IL-37b alleviates endothelial cell apoptosis and inflammation in Kawasaki disease through IL-1R8 pathway

Abstract

Kawasaki disease (KD) is an acute vasculitis of pediatric populations that may develop coronary artery aneurysms if untreated. It has been regarded as the principal cause of acquired heart disease in children of the developed countries. Interleukin (IL)-37, as one of the IL-1 family members, is a natural suppressor of inflammation that is caused by activation of innate and adaptive immunity. However, detailed roles of IL-37 in KD are largely unclear. Sera from patients with KD displayed that IL-37 level was significantly decreased compared with healthy controls (HCs). QRT-PCR and western blot analyses showed that the expression level of IL-37 variant, IL-37b, was remarkably downregulated in human umbilical vein endothelial cells (HUVECs) exposed to KD sera-treated THP1 cells. Therefore, we researched the role of IL-37b in the context of KD and hypothesized that IL-37b may have a powerful protective effect in KD patients. We first observed and substantiated the protective role of IL-37b in a mouse model of KD induced by Candida albicans cell wall extracts (CAWS). In vitro experiments demonstrated that IL-37b alleviated endothelial cell apoptosis and inflammation via IL-1R8 receptor by inhibiting ERK and NFκB activation, which were also recapitulated in the KD mouse model. Together, our findings suggest that IL-37b play an effective protective role in coronary endothelial damage in KD, providing new evidence that IL-37b is a potential candidate drug to treat KD.

Fig. 1. The expression level of IL-37b was decreased after KD sera treatment.

a The levels of IL-37 in sera from health controls (HCs, n = 17) and KD patients (n = 18) were evaluated by enzyme-linked immunosorbent assay (ELISA). Significance: *P < 0.05. b The mRNA expression levels of five IL-37 splice variants (IL-37a–e) were evaluated in KD-treated ECs by qRT-PCR analysis. Significance: *P < 0.05. c, d Protein expression of IL-37b was assessed in KD-treated ECs by western blot analysis. GAPDH was used as an internal control. Quantitative analysis of IL-37b expression was performed (d). All these experiments were repeated at least three times. Data are shown as mean ± SD (n = 3). Significance: *P < 0.05.

Fig. 2. The coronary arteritis was mitigated after Il-37b injection in a KD mouse model.

a Representative images from H&E staining at 28-day post-CAWS injection. Magnification: ×200. Scale bar = 100 μm. b–d The expression levels of vascular cell adhesion molecule 1 (VCAM-1), macrophage marker F4/80, and neutrophil marker were, respectively, determined using IHC staining in the endothelium of coronary arteries. Enlarged images of area of interesting (AOI) were indicated with a red arrow. Scale bar = 100 μm. e–g The expression levels of cytokines, adhesion molecules, and chemokines were detected in the mice heart section. Significance: *P < 0.05 vs. the PBS group, and ^#P < 0.05 vs. the CAWS group. N = 6 mice per group, and each experiment was conducted at least three times.

Fig. 3. IL-37b alleviated KD-treated endothelial cell apoptosis and inflammation.

a CCK8 assay was used to evaluate cell viability (n = 6). Significance: *P < 0.05. b DNA fragmentation was analyzed using TUNEL staining (n = 3). c Protein expression of BAX and Bcl-2 was determined by western blot analysis. d Quantitative analysis of BAX/Bcl-2 ratio was conducted. Data are presented as mean ± SD (n = 3). e, f The expression levels of cytokines, adhesion molecules, and chemokines were examined in KD-treated ECs after IL-37b treatment (n = 3). *P < 0.05 vs. the HC group, and ^#P < 0.05 vs. the KD group. At least three experiments were performed for each assay.

Fig. 4. IL-37b alleviated endothelial cell apoptosis and inflammation via IL-1R8 pathway.

a The mRNA expression of IL-1R8 was examined by qRT-PCR analysis (n = 3). Significance: *P < 0.05 vs. the HC group, and ^#P < 0.05 vs. the KD group. b, c The surface and intracellular expression levels of IL-1R8 were examined by western blot analysis (n = 3). GAPDH was used as an internal control of cytoplasmic proteins, and ATP1A1 was utilized as an internal control of cell surface membrane proteins. Significance: *P < 0.05, and **P < 0.01. d Endothelial cells were transiently transfected with nontargeting control siRNA (sictrl) or IL-1R8-specific siRNA (siIL-1R8). After that, the expression change of IL-1R8 was examined (n = 3). e–g Endothelial cells that were transiently transfected with siIL-1R8 were treated with KD sera-treated THP1, and then the IL-1R8 expression was determined at the mRNA level (e) and protein level (f, g). *P < 0.05 vs. the HC group, and ^#P < 0.05 vs. the KD group. h DNA fragmentation was analyzed using TUNEL staining after IL-1R8 expression was silenced (n = 3). Significance: *P < 0.05 vs. the HC group, ^#P < 0.05 vs. the KD group, and ^&P < 0.05 vs. the KD + IL-37b group. Magnification: ×200, Scale bar = 100 μm. i Protein expression of BAX and Bcl-2 was determined after IL-1R8 expression was silenced. j Quantitative analysis of the ratio of BAX/Bcl-2 performed. Data were expressed as mean ± SD (n = 3). k The expression levels of cytokines were assessed after silencing IL-1R8 expression (n = 3). Significance: *P < 0.05 vs. the HC group, ^#P < 0.05 vs. the KD group, and ^&P < 0.05 vs. the KD + IL-37b group. All these experiments were done at least three times.

Fig. 5. IL-37b inhibited the activation of ERK and NFκB in KD-treated ECs related to IL-1R8.

a Effects of IL-37b treatment on the phosphorylation of ERK, JNK, p38, and NFκB p65 were assessed in the treated endothelial cells. b–e Quantitative analysis of these above proteins was conducted. Data are presented as mean ± SD (n = 3). Significance: *P < 0.05 vs. the HC group, and ^#P < 0.05 vs. the KD group. f, g Effects of IL-1R8 silencing on the activation of ERK and NFκB p65 were observed. Significance: *P < 0.05 vs. the HC group, ^#P < 0.05 vs. the KD group, and ^&P < 0.05 vs. the KD + IL-37b group. i The nuclear translocation of NFκB p65 was observed after silencing IL-1R8 expression (n = 3). Magnification: ×200, Scale bar = 100 μm. Experiments were done at least three times in triplicate.

Fig. 6. Endothelial cell apoptosis and inflammation were alleviated in a KD mouse model after IL-37b treatment.

a, b The protein expression of BAX and Bcl-2 was examined in the KD mouse model after IL-37b treatment (n = 6). c Effects of IL-37b treatment on DNA fragmentation in the endothelial cells were evaluated by co-localized staining of TUNEL (green) and CD31 (an endothelial marker, red) (n = 5). Magnification: ×200. Scale bar = 50 μm. d TNF-α expression levels were examined in the coronary artery endothelial cells by double staining of TNF-α (green) and CD31 (red) (n = 5). Magnification: ×200. Scale bar = 50 μm. e–g The activation of ERK and NFκB was determined in the heart tissues by western blot analysis (n = 6). h The levels of p-ERK were examined in the coronary artery endothelial cells using p-ERK/CD31 double staining (n = 5). Magnification: ×200. Scale bar = 50 μm. Significance: *P < 0.05 vs. the PBS group, and ^#P < 0.05 vs. the CAWS group. Each experiment was conducted at least three times.

Fig. 7. Mitigation of IL-37b-mediated coronary artery endothelial cell apoptosis and inflammation was realized via IL-1R8 pathway.

a The expression of IL-1R8 were detected in the KD mouse model after IL-37b treatment (n = 6). b Effects of silencing of IL-1R8 on coronary arteritis were observed by H&E staining. Magnification: ×200. Scale bar = 100 μm. c–e The endothelial expression of VCAM-1, macrophage marker F4/80, and neutrophil marker was examined after IL-1R8 gene silencing using IHC staining. AOI were indicated with a red arrow. Scale bar = 100 μm. Right: The histograms respectively exhibited the percentage of VCAM-1, F4/80, and neutrophil marker-positive areas. Significance: *P < 0.05 vs. the PBS group, ^#P < 0.05 vs. the CAWS group, and ^&P < 0.05 vs. the CAWS + IL-37b group. f DNA fragmentation was examined using TUNEL staining. Scale bar = 100 μm. Below: Percentage of TUNEL-positive cells was shown in the histogram. Significance: *P < 0.05 vs. the PBS group, ^#P < 0.05 vs. the CAWS group, and ^&P < 0.05 vs. the CAWS + IL-37b group. g, h BAX/Bcl-2 ratio was analyzed by western blot analysis. i The mRNA expression levels of IL-6, TNF-α, and IL-1β were determined after silencing of IL-1R8. j–l Effects of IL-1R8 silencing on the phosphorylation level of ERK and NFκB p65 were assessed by western blotting. Significance: *P < 0.05 vs. the PBS group, ^#P < 0.05 vs. the CAWS group, and ^&P < 0.05 vs. the CAWS + IL-37b group. N = 6 mice per group, and each experiment was conducted at least three times.

Fig. 8. Schematic model for IL-37b against endothelial cell damage in Kawasaki disease.

IL-37b binds to IL-18α and IL-1R8, inhibits ERK and NFκB activation, and finally suppresses EC apoptosis and inflammation.