急性期川崎病IL-4基因组蛋白甲基化的改变及意义

Changes and significances of histone methylation of interleukin-4 gene in acute pediatric Kawasaki disease

目的探讨急性期川崎病(KD)患儿IL-4基因组蛋白甲基化改变及其在KD Th2细胞异常机制中的作用。方法 KD患儿急性期及IVIG治疗4~5 d后取血备检,同年龄健康儿童为对照组。采用流式细胞术检测外周血Th2比例及CD4^+ T细胞IL-4、pSTAT6和GATA3蛋白水平;染色质免疫共沉淀分析外周血CD4^+ T细胞IL-4基因CNS1、HSⅡ、HSVa组蛋白甲基化H3K4me3及GATA3、MLL1结合水平;荧光定量PCR分析CD4^+ T细胞IL-4、IL-5、IL-13、IL-4Rα、IL-2Rγ、SOCS5 mRNA水平;双抗体夹心ELISA检测血浆细胞因子IL-4、IL-5和IL-13蛋白浓度。结果 KD组42例,对照组36例。①KD患儿急性期Th2细胞比例,功能相关分子(IL-4、IL-5和IL-13)mRNA表达,血浆蛋白水平,IL-4基因CNS1、HSⅡ、HSVa位点组蛋白甲基化H3K4me3修饰水平均明显增加(P<0.05),且冠状动脉损伤组(CAL)前述指标均高于无冠状动脉损伤组(NCAL)(P<0.05),经IVIG治疗后明显降低(P<0.05);②KD患儿急性期外周血CD4^+ T细胞转录因子GATA-3、组蛋白甲基化酶MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平显著增高(P<0.05),且MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平与IL-4 mRNA表达显著正相关(r分别为0.42、0.33和0.39,P均<0.05),经IVIG治疗后均表现不同水平的恢复(P<0.05)。其中CAL组MLL1、GATA-3与IL-4基因CNS1、HSⅡ、HSVa结合水平明显高于NCAL组(P<0.05);③KD患儿急性期血浆IL-4浓度和CD4^+ T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA-3、SOCS5表达显著增高(P<0.05),其中CAL组血浆IL-4浓度和CD4+T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA3表达均高于NCAL组、SOCS5表达低于NCAL组(P<0.05),经IVIG治疗后均表现不同水平的下调(P<0.05)。结论 IL-4基因组蛋白甲基化修饰异常可能是导致KD患儿急性期Th2细胞异常的始动因素之一。

Objective To explore the changes of histone methylation of IL-4 gene and its roles in dysfunction of Th2 cells during acute pediatric Kawasaki disease(KD). Methods Forty-two children with KD were recruited in present study, while thirty-six age-matched healthy volunteer were as controls. Flow cytometry was performed to determine the frequencies of Th2 cells and levels of IL-4, phosphorylated STAT6(pSTAT6) and GATA3 protein. Chromatin immunoprecipitation(ChIP) and quantitative PCR were used to evaluate trimethylation of histone H3 at lysine 4 at CNS1, HSⅡ and HSVa of IL-4 gene and binding levels of GATA3 and MLL1 with loci afore-mentioned in CD4^+ T cells. Transcription levels of IL-4, IL-5, IL-13, IL-4 Rα, IL-2 Rγ and SOCS5 mRNA in CD4^+ T cells were quantitated by real-time PCR. Plasma concentrations of IL-4, IL-5 and IL-13 were analyzed by enzyme-linked immunosorbent assay. Results(1) In comparison with healthy controls, the frequencies of Th2 cells, mRNA levels and plasma concentrations of Th2 cytokines(IL-4, IL-5 and IL-13),and methylation levels of H3 K4 me3 associated with IL-4 loci(CNS1, HSⅡ and HSVa) increased significantly in patients with acute KD(P<0.05). The items mentioned afore in patients with coronary artery lesions(CAL) were found to be higher than those without coronary artery lesions(NCAL), and restored to some extent after IVIG therapy(P<0.05).(2) ChIP results showed binding abilities of GATA-3 and MLL1 proteins with IL-4 gene loci(CNS1, HSⅡ and HSVa), were up-regulated remarkably in CD4^+ T cells from patients with acute KD(P<0.05), and down-regulated after IVIG treatment(P<0.05). There are positive correlations between binding abilities of MLL1 protein with IL-4 loci(CNS1, HSⅡ and HSVa) and transcription level of IL-4 mRNA detected during acute KD(r=0.42, 0.33, 0.39, P<0.05). In parallel, binding abilities of GATA-3 and MLL1 with the three IL-4 loci in CAL group were higher than those in NCAL group(P<0.05). ⑶ During acute phase of KD, plasma levels of IL-4 protein, and expression levels of IL-4 signaling downstream molecules(IL-4 Rα, IL-2 Rγ、pSTAT6 and GATA-3) and negative regulator SOCS5 increased remarkably(P<0.05), and all the items decreased after IVIG treatment(P<0.05). Furthermore, the five items aforementioned in CAL group were higher than those in NCAL group, while the last items in CAL group was lower than that in NCAL group(P<0.05). Conclusion Excessive histone methylation of H3 K4 me3 with IL-4 gene might be one of initiative factors resulting in aberration of Th2 cells in KD.