维生素 D 通过 STAT3-CD23 轴抑制屋尘螨过敏患儿 B 细胞活化及 IgE 分泌

Vitamin D suppresses B cell activation and IgE secretion via STAT3-CD23 axis in children with house dust mite allergy

摘要

目的: 探讨维生素 D 对屋尘螨过敏患儿 B 细胞活化及总免疫球蛋白 E(immunoglobulin E,IgE)分泌的影响及其潜在的分子机制。 方法: 纳入 126 例屋尘螨过敏患儿(屋尘螨过敏组)及 81 例健康儿童(健康对照组),检测血清中 25-羟维生素 D[25-(OH)D]、IgE、人屋尘螨变应原(dermatophagoides pteronyssinus peptidase 1,Derp1)特异性 IgE 及可溶性 CD23(sCD23)水平;采用 Pearson 相关性分析 25-(OH)D 水平与总 IgE 水平、Derp1 特异性 IgE 水平、sCD23 水平以及外周血 CD19⁺CD23⁺B 细胞比例之间的关系;采用受试者工作特征(ROC)曲线分析血清 25-(OH)D 水平对屋尘螨过敏的诊断价值。 分离屋尘螨过敏患儿 CD19⁺ B 细胞,将其分为阴性对照组(常规培养)、阳性对照组(1 μg/ mL 脂多糖)和 10、50、100 nmol/ L 1,25-(OH)₂D₃组[分别用对应 10、50、100 nmol/ L 1,25-(OH)₂D₃处理],共培养 48 h;采用流式细胞术检测各组细胞中 CD23 膜表达,蛋白免疫印迹法检测信号转导和转录激活因子 3(signal transducer and activator of transcription 3,STAT3)、STAT5、p-STAT3 和 p-STAT5 水平;采用 ELISA 检测 IgE 分泌水平。通过时间动力学实验观察 100 nmol/ L 1,25-(OH)₂D₃ 处理不同时间点 STAT3 磷酸化和 IgE 分泌的动态变化。 将 CD19⁺B 细胞分为阴性对照组(常规培养)、1,25-(OH)₂D₃组[100 nmol/ L 1,25-(OH)₂D₃]、IL-6 组(20 ng/ mL IL-6)和 1,25-(OH)₂D₃+IL-6 组[100 nmol/ L 1,25-(OH)₂D₃+20 ng/ mL IL-6],检测各组细胞中 CD23 膜表达和 IgE 分泌水平。结果: 屋尘螨过敏组中 25-(OH)D 水平显著低于健康对照组(P<0. 001),且与 sCD23 水平呈弱负相关(r = -0. 248)。ROC 曲线结果显示,血清 25-(OH)D 水平对屋尘螨过敏患儿具有一定的诊断价值(AUC = 0. 769)。 体外实验结果显示,与阴性对照组相比,不同浓度 1,25-(OH)₂D₃组中 CD23 膜表达、p-STAT3 表达及 IgE 分泌均显著减少,呈浓度依赖性(P 均<0. 01)。 时间动力学实验结果显示,100 nmol/ L 1,25-(OH)₂D₃处理 30 min 即显著抑制 p-STAT3 表达,处理 48 h 显著降低 IgE 分泌(P<0. 01)。 与 1,25-(OH)₂D₃组相比,1,25-(OH)₂D₃+IL-6 组 CD23 膜表达和 IgE 分泌明显增加(P<0. 05)。 结论: 维生素 D 可能通过抑制 STAT3 磷酸化下调 CD23 表达,进而减少 B 细胞 IgE 分泌。

Abstract

Objective: To investigate the effect of vitamin D on B cell activation and total immunoglobulin E (IgE) secretion in children with house dust mite allergy and the underlying molecular mechanisms. Methods: A total of 126 children with house dust mite allergy (allergy group) and 81 healthy children (control group) were enrolled. Serum levels of 25-hydroxyvitamin D [25-(OH)D], total IgE, dermatophagoides pteronyssinus peptidase 1(Derp1)-specific IgE, and soluble CD23 (sCD23) were measured. Pearson correlation analysis was used to assess the relationships between serum 25-(OH)D levels and total IgE, Derp1-specific IgE, sCD23 levels, and the proportion of CD19 ⁺CD23 ⁺B cells in peripheral blood. The diagnostic value of serum 25-(OH)D for house dust mite allergy was assessed using receiver operating characteristic (ROC) curve analysis. CD19 ⁺ B cells isolated from allergic children were divided into negative control group (routine culture), positive control group (1 μg / mL lipopolysaccharide), and three treatment groups with 10, 50, and 100 nmol/ L 1,25-(OH) ₂D ₃, respectively; cells were co-cultured for 48 hours. Flow cytometry was used to detect membrane expression of CD23, while Western blotting was assessed levels of signal transducer and activator of transcription 3 (STAT3), STAT5, p-STAT3, and p-STAT5. ELISA was employed to measure IgE secretion. Time-course experiments were conducted to observe dynamic changes in STAT3 phosphorylation and IgE secretion at different time points after 100 nmol/ L 1,25-(OH) ₂D ₃ treatment. CD19 ⁺B cells were further divided into four groups: negative control group (routine culture), 1,25-(OH) ₂D ₃ group [100 nmol/ L 1,25-(OH) ₂D ₃], IL-6 group (20 ng / mL IL-6), and 1,25-(OH) ₂D ₃ + IL-6 group [100 nmol/ L 1,25-(OH) ₂D ₃+ 20 ng / mL IL-6]. Membrane expression of CD23 and IgE secretion were analyzed in each group. Results: Serum 25-(OH)D level in the allergy group was significantly lower than that in the control group (P<0. 001) and showed a weak negative correlation with sCD23 levels (r= -0. 248). ROC curve analysis indicated that serum 25-(OH)D had moderate diagnostic value for house dust mite allergy in children (AUC = 0. 769). In vitro experiments demonstrated that compared with the negative control group, CD23 membrane expression, p-STAT3 expression and IgE secretion were significantly reduced in the 1,25-(OH) ₂D ₃ groups in a concentration-dependent manner (all P<0. 01). Time-course experiments revealed that 100 nmol/ L 1,25-(OH) ₂D ₃ significantly inhibited p-STAT3 expression within 30 minutes and markedly reduced IgE secretion after 48 hours (P < 0. 01). Compared with the 1,25-(OH) ₂D ₃ group, the 1,25-(OH) ₂D ₃ +IL-6 group showed significantly increased CD23 membrane expression and IgE secretion (P < 0. 05). Conclusion: Vitamin D may downregulate CD23 expression by inhibiting STAT3 phosphorylation, thereby reducing IgE production from B cells in house dust mite allergy.

关键词

屋尘螨 / 维生素 D / B 细胞 / STAT3 / 免疫球蛋白 E

Key words

house dust mite / vitamin D / B cells / STAT3 / immunoglobulin E

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