Notch1信号调控调节性T细胞在川崎病中的作用

Effects of Notch1 signaling on regulatory T cells and coronary artery lesions in childhood Kawasaki disease

目的探讨Notch1信号对川崎病患儿调节性T细胞(Treg)的影响及在血管损伤机制中的作用。方法以2019年3月至2021年6月收治的42例川崎病患儿为研究对象,分别于急性期及输注丙种球蛋白(IVIG)治疗后取样送检,对照组为32名同年龄健康儿童。采用流式细胞术检测外周静脉血CD4^(+)CD25hiFoxp3^(+)Treg数量及叉头螺旋翼状转录因子(Foxp3)、细胞毒性T细胞相关抗原4(CTLA4)、糖皮质激素诱导的肿瘤坏死因子受体(GITR)、Notch1蛋白表达水平;免疫共沉淀-定量PCR技术鉴定CD4^(+)T细胞Foxp3基因启动子组蛋白H4乙酰化(H4Ac)及Notch1受体胞内结合域1(NICD1)、重组信号结合蛋白J(RBP-J)、p300结合水平;荧光定量PCR分析早老素1(PSEN1)、主导控制样蛋白1(MAML1)、RBP-J和Foxp3 mRNA表达;ELISA检测血浆中IL-10、TGF-β蛋白浓度。采用t检验、Pearson相关分析法进行统计分析。结果①与健康对照组相比,急性期川崎病患儿CD4^(+)CD25hiFoxp3^(+)Treg比例显著降低[(4.3±1.5)%和(7.9±2.9)%;t=6.41,P<0.001],分化及功能相关分子(Foxp3、CTLA4、GITR)表达水平和血浆IL-10、TGF-β蛋白浓度明显下降(t=6.87,P<0.001;t=4.26,P<0.001;t=7.88,P<0.001;t=8.42,P<0.001;t=13.01,P<0.001),其中合并冠状动脉损伤组(CAL)前述6项指标低于无冠状动脉损伤组(NCAL)[t=5.83,P<0.001;t=3.83,P<0.001;t=3.28,P=0.002;t=5.05,P<0.001;t=5.96,P<0.001;t=5.17,P<0.001],治疗后显著上调[t=7.13,P<0.001;t=6.10,P<0.001;t=4.31,P<0.001;t=6.55,P<0.001;t=7.40,P<0.001;t=7.84,P<0.001]。②急性期川崎病患儿Foxp3基因启动子H4Ac修饰及NICD1、p300结合水平明显低于同年龄对照组(t=10.25,P<0.001;t=6.93,P<0.001;t=6.75,P<0.001),其中CAL组Foxp3基因启动子H4Ac及NICD1、p300结合水平低于NCAL组(t=6.08,P<0.001;t=2.66,P=0.011;t=6.02,P<0.001),治疗后明显上调(t=7.72,P<0.001;t=4.16,P<0.001;t=5.76,P<0.001)。Pearson相关分析显示Foxp3基因启动子H4Ac与其mRNA水平呈正相关(r=0.47,P<0.001)。各组间Foxp3/RBP-J结合水平略有改变,但差异无统计学意义(t=0.57,P>0.005;t=0.61,P>0.05;t=1.20,P>0.05)。③急性期川崎病患儿CD4^(+)T细胞表面Notch1受体蛋白及下游信号分子PSEN1、MAML1及RBP-J表达水平显著下调[t=5.28,P<0.001;t=6.31,P<0.001;t=11.78,P<0.001;t=8.06,P<0.001],且CAL组前4项指标均低于NCAL组[t=3.16,P=0.003;t=4.13,P<0.001;t=5.42,P<0.001;t=4.05,P<0.001],治疗后呈不同程度回调(t=4.77,P<0.001;t=6.43,P<0.001;t=11.95,P<0.001;t=7.79,P<0.001)。经体外重组人Jagged1蛋白刺激48 h后,川崎病患儿及对照组CD4^(+)T细胞Foxp3基因H4Ac及NICD1、p300结合水平明显高于未处理组[(川崎病:t=15.36,P<0.001;t=7.25,P<0.001;t=14.29,P<0.001),(对照组:t=7.87,P<0.001;t=5.71,P<0.001;t=8.74,P<0.001)],RBP-J结合水平差异无统计学意义(川崎病:t=1.11,P>0.05;对照组:t=1.37,P>0.05),但川崎病患儿Foxp3基因H4Ac及NICD1、p300结合水平仍低于对照组(t=3.86,P<0.001;t=3.42,P=0.001;t=2.85,P=0.006)。④经川崎病血清刺激健康儿童外周血PBMCs建立炎症细胞模型,IVIG干预组Treg比例、Foxp3表达水平及CD4^(+)T细胞Notch1、RBP-J表达水平明显高于未处理组(t=7.10,P<0.001;t=10.16,P<0.001;t=8.06,P<0.001;t=9.77,P<0.001),Foxp3基因启动子H4Ac及NICD1结合水平亦高于后者(t=7.24,P<0.001;t=8.24,P<0.001)。结论Notch1信号低下所致Treg数量不足及功能障碍可能是导致川崎病患儿免疫功能异常活化及血管损伤的重要因素之一。

Objective To explore the effect of Notch1 signaling on regulatory T cells and its roles in vascular damage in patients with Kawasaki disease(KD).Methods A total of 42 children with KD were enrolled in the present study from March 2019 to June 2020,as 32 age-matched healthy children were recruited as control.The proportions of CD4^(+)CD25hiFoxp3^(+)regulatory T cells(Treg)and expressions of transcription factor forkhead box protein 3(Foxp3),cytotoxic T lymphocyte associated antigen-4(CTLA4),glucocorticoid-induced tumor necrosis factor receptor(GITR),and Notch1 protein were evaluated by flow cytometry.Chromatin immunoprecipitation was conducted to detect acetylation level of histone H4(H4Ac)associated with the promoter of Foxp3 gene and its binding abilities of Notch1 intracellular domain 1(NICD1),recombination signal binding protein for immunoglobulin kappa J region(RBP-J)and p300 in CD4^(+)T cells.Transcription levels of Foxp3,presenilin 1(PSEN1),mastermind like transcriptional coactivator 1(MAML1),and RBP-J in CD4^(+)T cells were determined by real-time polymerase chain reaction(PCR).Concentrations of interleukin(IL)-10 and transforming growth factor-β(TGF-β)in plasma and culture supernatant stimulated with Jagged1 were measured by enzyme linked immunosorbent assay.Independent-sample t-test,Pearson correlation analysis was used as the statistical method in this study.Results①The frequencies of Treg in acute KD patients decreased significantly[(4.3±1.5)%vs(7.9±2.9)%;t=6.41,P<0.001],as protein levels of Foxp3,CTLA4 and GITR and concentrations of IL-10 and TGF-βin plasma reduced remarkably in acute KD patients(t=6.87,P<0.001;t=4.26,P<0.001;t=7.88,P<0.001;t=8.42,P<0.001;t=13.01,P<0.001).All parameters afore-mentioned in patients combined with coronary artery lesions(CAL)were lower than those of patients without coronary artery lesions(NCAL)(t=5.83,P<0.001;t=3.83,P<0.001;t=3.28,P=0.002;t=5.05,P<0.001;t=5.96,P<0.001;t=5.17,P<0.001),and increased after therapy(t=7.13,P<0.001;t=6.10,P<0.001;t=4.31,P<0.001;t=6.55,P<0.001;t=7.40,P<0.001;t=7.84,P<0.001).②H4Ac associated with promoter of Foxp3 gene and the binding abilities of NICD1 and p300 in acute KD patients were lower than those of the controls(t=10.25,P<0.001;t=6.93,P<0.001;t=6.75,P<0.001),and increased remarkably after therapy(t=7.72,P<0.001;t=4.16,P<0.001;t=5.76,P<0.001).Meanwhile,the three items in CAL group were found to be less than those of NCAL group(t=6.08,P<0.001;t=2.66,P=0.011;t=6.02,P<0.001).Pearson correlation analysis showed a positive correlation between H4Ac associated with Foxp3 promoter and its mRNA level in acute KD patients(r=0.47,P<0.001).No statistical significant difference about the binding ability of RBP-J with Foxp3 promoter were found among the groups(t=0.57,P>0.05;t=0.61,P>0.05;t=1.20,P>0.05).③Protein level of Notch1 and the expressions of PSEN1,MAML1 and RBP-J mRNA in CD4^(+)T cells from acute KD patients were down-regulated remarkably(t=5.28,P<0.001;t=6.31,P<0.001;t=11.78,P<0.001;t=8.06,P<0.001),and restored after therapy(t=4.77,P<0.001;t=6.43,P<0.001;t=11.95,P<0.001;t=7.79,P<0.001).In parallel,the four indexes aforementioned of CAL group were lower than those of NCAL group(t=3.16,P=0.003;t=4.13,P<0.001;t=5.42,P<0.001;t=4.05,P<0.001).Upon rhJagged1 stimulation for 48 hours,H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in CD4^(+)T cells in KD patients and control group was significantly higher than those of untreated group[(KD:t=15.36,P<0.001;t=7.25,P<0.001;t=14.29,P<0.001),(Ctrl:t=7.87,P<0.001;t=5.71,P<0.001;t=8.74,P<0.001)],as the binding ability of RBP-J with Foxp3 promoter increased slightly without statistically significant difference(KD:t=1.11,P>0.05;Ctrl:t=1.37,P>0.05).Simultaneously,H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in KD group were still lower than those of the control group after stimulation(t=3.86,P<0.001;t=3.42,P=0.001;t=2.85,P=0.006).④After incubation of PBMC from heathy children with KD serum,the proportion of Treg cells,protein level of Foxp3 and expressions of Notch1 and RBP-J in CD4^(+)T cells in the group treated with IVIG increased significantly compared with the untreated group(t=7.10,P<0.001;t=10.16,P<0.001;t=8.06,P<0.001;t=9.77,P<0.001),as well as H4Ac level of Foxp3 promoter and its binding abilities with NICD1 in the group treat with IVIG were also higher than the latter(t=7.24,P<0.001;t=8.24,P<0.001).Conclusion Insufficiency and impaired function of Treg caused by aberrant Notch1 signaling may be the important factor contributing to immune dysfunction and vascular damage in KD.