低氧诱导因子-1α信号活化在川崎病患儿Th17／调节性T淋巴细胞失衡中的作用

The roles of activated hypoxia -inducible factor 1α signaling in Thl7/regulatory T cells imbalance in Kawasaki disease

目的探讨低氧诱导因子-1α（HIF-1α）信号活化在川崎病（KD）患儿Thl7／调节性T淋巴细胞（Treg）失衡机制中的作用。方法急性期KD患儿36例，健康对照组32例。KD患儿分别于IVIG治疗前、后直接取血备检。采用流式细胞术检测Thl7细胞、Treg比例及叉头蛋白P3（Foxp3）、IL-10、IL-17A、HIF-1理、磷酸化信号转导和转录活化因子3（pSTAT3）蛋白表达水平；实时荧光定量PCR检测CD4＋T淋巴细胞Toll样受体4（TLR4）、髓样分化因子88（MyD88）、受体相互作用蛋白1（RIP1）、IL-17A、RAR相关孤儿受体1t（RORγt）、p300mRNA表达；免疫共沉淀一定量PCR及免疫印迹法检测外周血CD4细胞IL-17A基因组蛋白H3乙酰化、Foxp3蛋白泛素化水平；ELISA检测血浆中IL．1B、IL-6、IL．21、IL-23、TNF-α水平。结果1．急性期KD患儿Th17细胞比例及IL-17A表达显著上升（t=7．62、9．60，P均〈0．05），Treg细胞比例及转录因子Foxp3、IL-10表达明显降低（t=5．72、6．82、5．83，P均〈0．05），Th17／Treg细胞比值远高于健康对照组（t=12．21，P〈0．05），经静脉用丙种球蛋白（IVIG）治疗后均有明显恢复（t=6．28、8．30、4．75、5．02、3．95、9．39，P均〈0．05）。2．急性期KD患儿CD4＋T细胞IL．17A基因组蛋白H3乙酰化水平及Foxp3蛋白泛素化水平显著上升[H3：（7．32±2．53）％比（2．60±1．08）％；Foxp3：（42．94±15．91）％比（11．01±3．60）％，t=10．20、11．71，P〈0．05]，HIF-1α、RO脚t及p300表达亦明显高于健康对照组（t=6．02、6．92、6．01，P均〈0．05），经IVIG治疗后均显著降低[H3：（4．63±2．07）％比（7．32±2．07）％；Foxp3：（26．37±11．58）％比（42．94±15．91）％；t=4．94、5．05、3．40、5．31、4．29，P均〈0．05]。其中，HIF-1理表达与IL-17A基因组蛋白H3乙酰化水平、Foxp3蛋白泛素化水平呈正相关（r=0．65、0．52，P均〈0．05）。3．急性期KD患儿血浆炎性细胞因子IL-1B、IL-6、IL-21、IL-23、TNF-α水平及TLR4表达显著增高（t=11．00、15．18、7．21、11．09、9．09、3．84，P均〈0．05），其下游信号分子MyD88、pSTAT3、RIP1表达亦明显高于健康对照组（t=10．07、5．41、8．13，P均〈0．05），经IVIG治疗后均显著降低（t=7．81、10．59、4．70、6．73、6．49、3．03、6．50、3．63、5．22，P均〈0．05）。结论HIF-1d信号异常活化可能是导致急性期KD患儿Th17／Treg细胞失衡的重要因素之一。

Objective To investigate the roles and significance of hypoxia - inducible factor 1 ct （ HIF - 1α） signaling in Thl7/Treg imbalance in Kawasaki disease（KD）. Methods Thirty - six children with KD and 32 age - matched healthy children consented to participate in the study. The proportions of Th17, Treg and expression levels of forkhead box protein P3 （Foxp3）, IL - 10, IL - 17A, HIF - 1 ot and phosphated signal transducer and activator of tran- scription 3 （pSTAT3） in CD4 ＋ T cells, were evaluated by flow cytometry. Quantitative real - time PCR was used to as- sess the transcription levels of Toll - like receptor 4 （ TLR4 ）, myeloid differentiation factor 88 （ MyD88 ） , receptor interacting protein - 1 （ RIP1 ）, IL - 17A, RAR - related orphan receptor γt（RORγt） and p300 in CD4＋ T cells. Immu- noprecipitation combined with quantitative PCR and Western blot was performed to determine histone acetylation level of IL - 17A gene and poly - ubiquitination status of Foxp3 protein in CD4＋ T cells. Plasma concentrations of IL - 1β, IL -6, IL - 21, IL - 23 and tumor necrosis factor α （TNF - α） were measured by enzyme - linked immunosorbent assay. Results （ 1 ） The proportion of Thl7 and expression of IL - 17A in patients with acute KD increased remarkably while the proportion of Treg and expressions of Foxp3 and IL - 10 were found to be lower than those of the healthy con-trols （ t = 7.62,9.60,5.72,6.82,5.83 ;all P 〈 0.05 ）, which resulted in a higher ratio of Thl 7/Treg during acute KD （t = 12.21, P 〈 0.05 ）, and all the data mentioned above restored significantly after intravenous immunoglobulin（IVIG） treatment （ t = 6.28,8.30,4.75,5.02,3.95,9.39 ; all P 〈 0.05 ）. （ 2 ） Aeetylation of the histone H3 associated with IL- 17A gene and poly- ubiquitination of Foxp3 protein in CD4＋T cells increased significantly, and more higher expressions of HIF - 1α, ROR3,t and p300 were detected during acute KD [ H3 ： （ 7.32± 2.53 ） % vs （ 2.60 ±1.08 ） % ; Foxp3 ： （42.94±15.91 ） % vs （ 11.01±3.60）%;t = 10.20,11±71,6.02,6.92,6.01 ; all P 〈 0.05 ], and the 5 items decreased remarkably after IVIG therapy[ H3 ： （ 4.63±2.07 ） % vs （ 7.32 ±2.07 ） % ; Foxp3 ： （ 26.37±11.58 ） % vs （42.94±15.91 ） % ; t = 4.94,5.05,3.40,5.31,4.29 ; all P 〈 0.05 ]. Meanwhile, positive correlations between expression of HIF -1a with histone acetylation of IL - 17A gene and poly - ubiquitination of Foxp3 protein were observed in patients with acute KD （r = 0.65,0.52;all P 〈 0.05 ）. （3） Compared with the healthy controls, plasma concentrations of inflammatory cytokines （ IL - 1β, IL - 6, IL - 21, IL - 23 and TNF - or） and expressions of TLR4 were elevated while expression levels of its downstream molecules（ MyD88 ,pSTAT3 and RIP1 ）in CD4 ＋ T cells were up-regulated during acute KD （ t = 11.00,15.18,7.21,11.09,9.09,3.84,10.07,5.41,8.13 ; all P 〈 0.05 ）, and restored remarkably after therapy（t=7.81,10.59,4.70,6.73,6.49,3.03,6.50,3.63,5.22;all P〈0.05）. Conclusion Excess activation of HIF - 1α signaling, may be one of the important factors contributing to the imbalance of Th17/Treg in KD.