桥本甲状腺炎小鼠中性粒细胞胞外捕获网与T细胞分化相关性及维生素D的缓解作用

Correlation of neutrophil extracellular traps with T cells differentiation in Hashimoto′s thyroiditis mice and vitamin D′s relief effect

目的探讨实验性自身免疫性甲状腺炎(EAT)小鼠中性粒细胞胞外捕获网(NETs)与T淋巴细胞的相关性及活性维生素D的干预作用。方法6周龄雌性BALB/c小鼠分为对照组、EAT组和骨化三醇干预组(VitD组;n=6/组)。HE染色观察甲状腺病理,ELISA法检测血浆甲状腺球蛋白抗体(TGAb)、甲状腺过氧化物酶抗体(TPOAb)、1,25(OH)2D3;流式细胞术检测外周血NETs形成率、脾脏Th1、Th2及Th17细胞比例;分析NETs形成率与Th1、Th2、Th17细胞比例的相关性。结果与对照组相比,EAT组甲状腺炎症评分、发病率、TPOAb、TGAb、NETs形成率、Th2(CD4^(+)IL-4^(+)或CD4^(+)IL-13^(+))和Th17细胞比例显著升高(P分别<0.001、0.002、0.007、<0.001、<0.001、0.003、0.001、0.002),1,25(OH)2D3水平、Th1细胞比例、Th1/Th2(CD4^(+)IL-4^(+))、Th1/Th2(CD4^(+)IL-13^(+))、Th1/Th17比值显著降低(P分别0.010、0.018、0.010、0.005、0.007)。与EAT组相比,VitD组甲状腺炎症评分、TPOAb、TGAb水平、NETs形成率、Th2(CD4^(+)IL-4^(+)或CD4^(+)IL-13^(+))和Th17细胞比例显著下降(P分别0.044、0.007、<0.001、0.001、0.014、0.008、0.001),Th1细胞比例、Th1/Th2(CD4^(+)IL-13^(+))及Th1/Th17比值明显上升(P分别0.011、0.009、0.003),Th1/Th2(CD4^(+)IL-4^(+))升高但差异无统计学意义(P=0.174)。NETs形成率与Th2(CD4^(+)IL-4^(+)或CD4^(+)IL-13^(+))、Th17细胞比例呈正相关(r分别0.65、0.59、0.61,相应P分别为0.004、0.010、0.007),与Th1细胞比例无相关性(r=-0.47,P=0.051)。结论EAT小鼠NETs形成增多,活性维生素D可能通过减少NETs形成进而改善EAT小鼠Th2和Th17细胞升高、Th1细胞下降的全身免疫失衡状态,并减轻甲状腺损伤及降低甲状腺抗体水平。

Objective To investigate correlation between neutrophil extracellular traps(NETs)formation and T cell subsets in mice with experimental autoimmune thyroiditis(EAT)and the impact of active vitamin D intervention.Methods Six-week-old female BALB/c mice were randomly divided into Control group,EAT group and 1,25 dihydroxy vitamin D3[1,25(OH)2D3]treatment group(VitD group;n=6/group).HE staining was used to observe thyroid pathology.Plasma thyroglobulin antibody(TGAb),thyroid peroxidase antibody(TPOAb),and 1,25(OH)2D3 were measured by ELISA.Peripheral NETs formation,Th1,Th2,and Th17 cell ratio from spleen were measured by flow cytometry.Correlation between NETs formation rate and Th1,Th2,and Th17 cell ratio was analyzed.Results Compared with Control group,mice in EAT group had significantly increased thyroid inflammation scores,thyroiditis morbidity,TPOAb,TGAb levels,NETs formation rate,Th2(CD4^(+)IL-4^(+)or CD4^(+)IL-13^(+))and Th17 cell proportions(P were<0.001,0.002,0.007,<0.001,<0.001,0.003,0.001,and 0.002,respectively),and significant decreased 1,25(OH)2D3,Th1 cell proportions,Th1/Th2(CD4^(+)IL-4^(+)),Th1/Th2(CD4^(+)IL-13^(+)),and Th1/Th17 ratios(P were 0.010,0.018,0.010,0.005,and 0.007,respectively).Compared with the EAT group,the VitD group had lower thyroid inflammation scores,TPOAb,TGAb levels,NETs formation rate,Th2(CD4^(+)IL-4^(+)or CD4^(+)IL-13^(+))and Th17 cell proportions(P were 0.044,0.007,<0.001,0.001,0.014,0.008,and 0.001,respectively),and significant higher Th1 cell ratio,Th1/Th2(CD4^(+)IL-13^(+))and Th1/Th17 ratio(P were 0.011,0.009,and 0.003,respectively).The Th1/Th2(CD4^(+)IL-4^(+))was not significantly increased in VitD group compared with EAT group(P=0.174).NETs formation rate was positively correlated with Th2(CD4^(+)IL-4^(+)or CD4^(+)IL-13^(+))and Th17 cell proportion(r were 0.65,0.59,and 0.61;and P were 0.004,0.010,and 0.007,respectively),but not with Th1 cell proportion(r=-0.47,P=0.051).Conclusion EAT mice were more prone to NETs formation.Active vitamin D may relieve immune imbalance with increased Th2 and Th17 cell ratio and decreased Th1 cell ratio by reducing the formation of NETs in EAT mice.Vitamin D played the protective role in thyroid by reducing thyroid pathological damage and thyroid autoantibody levels,and relived overall lymphocyte imbalance.