CD4^＋白细胞介素-17^＋辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

Effect of interleukin-17-producing CD4＋ T helper lymphocytes on cigarette smoke-induced lung inflammation and emphysema in mice

目的 观察香烟烟雾暴露小鼠肺实质中CD4＋白细胞介素（IL）-17＋辅助性T细胞（Th17）数量及活性的表达,探讨其在香烟暴露小鼠肺部CD4＋ γ-干扰素＋（Th1）炎症及肺气肿中的作用及相关机制.方法 将40只雄性Balb/c小鼠按随机数字表法分为4组：对照12周（C12）组、对照24周（C24）组、烟雾暴露12周（S12）组、烟雾暴露24周（S24）组,每组10只.香烟烟雾暴露法建立小鼠肺气肿模型.HE染色观察小鼠肺气肿的改变,计算平均内衬间隔和肺泡破坏指数（DI）;流式细胞术检测小鼠肺实质中CD4＋IL-17＋T（Th17）细胞、CD4＋γ-干扰素＋T（Th1）细胞、CD4＋IL-17＋γ-干扰素＋T（Th17/Th1）细胞、CD8＋γ-干扰素＋T（Tc1）细胞、CD8＋IL-21R＋细胞及CD4＋IL-17＋IL-21＋细胞比例;荧光定量PCR法检测小鼠肺实质中维甲酸相关孤独受体（RORγt）和IL-17的mRNA表达,并分析这些指标的相互关系.结果 S12组和S24组的平均内衬间隔[（39±4）μm和（47±7）μm]和DI（39.1±1.6和45.2±3.1）明显高于C12组[（32±4）μm和28.2±1.6]和C24组[（33±3）μm和28.9±2.1],且以S24组的增高更为明显,差异均有统计学意义（t值为4.378～15.188,均P＜0.05）;S12组和S24组Th17细胞比例[（3.3±1.1）%和（7.2±2.2）%]均明显高于C12组和C24组[（1.8±0.8）%和（2.0±0.6）%];S12组和S24组RORγtmRNA表达量[（25±4）和（35±3）]及IL-17的mRNA表达量[（26±3）和（36±3）]亦明显高于C12组[（10±5）和（13±5）]和C24组[（11±7）和（8±6）],以S24组增高更为明显,差异均有统计学意义（P＜0.05）;S12组和S24组Th1细胞比例[（10.0±3.7）%和（26.2 ±6.0）%]、Th17/Th1细胞比例[（0.61±0.30）%和（1.82±0.52）%]及Tc1细胞比例[（17.0±4.5）%和（26.8±8.5）%]均明显高于C12组[（3.8±1.7）%、（0.27±0.17）%和（4.8±1.9）%]和C24组[（4.2±1.3）%、（0.28±0.11）%和（5.2±1.0）%],以S24组增高更为明显,差异均有统计学意义（P＜0.05）;S12组和S24组小鼠Th17细胞与Th1、Tc1细胞比例、平均内衬间隔、DI值均呈显著正相关（r值为0.519～0.797,均P＜0.01）;Th17/Th1细胞比例与平均内衬间隔、DI值呈显著正相关（r值分别为0.742和0.802,均P＜0.01）;S12组和S24组CD4＋IL-17＋IL-21＋细胞比例[（0.19±0.04）和（0.55±0.24）]明显高于C12组和C24组[（0.07±0.03）和（0.08±0.03）],S24组增高更为明显,差异均有统计学意义（P＜0.05）.S12组和S24组的CD8＋IL-21R＋细胞比例[（2.94±1.26）和（4.12±2.26）]高于C12组和C24组[（1.22±0.31）和（1.34±0.18）]（P＞0.05）;S12组及S24组小鼠CD4＋IL-17＋IL-21＋细胞比例与Th1、Tc1细胞比例、平均内衬间隔和DI值均呈显著正相关（r值为0.694～0.754,均P＜0.05）;S12及S24组小鼠CD8＋IL-21R＋细胞比例与平均内衬间隔和DI呈显著正相关（r值分别为0.516和0.725均P＜0.05）.结论 香烟暴露导致肺气肿小鼠肺内Th17细胞数量及活性上调,并随烟雾暴露时间延长而增强;Th17细胞通过IL-21及IL-21R在肺部Th1/Tc1炎症中起重要促进作用;这对探讨COPD肺部炎症和肺气肿发生机制以及新的治疗靶点具有重要意义.

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group （S12） and a smoke-exposure 24 week group S24 （n = 10 each）.Morphological changes were evaluated by mean linear intercepts and destructive index （DI）.The proportion of CD4＋ IL-17 ＋ Th17, CD4＋ IFN-γ＋ Th1, CD4＋ IL-17 ＋IFN-γ＋ T（ Th17/ Th1 ）, CD8＋ IFN-γ＋ Tc1, CD8＋ IL-21R ＋ and CD4＋ IL-17 ＋ IL-21 ＋ T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [（39 ± 4）μm, （47 ±7） μm], （39.1 ± 1.6, 45.2 ±3.1 ） as compared to C12[（32 ±4） μm,28.2 ± 1.6] and C24groups [（33 ± 3 ） μm ,28.9 ± 2.1], all P 〈 O.05.The percentage of Th17 of S12 and S24 groups [（3.3 ±1.1 ）%, （7.2 ±2.2）%] was significantly increased as compared with that of C12 and C24 groups [（ 1.8± 0.8） %, （2.0 ± 0.6） %], all P 〈 0.05.The mRNA levels of RORγt [（ 25 ± 4）, （ 35 ± 3 ）] and IL-17 [（26 ± 3）, （36 ± 3 ）] in S12 and S24 groups were higher than in C12 [（10 ± 5 ）, （13 ± 5 ）] and C24 groups [（ 11 ± 7 ）, （8 ± 6）], all P 〈 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [（10.0 ±3.7）%, （26.2 ±6.0）%], [（0.61 ±0.30）%, （1.82 ±0.52）%], [（17.0±4.5 ） %, （ 26.8 ± 8.5 ） %] was significantly increased as compared with that of C12 [（ 3.8 ± 1.7 ） %,（0.27±0.17）%, （4.8 ±1.9）%] and C24 groups [（4.2±1.3）%, （0.28±0.11）%, （5.2±1.0）%], all P〈0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions （ r =0.519 - 0.797, all P 〈 0.01 ）.In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found （r =0.742, 0.802, all P 〈0.01 ）.The percentage of CD4＋ IL-17＋ IL-21 ＋T cells was significantly increased in S12 and S24 groups [（0.19 ±0.04） %, （0.55 ± 0.24） %] compared to controls [（0.07 ± 0.03 ） %, （0.08 ± 0.03 ） %], all P 〈 0.05.Meanwhile, as compared with that of the controls [（ 1.22 ± 0.31 ）, （ 1.34 ± 0.18 ）], the percentage of CD8＋ IL-21 R ＋ T cells was also increased in SI 2 and S24 groups [（ 2.94 ± 1.26 ）, （4.12 ± 2.26 ）], but there were no differences among smoke-exposure groups （ P 〉0.05 ）.The frequency of CD4＋ IL-17 ＋ IL-21 ＋ T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions （r = 0.694 -0.754, all P 〈0.05）.And the frequency of CD8＋ IL-21R＋ T cells also had a positive correlation with emphysematous lesions （ r = 0.516, 0.725, all P 〈 0.05）.Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential （active） role in the development of lung inflammation through IL-21/IL-21R pathway.