Ash2样蛋白和含Jumonji结构域的蛋白3对川崎病患儿干扰素γ组蛋白甲基化的影响及其在血管损伤中的作用

Role of ash2 （absent, small, or homeotic）-like and Jumonji domain-containing protein 3 on histone methylation of interferon-gamma gene and their associations with vascular damage of Kawasaki disease

目的 探讨Ash2样蛋白（Ash2L）和含Jumonji结构域的蛋白3（Jmjd3）对急性期川崎病患儿干扰素γ（IFN-γ）基因组蛋白甲基化的影响及其在血管损伤中的作用.方法 采用前瞻性研究方法,选取2015年2月至2016年6月就诊于深圳市儿童医院并诊断为川崎病的患儿36例（川崎病组）和健康同年龄儿童28名（对照组）.另外,根据是否合并冠状动脉损伤,将川崎病患儿分为冠状动脉损伤组（16例）和无冠状动脉损伤组（20例）.川崎病患儿均接受人丙种球蛋白治疗.采用流式细胞术检测外周血1型辅助T细胞（Th1）细胞比例及IFN-γ、T细胞表达的盒状蛋白（T-bet）、磷酸化的信号转导及转录活化因子（pSTAT）1、pSTAT4蛋白水平;采用染色质免疫共沉淀检测外周血CD4＋T细胞IFN-γ基因组蛋白甲基化[组蛋白H3亚基4号赖氨酸残基三甲基化（H3K4me3）和组蛋白H3亚基27号赖氨酸残基三甲基化（H3K27me3）]及相关酶Ash2L、Jmjd3和zeste基因增强子同源物2（Ezh2）结合水平;采用实时荧光定量PCR检测外周血CD4＋T细胞IFN-γ、IFN-γ受体（IFN-γR）1、IFN-γR2、白细胞介素（IL）-12受体亚基（IL-12R）β1、IL-12Rβ2、IL-18受体亚基（IL-18R）α、IL-18Rβ、肿瘤坏死因子受体1（TNFR1）、Toll样受体4（TLR4）、受体交互丝氨酸/苏氨酸蛋白1（RIP-1）和髓性分化原发反应蛋白88（MyD88） mRNA水平;采用酶联免疫吸附试验检测血浆中IFN--γ、IL-12、IL-18和肿瘤坏死因子α（TNF-α）水平.结果 （1）川崎病组Thl细胞比率和IFN-γ蛋白水平均高于对照组（P均＜0.05）.冠状动脉损伤组前述两项指标水平也均高于无冠状动脉损伤组（P均＜0.05）.川崎病患儿治疗后前述两项指标水平均低于治疗前（P均＜0.05）.（2）川崎病组CD4＋T细胞IFN-γ mRNA水平及H3K4me3水平均高于对照组,而H3K27me3水平低于对照组（P均＜0.05）;川崎病组H3 K4me3/H3 K27 me3高于对照组（P＜0.05）,且与IFN-γ mRNA水平呈正相关（r =0.55,P＜0.05）.冠状动脉损伤组IFN-γmRNA、H3 K4 me3水平及H3 K4 me3/H3 K27 me3均高于无冠状动脉损伤组,而H3 K27 me3水平则低于无冠状动脉损伤组（P均＜0.05）.川崎病患儿治疗后H3 K27 me3水平高于治疗前,而IFN-y mRNA、H3 K4 me3水平及H3 K4me3/H3 K27 me3均低于治疗前（P均＜0.05）.（3）川崎病组IFN-y基因Ash2L、Jmjd3结合水平和CD4＋T细胞T-bet蛋白水平均高于对照组（P均＜0.05）.冠状动脉损伤组前述各项指标水平也均高于无冠状动脉损伤组（P均＜0.05）.川崎病患儿治疗后前述各项指标水平均低于治疗前（P均＜0.05）.川崎病组与对照组、冠状动脉损伤组与无冠状动脉损伤组、川崎病患儿治疗前与治疗后之间的IFN-γ基因Ezh2结合水平差异均无统计学意义（P均＞0.05）.（4）川崎病组CD4＋T细胞表面受体IFN-γR1、IFN-γR2、IL-12Rβ1、IL-12Rβ2、IL-18Rα、IL-18R3、TNFR1、TLR4 mRNA水平、下游信号分子RIP-1、MyD88 mRNA水平和pSTAT1、pSTAT4蛋白水平均高于对照组（P均＜0.05）;川崎病组血浆IFN-γ、IL-12、IL-18和TNF-α浓度均高于对照组（P均＜0.05）.冠状动脉损伤组前述各项指标均高于无冠状动脉损伤组（P均＜0.05）.川崎病患儿治疗后前述各项指标均低于治疗前（P均＜0.05）.结论 Ash2L与Jmjd3过度结合所致的IFN-γ基因组蛋白甲基化修饰异常改变可能与急性期川崎病患儿免疫功能紊乱及血管损伤有关.

Objective To investigate the impacts of ash2 （absent,small,or homeotic）-like （Ash2L） and Jumonji domain-containing protein 3 （Jmjd3） on histone methylation of interferon-gamma （IFN-γ） gene and association with vascular damage of Kawasaki disease （KD） in acute phase.Methods This study was performed among 36 children with KD in acute phase （KD group） and 28 age-matched health children （control group）,who were treated or underwent physical examination in our hospital between February 2015 and June 2016.Patients were further divided into KD groups with or without coronary artery lesions （KD-CAL＋,16 cases;KD-CAL-,20 cases）.All KD patients were treated with intravenous immunoglobulin.The proportion of type 1 helper T（Th1） cells and protein levels of IFN-γ,T-box expressed in T cells （T-bet）,phosphorylated signal transducer and activator of transcription 1 （pSTAT1） and phosphorylated signal transducer and activator of transcription 4 （pSTAT4） were analyzed by flow cytometry.Chromatin immunoprecipitation was performed to determine histone methylation （histone H3 tri-methyl K4（H3K4me3）,histone H3 tri-methyl K27 （H3K27me3）） and binding levels of Ash2L,Jmjd3 and Ezh2 associated with IFN-γ in CD4 ＋ T cells.Quantitative real-time PCR was used to determine mRNA levels of IFN-γ,interferon γ receptor 1 （IFN-γR1）,interferon γ receptor 2 （IFN-γR2）,interleukin 12 receptor subunit beta 1 （IL-12R31）,interleukin 12 receptor subunit beta 2 （IL-12R32）,interleukin 18 receptor subunit beta α（IL-18Rα）,interleukin 18 receptor subunit beta β（IL-18Rβ）,tumor necrosis factor receptor 1 （TNFR1）,toll-like receptor 4 （TLR4）,receptor interacting serine/threonine kinase 1 （RIP-1） and myeloid differentiation primary response gene 88 （MyD88） in CD4 ＋ T cells.Plasma concentrations of IFN-γ,interleukin 12 （IL-12）,i nterleukin 18 （IL-18） and tumor necrosis factor α （TNF-α） were measured by enzyme-linked Immunosorbent assay.Results （1）The proportion of Th1 and its protein level of IFN-γwere significantly higher in KD group than those in control group and higher in KD-CAL ＋ group than in KD-CAL-group （all P ＜ 0.05）,and lower after treatment than before treatment （all P ＜ 0.05）.（2） Compared with control group,mRNA level of IFN-γ and IFN-γ-associating H3K4me3 was increased,while level of IFN-γ associating H3K27me3 in CD4 ＋ T cells was reduced in KD group （all P ＜ 0.05）,which resulted in a higher rate of H3K4me3/H3 K27me3 （P ＜ 0.05） in KD group,which was positively correlated with IFN-γ mRNA in KD group（r =0.55,P ＜0.05）.Similar results were found between KD-CAL＋ group and KD-CAL-group （all P ＜ 0.05）.Level of IFN-γ associating H3K27me3 was increased,and mRNA level of IFN-γ and IFN-γ associating H3K4me3 was decreased after treatment than before treatment （all P ＜ 0.05）.（3）Expression of T-bet protein and binding levels of Ash2L and Jmjd3 with IFN-γ gene were significantly higher in KD group than those in control group（all P ＜0.05）,higher in KD-CAL＋ group than those in KD-CAL-group （all P ＜ 0.05）.These parameters were significantly lower after treatment than before treatment （all P ＜ 0.05）.Binding level of Ezh2 with IFN-γ gene was similar among various groups （all P ＞ 0.05）.（4） In comparison with control or after treatment,surface receptors （IFN-γR1/2,IL-12Rβ1/2,IL-18Rα/β,TNFR1 and TLR4） and its downstream molecules（pSTAT1,pSTAT4,RIP1 and MyD88） in CD4 ＋ T cells,and plasma concentrations of inflammatory cytokines（IFN-γ,IL-12,IL-18 and TNF-α） were found to be higher in KD group （all P＜ 0.05）.These parameters were also higher in KD-CAL ＋ group than in KD-CAL-group （all P ＜ 0.05）.Conclusion Aberrant histone methylation of IFN-γ associating H3K4me3 and H3K27me3 caused by over-binding of Ash2L and Jmjd3 might be involved in immune dysfunction and vascular damage in KD in the acute phase.