急性期川崎病Foxp3蛋白泛素化改变及意义

Changes and significance of ubiquitination of Foxp3 protein during the acute phase of Kawasaki disease

目的探讨急性期川崎病（KD）Foxp3蛋白泛素化水平及其在KD免疫发病机制中的作用。方法急性期KD患儿48例，正常同年龄对照组28例，KD患儿分别于丙种球蛋白（IVIG）治疗前、后直接取血备检。采用免疫共沉淀-印迹法检测外周血CIM细胞Foxp3蛋白泛素化水平；流式细胞术检测CD4＋CD25highFoxp3＋细胞（Treg）比例及IL-10、TGF-β、CTLA4、STUB1、HSP70、USP7、pSTAT3蛋白表达水平；实时荧光定量PCR检测CIM＋T细胞IL-1R1、IL-1RAP、IL-6Ra、gp130、TNFR1、MyD88、RIP1 mRNA表达；ELISA检测血浆中IL-1β、IL-6、TNF-α浓度。结果（1）急性期KD患儿Treg细胞比例及IL-10、TGF-β、CTLA4、Foxp3蛋白表达水平显著降低（P〈0．05），Foxp3蛋白泛素化水平明显高于对照组[（0．52±0．19）VS（0．08±0．02），P〈0．05]，其中KD合并冠状动脉损伤组（KD-CAL＋）前述5项均低于无冠状动脉损伤组（KD-CAL-）（P〈0．05），Foxp3蛋白泛素化水平则高于后者[（0．70±0．28）VS（0．43±0．17），P〈0．05]，经IVIG治疗后均明显恢复[Ubi-Foxp3：（0．24±0．10）VS（0．52±0．19），P〈0．05]。（2）急性期KD患儿CD4＋T细胞STUB1、HSP70表达显著上调（P〈0．05），USP7表达明显低于正常对照组（P〈0．05），其中STUB1／USP7二者比值显著增高且与其Foxp3蛋白表达呈负相关关系（r=-0．56，P〈0．05），经IVIG治疗后均明显恢复（P〈0．05）。其中KD-CAL＋组STUB1、HSP70表达及STUB1／USP7比值均高于KD-CAL-组（P〈0．05），而USP7表达则明显低于后者（P〈0．05）。（3）急性期KD患儿血浆IL-1β、IL-6、TNF-α浓度及CD4＋T细胞IL-1R1／IL-1RAP／TLR4／MyD88、IL-6Rα／gp130／pSTAT3、TNFR1／RIP1表达均显著上调（P〈0．05），其中KD-CAL＋组前述各项均高于KD-CAL-组（P〈0．05），经IVIG治疗后明显降低（P〈0．05），其他TLR表达无改变（P〉0．05）。结论Voxp3蛋白泛素化异常可能与急性期KD患儿免疫调节功能紊乱有关。

Objective To investigate the changes and significance of uhiquitination of Foxp3 pro- tein during the acute phage of Kawasaki disease （KD）. Methods Forty-eight children with KD and twenty- eight age-matched healthy children were recruited in this study. Co-immunoprecipitation and Western blot assays were performed to determine the poly-ubiquitination status of Foxp3 in CD4＋ T cells. The percentages of CD4＋CD25highFoxp3＋ regulatory T cells （Treg） and the levels of IL-10, transforming growth factor-J3 （TGF-β）, cytotoxic T lymphocyte-associated protein-4 （CTLA4） , STIP1 homology and U-Box containing protein 1 （STUBI） , heat shock protein 70 （HSPTO） , ubiquitin-specific-processing protease 7 （USP7） and pSTAT3 were analyzed by flow cytometry analysis. Quantitative real-time PCR was used to evaluate the tran- scription levels of TLRI-10, IL-1R1, IL-1RAP, IL-6Rα, gpl30, TNFR1, MyD88 and RIP1 in CD4＋ T cells. Plasma concentrations of IL-1 β, IL-6 and TNF-α were measured by enzyme-linked immunosorbent as- say. Results （ 1 ） The percentages of Treg cells and the levels of IL-10, TGF-β, CTLA4 and forkhead box P3 （ Foxp3 ） in patients with acute KD were lower than those of healthy subjects （ P〈0.05 ）, while the poly- ubiquitination of Foxp3 protein was significantly enhanced in patients with acute KD [ （0.52±0. 19 ） vs （0.08±0.02）, P〈0.05 ]. Meanwhile, the four former items in KD patients with coronary artery lesions （ KD-CAL＋ ） were lower than those of KD patients without coronary artery lesions （ KD-CAL- ） （ P〈0.05 ） , while the polyubiquitination level of Foxp3 protein in KD-CAL＋ group was much higher than that of KD-CAL- group [ （0.70±0.28） vs （0.43±0.17） , P〈0.05 ]. The levels of Treg cells, IL-10, TGF-β and CTLA4 in patients with KD were increased and the ubiquitination of Foxp3 protein was inhibited [ （0.24±0. 10） vs （0.52±0.19）, P〈0.05] upon the treatment with IVIG. （2） The levels of STUB1 and HSP70 in CD4＋ T cells were significantly elevated during acute KD, while the levels of USP7 were decreased （ P〈0.05 ）. The ratios of STUB1/USP7 in patients with acute KD were much higher than those of the control group [ （2.65± 0.92） vs （ 1.09±0.37 ） , P〈0.05 ] , but were significantly decreased after IV1G therapy [ （ 1.46±0.53 ） vs （2.65±0.92）, P〈0.05 ]. A negative correlation was found between STUB1/USP7 ratio and Foxp3 level during acute KD （r=-O. 56, P〈0. 05）. Moreover, KD patients with CAL＋ showed higher levels of STUB1 and HSP70 and higher ratios of STUB1/USP7 （ P〈0.05 ） , but lower levels of USP7 as compared with those of KD-CAL- group （ P〈0.05 ）. （3） The plasma concentrations of inflammatory cytokines （ IL-1β, IL-6 and TNF-α） , the levels of surface receptors （IL-1R1/IL-1RAP/TLR4, IL-6Rα/gp130 and TNFR1 ） and its downstream molecules （ MyD88, pSTAT3 and RIP1 ） in CD4＋ T cells were up-regulated during acute KD （ P 〈0.05 ） , especially in patients with CAL＋, but were down-regulated upon the IVIG therapy （ P〈0.05 ）. No significant differences with other TLRs were found among the groups （ P〉0.05 ）. Conclusion Hyper-ubiq- uitination of Foxp3 protein might be involved in the immune dysfunction during Kawasaki disease.