] between 70% and 130% (r=0.8, P=0.05).²⁴ The panels showed the cytokines listed in the Supplementary Table.

Statistical Analysis

Differences in the results were analyzed using non-repeated-measures ANOVA. For all statistical analyses performed, the statistical significance was set at P<0.05. Analyses and calculations were performed using Excel Statistics for Windows, v.3.00 (BellCurve, Tokyo, Japan).

Results

IL-33 Released From Necrotic HCASMCs

Because we hypothesized that edematous dissociation of the tunica media might contribute to worsening of KD vasculitis through IL-33 release,⁶ we evaluated the effects of primary necrosis on HCASMCs using the freeze-thaw procedure. As shown in Figure 1, primary necrosis of HCASMCs induced a significant release of IL-33. Moreover, pretreatment with IL-1β, but not TNF-α, enhanced the production of IL-33 in HCASMCs, which was consistent with the results of our previous study.⁶

Figure 1.

Primary necrosis of HCASMCs can induce IL-33 release. HCASMCs were exposed to recombinant human TNF-α or IL-1β at a concentration of 10 ng/mL for 4 h to mimic KD conditions. After the stimulation, the HCASMCs were frozen at −80℃ and thawed to cause primary necrosis, which induced significant release of IL-33. Moreover, pretreatment with IL-1β, but not TNF-α, enhanced the production of IL-33 by HCASMCs. HCASMCs, human coronary smooth muscle cells; IL, interleukin; KD, Kawasaki disease; TNF, tumor necrosis factor.

Necrotic HCASMCs Induced Cytokine Production by HCAECs

We evaluated the impact of edematous dissociation of the tunica media, which may be involved in the pathogenesis of KD vasculitis, on cytokine production by HCAECs. ELISA of the cell culture supernatant of HCAECs demonstrated that necrotic HCASMCs increased the levels of IL-6 (Figure 2). Moreover, pretreatment of the HCASMCs with IL-1β further elevated cytokine production.

Figure 2.

Necrotic HCASMCs induce cytokine production in HCAECs. HCASMCs were cocultured with HCAEC using the Transwell^® cell culture insert system. At 24 h after the coculture, IL-6 levels in the supernatant of HCAEC were measured by ELISA. The necrotic HCASMCs pretreated by IL-1β induced the most significant increase in IL-6 production by the HCAECs. *P<0.05 compared to bar graph left. ELISA, enzyme-linked immunosorbent assay; HCAECs, human coronary endothelial cells; HCASMCs, human coronary smooth muscle cells; IL, interleukin.

Anti-Inflammatory Effects of Anti-IL-33/ST2 Axis Antibodies in Comparison With Conventional Treatments In Vitro

Figure 3 shows the inhibitory effects of coculture with HCASMCs on cytokine production by HCAECs. A total of 77 cytokines were analyzed. All the treatments showed significant anti-inflammatory effects on the production of various cytokines. Among the treatments, high-dose IgG appeared to have the strongest anti-inflammatory effect. However, anti-IL-33, anti-ST2, and anti-TNF-α antibodies also showed unique anti-inflammatory effects compared with high-dose IgG; anti-IL-33, anti-ST2, and anti-TNF-αantibodies but not high-dose IgG inhibited the production of platelet-derived growth factor (PDGF)-BB in HCAECs. Moreover, the anti-ST2 and anti-TNF-α antibodies showed a significant inhibitory effect on IL-12(p70), and RANTES and stem cell growth factor-β production, respectively.

Figure 3.

(A) Comprehensive cytokine assay by Bio-Plex 3D system to assess drug efficacy. The heat map color scale corresponds to the inhibition ratio for cytokine production by HCAECs with each treatment. Red and white indicate the highest (100%) and lowest (0%) inhibition rates compared with MOCK (all P<0.05). Gray represents no significant difference. (B–E) All of the anti-IL-33, anti-ST2, and anti-TNF-α antibodies had unique anti-inflammatory effects on HCAECs compared with high-dose IgG. *P<0.05 vs. MOCK. **P<0.01 vs. MOCK. ***P<0.001 vs. MOCK. HCAECs, human coronary endothelial cells; IL, interleukin; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T cell expressed and secreted; SCGF, somnogenic cytokine growth factor; TNF, tumor necrosis factor. A total of 77 cytokines are listed in the Supplementary Table.

Discussion

In this study, necrotic HCASMCs induced a significant increase in proinflammatory cytokine production by HCAECs. Additionally, we demonstrated that inhibition of the IL-33/ST2 axis showed unique anti-inflammatory effects on HCAECs compared with conventional therapies. Although high-dose IVIG appeared to show strong anti-inflammatory effects, the unique anti-inflammatory effects of PDGF-BB and IL-12(p70) production might be beneficial for KD vasculitis.

Since Furusho et al. first described the effectiveness of IVIG for KD in 1984,²⁵ the incidence of CALs has dramatically decreased. In recent years, prednisolone, anti-TNF-α monoclonal antibody (Remicade^®), and cyclosporine have been approved as therapeutic drugs for KD by the Japanese Ministry of Health, Labour and Welfare.^26–28 However, the incidence rate of cardiac complications remains unchanged, despite the addition of novel therapies; within 30 days of KD onset, approximately 9.0% of patients are diagnosed with cardiac complications and 2.6% of patients develop cardiac sequelae.²⁹ Thus, new therapeutic targets and agents are required to prevent CAL development. In our previous study, we demonstrated that the anti-IL-33/ST2 axis may be a therapeutic target in KD.⁶ In the present study, we demonstrated that not only immunocompetent cells and cytokines, but also SMCs in the tunica media could be novel therapeutic targets for KD vasculitis. Furthermore, anti-IL-33/ST2 axis therapy has distinct anti-inflammatory effects compared with conventional therapies. From this viewpoint, novel therapeutic agents for the anti-IL-33/ST2 axis might be a “game changer” for preventing CALs in patients with KD.

IL-33 is released by activated macrophages and plays an important role in acute KD.³⁰ The IL-33/ST2 complex initiates the recruitment of adaptor molecules, including myeloid differentiation primary response protein 88 (MyD88), IL-1 receptor-associated kinases (IRAK-1 and IRAK-4), and TNF receptor-associated factor 6 (TRAF6), which leads to subsequent activation of the p38 MAPK, c-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK), and NF-κB pathways.⁹ In our study, anti-ST2 antibody suppressed the production of IL-12(p70) by HCAECs, which might be due to the blockade of the MyD88 pathway that plays a critical role in the production of IL-12(p70).^31,32 IL-12 is a T-cell-stimulating factor essential for the growth and function of T cells. It stimulates the production of TNF-α and IFN-γ from immunocompetent cells and activate natural killer cells and cytotoxic T lymphocytes.³³ In previous reports, the activation of cytotoxic T lymphocytes with enhanced IFN-γ production might be involved in IVIG-resistant KD.^22,34–36 From this perspective, anti-ST2 therapy may be beneficial as second-line therapy for IVIG-resistant KD. Recently, the efficacy and feasibility of anti-ST2 therapy in adults have been reported in adult patients with food allergy, asthma, chronic obstructive pulmonary disease, and endometriosis.^37–40 Thus, IL-33/ST2 is a potential therapeutic target for KD.

In the present study, the production of PDGF-BB by HCAECs was also decreased by anti-IL-33/ST2 or anti-TNF-α treatment. Although the specific mechanism remains unknown, it is possible that anti-IL-33/ST2 or anti-TNF-α secondarily inhibit certain signaling pathways, such as the TGF-β/Smad pathway, followed by the ERK or Akt pathways.⁹ PDGF-BB stimulates the proliferation of various cells, such as vascular SMCs, mesenchymal stem cells, fibroblasts, and epithelial-like cells, and plays a role in wound healing.⁴¹ Meanwhile, PDGF signaling is involved in cardiovascular diseases, such as vascular inflammation and atherosclerosis; additionally, inhibition of anti-PDGF might have the potential to prevent atherosclerosis.⁴² Based on previous findings, the inhibition of PDGF-BB also may be beneficial in preventing CALs.

One of the concerns regarding this study is whether the coculture assay using HCAECs and HCASMCs reflects the pathogenesis of KD vasculitis. Although there have been no reports on coculture assays of HCAECs and HCASMCs mimicking KD vasculitis, the system has been well used as one of the ways to understand vascular biology, particularly in the context of atherosclerosis, restenosis, and vascular tissue engineering.^13–15 Moreover, coculture assays using the Transwell^® cell culture insert system have been conducted to investigate the relationships between HCAECs and immunocompetent cells or endothelial cell migration in KD.^43,44 Against this background, we believe the present assay does reflect one of the aspects of KD pathogenesis.

Study Limitations

We did not evaluate the impact of the therapeutic agents on immunocompetent cells, such as monocytes/macrophages or T cells, because we specifically focused on studying the relationship between edematous dissociation of the tunica media and worsening KD vasculitis, which has not received much attention. In addition, we did not assess the optimal dosage of the anti-IL-33/ST2 axis agents. Nevertheless, this study provides new insights into immunomodulatory therapies for patients with KD.

In conclusion, necrotic changes in SMCs, also known as edematous dissociation of the tunica media, and the IL-33/ST2 axis, may play key roles in the development of CALs in KD. The findings of the present study also suggest that anti-IL-33/ST2 antibodies may be a beneficial therapy for KD vasculitis. However, further studies are needed to confirm this hypothesis.

Statements & Declarations

Funding: This work was supported by JSPS KAKENHI (Grant number, JP21K15906) (S.O.), AMED (Grant number, 22ek0109606 h) (S.O.), AMED (Grant number, 21fk0108572 h) (A.S.), and a grant from Japan Blood Products Organization.

Author Contributions: S.O. contributed to conception, design, acquisition, analysis, and interpretation of data, and drafting the article. A.S, Y.O., T.M., T.W., and M.S. contributed to acquisition and analysis of data. H.Y. and R.F. contributed to analysis and interpretation of data. S.H. contributed to revising it critically for important intellectual content and final approval of the version to be published. The first draft of the manuscript was written by S.O. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Disclosure

The authors have no relevant financial or non-financial interests to disclose.

IRB Information

The present study was granted an exemption from requiring ethics approval by Yamaguchi University because it did not involve human subjects.

Supplementary Files

Please find supplementary file(s);

https://doi.org/10.1253/circj.CJ-24-0295

References

• 1.   Mori M, Matsubara T. Overview of Guidelines for the medical treatment of acute Kawasaki disease in Japan (2020 Revised Version) and positioning of plasma exchange therapy in the acute phase. Pediatr Infect Dis J 2023; 42: e328–e332, doi:10.1097/INF.0000000000003974.
• 2.   Broderick C, Kobayashi S, Suto M, Ito S, Kobayashi T. Intravenous immunoglobulin for the treatment of Kawasaki disease. Cochrane Database Syst Rev 2023; 1: CD014884, doi:10.1002/14651858.CD014884.pub2.
• 3.   Fukazawa R, Kobayashi J, Ayusawa M, Hamada H, Miura M, Mitani Y, et al; Japanese Circulation Society Joint Working Group. JCS/JSCS 2020 Guideline on diagnosis and management of cardiovascular sequelae in Kawasaki disease. Circ J 2020; 84: 1348–1407, doi:10.1253/circj.CJ-19-1094.
• 4.   Hara T, Yamamura K, Sakai Y. The up-to-date pathophysiology of Kawasaki disease. Clin Transl Immunol 2021; 10: e1284, doi:10.1002/cti2.1284.
• 5.   Shahi A, Afzali S, Firoozi Z, Mohaghegh P, Moravej A, Hosseinipour A, et al. Potential roles of NLRP3 inflammasome in the pathogenesis of Kawasaki disease. J Cell Physiol 2023; 238: 513–532, doi:10.1002/jcp.30948.
• 6.   Okada S, Yasudo H, Ohnishi Y, Matsuguma C, Fukano R, Motonaga T, et al. Interleukin-33/ST2 axis as potential biomarker and therapeutic target in Kawasaki disease. Inflammation 2023; 46: 480–490, doi:10.1007/s10753-022-01753-7.
• 7.   Nakashima Y, Sakai Y, Mizuno Y, Furuno K, Hirono K, Takatsuki S, et al. Lipidomics links oxidized phosphatidylcholines and coronary arteritis in Kawasaki disease. Cardiovasc Res 2021; 117: 96–108, doi:10.1093/cvr/cvz305.
• 8.   Hara T, Nakashima Y, Sakai Y, Nishio H, Motomura Y, Yamasaki S. Kawasaki disease: A matter of innate immunity. Clin Exp Immunol 2016; 186: 134–143, doi:10.1111/cei.12832.
• 9.   Li W, Liu M, Chu M. Strategies targeting IL-33/ST2 axis in the treatment of allergic diseases. Biochem Pharmacol 2023; 218: 115911, doi:10.1016/j.bcp.2023.115911.
• 10.   Takahashi K, Oharaseki T, Yokouchi Y, Hiruta N, Naoe S. Kawasaki disease as a systemic vasculitis in childhood. Ann Vasc Dis 2010; 3: 173–181, doi:10.3400/avd.sasvp01003.
• 11.   Sato W, Yokouchi Y, Oharaseki T, Asakawa N, Takahashi K. The pathology of Kawasaki disease aortitis: A study of 37 cases. Cardiovasc Pathol 2021; 51: 107303, doi:10.1016/j.carpath.2020.107303.
• 12.   Janko C, Munoz L, Chaurio R, Maueröder C, Berens C, Lauber K, et al. Navigation to the graveyard-induction of various pathways of necrosis and their classification by flow cytometry. Methods Mol Biol 2013; 1004: 3–15, doi:10.1007/978-1-62703-383-1_1.
• 13.   Zhang Z, Chen Y, Zhang T, Guo L, Yang W, Zhang J, et al. Role of myoendothelial gap junctions in the regulation of human coronary artery smooth muscle cell differentiation by laminar shear stress. Cell Physiol Biochem 2016; 39: 423–437, doi:10.1159/000445636.
• 14.   Xia Y, Bhattacharyya A, Roszell EE, Sandig M, Mequanint K. The role of endothelial cell-bound Jagged1 in Notch3-induced human coronary artery smooth muscle cell differentiation. Biomaterials 2012; 33: 2462–2472, doi:10.1016/j.biomaterials.2011.12.001.
• 15.   Isakson BE, Duling BR. Heterocellular contact at the myoendothelial junction influences gap junction organization. Circ Res 2005; 97: 44–51, doi:10.1161/01.RES.0000173461.36221.2e.
• 16.   Chen YL, Gutowska-Owsiak D, Hardman CS, Westmoreland M, MacKenzie T, Cifuentes L, et al. Proof-of-concept clinical trial of etokimab shows a key role for IL-33 in atopic dermatitis pathogenesis. Sci Transl Med 2019; 11: eaax2945, doi:10.1126/scitranslmed.aax2945.
• 17.   Kamekura R, Kojima T, Takano K, Go M, Sawada N, Himi T. The role of IL-33 and its receptor ST2 in human nasal epithelium with allergic rhinitis. Clin Exp Allergy 2012; 42: 218–228, doi:10.1111/j.1365-2222.2011.03867.x.
• 18.   Nomura K, Kojima T, Fuchimoto J, Obata K, Keira T, Himi T, et al. Regulation of interleukin-33 and thymic stromal lymphopoietin in human nasal fibroblasts by proinflammatory cytokines. Laryngoscope 2012; 122: 1185–1192, doi:10.1002/lary.23261.
• 19.   Ganesan S, Pham D, Jing Y, Farazuddin M, Hudy MH, Unger B, et al. TLR2 activation limits rhinovirus-stimulated CXCL-10 by attenuating IRAK-1-dependent IL-33 receptor signaling in human bronchial epithelial cells. J Immunol 2016; 197: 2409–2420, doi:10.4049/jimmunol1502702.
• 20.   Zhou Q, Wu X, Wang X, Yu Z, Pan T, Li Z, et al. The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis. Oncogene 2020; 39: 1414–1428, doi:10.1038/s41388- 019-1078-x.
• 21.   Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol 2008; 121: 1484–1490, doi:10.1016/j.jaci.2008.04.005.
• 22.   Makata H, Ichiyama T, Uchi R, Takekawa T, Matsubara T, Furukawa S. Anti-inflammatory effect of intravenous immunoglobulin in comparison with dexamethasone in vitro: Implication for treatment of Kawasaki disease. Naunyn Schmiedebergs Arch Pharmacol 2006; 373: 325–332, doi:10.1007/s00210-006-0084-z.
• 23.   Pietikäinen A, Maksimow M, Kauko T, Hurme S, Salmi M, Hytönen J. Cerebrospinal fluid cytokines in Lyme neuroborreliosis. J Neuroinflamm 2016; 13: 273, doi:10.1186/s12974-016-0745-x.
• 24.   Sebitloane HM, Naicker T, Moodley J. The impact of the duration of HAART on cytokine profiles in pregnancy. Inflamm Res 2020; 69: 1053–1058, doi:10.1007/s00011-020-01375-5.
• 25.   Furusho K, Kamiya T, Nakano H, Kiyosawa N, Shinomiya K, Hayashidera T, et al. High-dose intravenous gammaglobulin for Kawasaki disease. Lancet 1984; 2: 1055–1058, doi:10.1016/s0140-6736(84)91504-6.
• 26.   Kobayashi T, Saji T, Otani T, Takeuchi K, Nakamura T, Arakawa H, et al; RAISE study group investigators. Efficacy of immunoglobulin plus prednisolone for prevention of coronary artery abnormalities in severe Kawasaki disease (RAISE study): A randomised, open-label, blinded-endpoints trial. Lancet 2012; 379: 1613–1620, doi:10.1016/S0140-6736(11)61930-2.
• 27.   Mori M, Hara T, Kikuchi M, Shimizu H, Miyamoto T, Iwashima S, et al. Infliximab versus intravenous immunoglobulin for refractory Kawasaki disease: A phase 3, randomized, open-label, active-controlled, parallel-group, multicenter trial. Sci Rep 2018; 8: 1994, doi:10.1038/s41598-017-18387-7.
• 28.   Hamada H, Suzuki H, Onouchi Y, Ebata R, Terai M, Fuse S, et al; KAICA trial Investigators. Efficacy of primary treatment with immunoglobulin plus ciclosporin for prevention of coronary artery abnormalities in patients with Kawasaki disease predicted to be at increased risk of non-response to intravenous immunoglobulin (KAICA): A randomised controlled, open-label, blinded-endpoints, phase 3 trial. Lancet 2019; 393: 1128–1137, doi:10.1016/S0140-6736(18)32003-8. Erratum in: Lancet 2019; 393: 1298; Lancet 2019; 393: 1504.
• 29.   Ae R, Makino N, Kosami K, Kuwabara M, Matsubara Y, Nakamura Y. Epidemiology, treatments, and cardiac complications in patients with Kawasaki disease: The nationwide survey in Japan, 2017–2018. J Pediatr 2020; 225: 23–29.e2, doi:10.1016/j.jpeds.2020.05.034.
• 30.   De la Fuente M, MacDonald TT, Hermoso MA. The IL-33/ST2 axis: Role in health and disease. Cytokine Growth Factor Rev 2015; 26: 615–623, doi:10.1016/j.cytogfr.2015.07.017.
• 31.   Delale T, Paquin A, Asselin-Paturel C, Dalod M, Brizard G, Bates EE, et al. MyD88-dependent and -independent murine cytomegalovirus sensing for IFN-alpha release and initiation of immune responses in vivo. J Immunol 2005; 175: 6723–6732, doi:10.4049/jimmunol175.10.6723.
• 32.   Ichikawa S, Miyake M, Fujii R, Konishi Y. MyD88 associated ROS generation is crucial for Lactobacillus induced IL-12 production in macrophage. PLoS One 2012; 7: e35880, doi:10.1371/journal.pone.0035880.
• 33.   Cheng EM, Tsarovsky NW, Sondel PM, Rakhmilevich AL. Interleukin-12 as an in situ cancer vaccine component: A review. Cancer Immunol Immunother 2022; 71: 2057–2065, doi:10.1007/s00262-022-03144-1.
• 34.   Wakiguchi H, Hasegawa S, Suzuki Y, Kudo K, Ichiyama T. Relationship between T-cell HLA-DR expression and intravenous immunoglobulin treatment response in Kawasaki disease. Pediatr Res 2015; 77: 536–540, doi:10.1038/pr.2015.12.
• 35.   Okada S, Ohnishi Y, Furuta T, Suzuki Y, Kawakami-Miyake A, Matsuguma C, et al. Circulating immunocompetent cell profiles during oral cyclosporin therapy for immunoglobulin-resistant Kawasaki disease. World J Pediatr 2021; 17: 671–673, doi:10.1007/s12519-021-00468-3.
• 36.   Okada S, Azuma Y, Suzuki Y, Yamada H, Wakabayashi-Takahara M, Korenaga Y, et al. Adjunct cyclosporine therapy for refractory Kawasaki disease in a very young infant. Pediatr Int 2016; 58: 295–298, doi:10.1111/ped.12778.
• 37.   Chinthrajah S, Cao S, Liu C, Lyu SC, Sindher SB, Long A, et al. Phase 2a randomized, placebo-controlled study of anti-IL-33 in peanut allergy. JCI Insight 2019; 4: e131347, doi:10.1172/jci.insight.131347.
• 38.   Kelsen SG, Agache IO, Soong W, Israel E, Chupp GL, Cheung DS, et al. Astegolimab (anti-ST2) efficacy and safety in adults with severe asthma: A randomized clinical trial. J Allergy Clin Immunol 2021; 148: 790–798, doi:10.1016/j.jaci.2021.03.044.
• 39.   Yousuf AJ, Mohammed S, Carr L, Yavari Ramsheh M, Micieli C, Mistry V, et al. Astegolimab, an anti-ST2, in chronic obstructive pulmonary disease (COPD-ST2OP): A phase 2a, placebo-controlled trial. Lancet Respir Med 2022; 10: 469–477, doi:10.1016/S2213-2600(21)00556-7.
• 40.   Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell 2021; 184: 2807–2824, doi:10.1016/j.cell.2021.04.041.
• 41.   Hu W, Huang Y. Targeting the platelet-derived growth factor signalling in cardiovascular disease. Clin Exp Pharmacol Physiol 2015; 42: 1221–1224, doi:10.1111/1440-1681.12478.
• 42.   Rutherford C, Martin W, Salame M, Carrier M, Anggård E, Ferns G. Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor. Atherosclerosis 1997; 130: 45–51, doi:10.1016/s0021-9150(96)06042-x.
• 43.   Qin J, Zheng Y, Ding Y, Huang C, Hou M, Li M, Qian G, Lv H. Co-culture of peripheral blood mononuclear cell (PBMC) and human coronary artery endothelial cell (HCAEC) reveals the important role of autophagy implicated in Kawasaki disease. Transl Pediatr 2021; 10: 3140–3150, doi:10.21037/tp-21-344.
• 44.   Weng KP, Chien KJ, Huang SH, Huang LH, Lin PH, Lin Y, et al. iTRAQ proteomics identified the potential biomarkers of coronary artery lesion in Kawasaki disease and in vitro studies demonstrated that S100A4 treatment made HCAECs more susceptible to neutrophil infiltration. Int J Mol Sci 2022; 23: 12770, doi:10.3390/ijms232112770.