2型固有淋巴样细胞在MC903诱导的小鼠特应性皮炎样反应中的作用

Role of group 2 innate lymphoid cells in mice with atopic dermatitis-like inflammatory response induced by MC903

目的探讨2型固有淋巴样细胞(ILC2)在特应性皮炎(AD)中的作用。方法以C57BL/6J和Rag1-/-小鼠为研究对象。C57BL/6J小鼠分为模型组和对照组,模型组小鼠两侧耳部连续14 d每日涂抹卡泊三醇(MC903)以制备AD样小鼠模型,对照组仅涂抹无水乙醇,第15天时收集小鼠外周血血清,处死小鼠后,取耳部皮肤行组织病理检查,摘取脾脏,用实时荧光定量PCR检测皮肤和脾中炎症因子表达水平,同时采用流式细胞仪检测皮肤中ILC2比例。Rag1-/-小鼠分为模型组、对照组和实验组,模型组和对照组处理及评估同C57BL/6J小鼠,实验组小鼠在涂抹MC903前2 d开始腹腔注射单克隆抗体CD90.2拮抗ILC2功能,隔2 d 1次,共计7次,每次300μg/150μl,其他处理同模型组,第15天时收集小鼠外周血血清,处死小鼠后,取耳部皮肤行组织病理检查,用实时荧光定量PCR检测皮肤中炎症因子表达水平,同时采用流式细胞仪检测皮肤中ILC2比例。采用两独立样本t检验进行两组间比较,单因素方差分析进行多组间比较。结果模型组C57BL/6J小鼠耳部皮肤可见明显的红肿、干燥、结痂,HE染色可见表皮厚度增加,真皮嗜酸性粒细胞浸润,血清IgE水平[(6751.016±282.324)μg/L]高于对照组[(6387.038±267.853)μg/L,P=0.007),且皮肤白细胞介素(IL)-4、IL-13和干扰素γ(IFN-γ)的表达水平较对照组明显增高(P值分别为0.005、0.012,<0.001),而IL-5差异无统计学意义(P=0.190),脾脏IL-4、IL-5和IL-13的表达较对照组明显增高(均P<0.001),而IFN-γ差异无统计学意义(P=0.278);模型组ILC2比例(5.604±2.105)%较对照组(1.750±1.104)%明显增高(P=0.003)。在Rag1-/-小鼠中,模型组耳部皮肤也可见明显红肿、干燥、结痂,HE染色可见表皮厚度增加,真皮嗜酸性粒细胞浸润,模型组皮肤IL-4、IL-5、胸腺基质淋巴生成素和IL-33的表达较对照组明显增高(P值分别为0.010、0.043、0.034、0.007),而IL-13和IFN-γ差异无统计学意义(P值分别为0.274,0.697);模型组ILC2比例(5.165±2.436)%高于对照组[(0.835±0.578)%,P=0.014];抗体拮抗Rag1-/-小鼠ILC2后,实验组小鼠皮损较模型组明显减轻,HE染色可见表皮增厚减轻,真皮嗜酸性粒细胞浸润减少,而细胞因子表达分析显示,实验组IL-4、IL-5、IL-13、TSLP、IL-33水平与模型组差异均无统计学意义(均P>0.05)。结论ILC2参与了MC903诱导的AD样小鼠炎症反应,且该反应不依赖于适应性免疫。

Objective To explore the role of group 2 innate lymphoid cells(ILC2)in atopic dermatitis(AD).Methods C57BL/6J and Rag1-/-mice served as research objects.The C57BL/6J mice were divided into 2 groups:model group topically treated with calcipotriol(MC903)on both ears every day for 14 consecutive days,control group topically treated with anhydrous ethanol alone at the same time.On day 15,peripheral blood samples were collected from the mice.After the sacrifice,the ear skin tissues were obtained for histopathological examination,and the spleens were resected.Real-time fluorescence-based quantitative PCR was performed to determine the expression of inflammatory factors in the skin and spleen tissues,and flow cytometry to determine the proportion of ILC2 in the skin tissues.The Rag1-/-mice were divided into model group,control group and experimental group:the Rag1-/-mice in the model group and control group received the same treatment and evaluation as the C57BL/6J mice;two days before the topical treatment with MC903,the Rag1-/-mice in the experimental group started to be intraperitoneally injected with the monoclonal antibody CD90.2 at a dose of 300μg/150μl once every other 2 days for 7 sessions,with the purpose of antagonizing the function of ILC2,and other treatments were the same as those in the model group.Skin manifestations were observed,and histopathological features were evaluated.Two-independent-sample t test was used for comparisons between 2 groups,and one-way analysis of variance for comparisons among multiple groups.Results In the model group,the ear skin of the C57BL/6J mice was apparently red,swollen and dry with crusts,and hematoxylin and eosin(HE)staining showed increased thickness of the epidermis and dermal infiltration of eosinophils;the serum level of IgE(6751.016±282.324μg/L)was significantly higher in the model group than in the control group(6387.038±267.853μg/L,P=0.007),so were the expression of interleukin(IL)-4,IL-13 and interferon(IFN)-γin the skin tissues(P=0.005,0.012,<0.001,respectively),but there was no significant difference in IL-5 expression(P=0.190);the expression of IL-4,IL-13 and IFN-γin the spleen was significantly higher in the model group than in the control group(all P<0.001),but there was no significant difference in IFN-γexpression(P=0.278);moreover,the model group showed significantly increased proportion of ILC2(5.604%±2.105%)compared with the control group(1.750%±1.104%,P=0.003).In the Rag1-/-mice,the ear skin was obviously red,swelling and dry with crusts in the model group,and HE staining showed increased epidermal thickness and eosinophil infiltration in the dermis;the model group showed significantly increased expression of IL-4,IL-5,thymic stromal lymphopoietin(TSLP)and IL-33 in skin tissues(P=0.010,0.043,0.034,0.007,respectively),but no significant difference in the expression of IL-13 or IFN-γ(P=0.274,0.697,respectively)compared with the control group;the proportion of ILC2 was significantly higher in the model group(5.165%±2.436%)than in the control group(0.835%±0.578%,P=0.014);the experimental group showed markedly attenuated skin lesions,reduced epidermal thickness and number of eosinophils infiltrating in the dermis,but no significant difference in the expression of IL-4,IL-5,IL-13,TSLP or IL-33 compared with the model group(all P>0.05).Conclusion ILC2 play a role in the mice with AD-like inflammatory response induced by MC903,which dose not depend on adaptive immunity.