1，25-二羟维生素D3处理的树突细胞在变应性气道炎症中的免疫调节作用

Immunoregulatory role of 1, 25-dihydroxyvitamin D3-treated dendritic cells in allergic airway inflammation

目的探讨1，25-二羟维生素D，[1，25（OH）2D3]处理的树突细胞（DC）在变应性气道炎症中的免疫调节作用及其机制。方法小鼠骨髓来源DC分2组，分别用1，25（OH）2D3和磷酸盐缓冲液（PBS）处理，RT—PCR和蛋白质印迹法检测2组DC Notch配体mRNA和蛋白表达。将2组DC和Notch配体中和抗体封闭的1，25（OH）2D3处理DC分别与小鼠脾脏来源CD4＋T细胞共培养，流式细胞仪检测CD4＋T细胞中CD4＋CD25＋Foxp3＋T细胞百分比。将卵清蛋白（OVA）致敏小鼠分2组，每组5只，分别过继转移1，25（OH）2D3组DC[1，25（OH）2D3-DC组]和PBS组DC（PBS—DC组），以OVA激发气道炎症后行肺脏病理、支气管肺泡灌洗液（BALF）中白细胞介素4（IL-4）、IL-5、IL-13和干扰素γ（IFN-γ）水平以及脾脏CD4＋T细胞中CD4＋CD25＋Foxp3＋T细胞百分比检查。结果1，25（OH）2D3组DCNotch配体Jagged1和Jagged2 mRNA表达（0．3764±0．029、0．5644±0．018）和蛋白表达（0．786±0．034、0．632±0．026）均明显高于PBS组（分别为0．146±0．032、0．267±0．012和0．124±0．025、0．098±0．012，均P〈0．01）。与CD4＋T细胞共培养后，1，25（OH）2D3组CD4＋T细胞中CD4＋CD25＋Foxp3＋T细胞百分比（22．49％±0．56％）明显高于PBS组（6．67％±0．60％，P〈0．01）；经Jagged2中和抗体封闭组CD4＋CD25＋Foxp3＋T细胞百分比（6．56％±1．89％）明显低于未封闭组（20．37％±1．64％，P〈0．01）。1，25（OH）2D3-DC组小鼠气道炎症明显轻于PBS-DC组；BALF中IL-4、IL-5、IL．13和IFN-γ水平（pg／ml）分别为33±5、134±23、91±11和未检测到（〈12．5），均明显低于PBS—DC组（分别为55±7、332±49、152±19、23±6，均P〈0．01）；脾脏CD4＋T细胞中CD4＋CD25＋Foxp3＋T细胞百分比（14．69％±1．14％）明显高于PBS—DC组（2．38％±0．14％，P〈0．01）。结论1，25（OH）2D3处理DC对变应性气道炎症有抑制作用，这种作用可能与1，25（OH）2D3处理DC通过Jagged2介导的Notch信号途径诱导CD4＋CD25＋Foxp3＋T细胞生成有关。

Objective To explore the mechanism and immunoregulatory role of 1,25-dihydroxy vitamin D3 [ 1,25 （ OH ）2D3 ] -treated dendritic cells （ DCs ） in allergic airway inflammation. Methods Mouse bone marrow-derived DCs were treated by 1,25 （OH） 2 D3 for 72 h. The expression levels of different Notch ligands： Jagged1, Jagged2, Delta1, Delta3, and Delta4 in these DCs were detected by RT-PCR and Western blotting. Mouse spleen CD4＋ T cells were cultured with 1, 25 （OH）2D3-treated DCs or 1, 25 （OH）2D3-treated DCs blocked by polyclone antibody of Jaggedl or Jagged2 （control group） for 48 h. The percentage of CD4＋CD25＋Foxp3＋ T cells in CD4＋ T cells was detected by flow cytometry （FCM）. Ten mice underwent intraperitoneal injection of ovalbumin （OVA） to be sensitized and then divided into 2 groups to inhale 1, 25 （OH）2D3-treated DC suspension and PBS-DC suspension respectively for 6 successive days. Then the mice were killed. Bronchoalveolar lavage fluid was obtained to detect the eosinophil count and the levels of interleukin （IL）-4, IL-6, IL-13, and interferon （IFN）-γ, and pathological examination of lung was conducted. Spleens were taken out to isolate the CD4＋ T cells, and immunolabeling and FCM were used to detect the percentage of CD4＋CD25＋Foxp3＋ T cells. Results The mRNA and protein expression levels of Jaggedl and Jagged2 in the 1,25（OH）2D3-treated DCs were （0. 376±0. 029） and （0. 786±0. 034）, and （ 0. 564 ± 0. 018 ） and （ 0. 632 ± 0. 026） respectively, all significantly higher than those of the control group [ （0. 146 ± 0. 032 ） and （0. 124 ± 0. 025 ）, and （0. 267 ±0. 012 ） and （ 0. 098 ±0. 012） respectively, all P 〈 0. 01 ） ]. The percentage of CD4 ＋CD25 ＋Foxp3 ＋ T cells in the CD4 ＋ T cells cultured with 1,25（OH）2D3-treated DCs was （22.49% ±0. 56% ）, significantly higher than that of the a PBS control group [ （6.67% ±0. 60% ）, P 〈 0. 01 ]. The percentage of CD4＋CD25 ＋Foxp3＋ T cells in CD4＋ T cells after cultured with 1,25 （OH）2 D3-treated DC blocked by polyelone antibody of Jagged2 was （6. 56% ± 1.89% ）, significantly lower than that of the un-blocked control group [ （20. 37% ± 1.64% ）, P 〈0. 01 ]. The levels of IL-4, IL-5, IL-13, and IFN-γ, and eosinophil count in the BALF of the 1,25 （OH）2 D3-treated DC group were （ 33 ± 5 ） pg/ml, （ 134 ± 23 ） pg/ml, （ 91 ±11 ） pg/ml, and undetectable（ 〈 12. 5 pg/ml）, and （ 236 ±29） × 10^3/ml,all significantly lower than those of the PBS-DC group [ （55 ±7）pg/ml, （332 ±49）pg/ml, （ 152 ± 19）pg/ml, and （23±6）pg/ml,and （588 ±56）× 10^3/ml, all P 〈0. 01 ]. The percentage of CD4＋ CD25＋Foxp3＋ T cells in the spleens of the 1,25 （ OH ） 2 D3-treated DC group was （ 14. 69% ± 1.14% ）, significantly higher than that of the PBS-treated DC group [ （2. 38% ±0. 14%, P 〈0. 01 ）. Conclusion Treatment of the DCs with 1, 25 （OH）2D3 inhibits the allergic inflammation in the airway, maybe via the induction of CD4＋CD25＋Foxp3＋ regulatory T cells by 1,25 （OH）2D3-treated DCs through Jagged2-mediated Notch signal pathway.