摘要
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.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第6期436-441,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金(81102227,81300124)
广东省医学科学技术研究基金(A2012582,A2013604)