Treg分化对放射性肺损伤的影响

Effect of Tregs differentiation on radiation-induced lung injury

目的探讨调节性T细胞(Treg)的分化对放射性肺损伤的影响及其作用机制。方法建立Treg抑制小鼠模型,按随机数字表法将C57BL/6小鼠分成4组:空白对照组、单纯照射组、照射+免疫球蛋白G(IgG)组和照射+CD25组,每组12只,除空白对照组外其余3组小鼠给予单次20 Gy X射线全胸照射,照射+IgG组和照射+CD25组小鼠每周腹腔注射IgG抗体和CD25抗体。分别于照射后第4周和第8周各处死小鼠6只,采用流式细胞术检测小鼠肺组织内CD25+Foxp3+Treg(Foxp3:叉头样转录因子3)的百分比以鉴定模型是否建立成功;采用Western blot法检测单纯照射组小鼠肺组织内神经纤毛蛋白1(NRP1)的表达;采用免疫荧光法检测每组小鼠肺组织内CD25+NRP1+Treg的百分比;拍照并观察每组小鼠皮肤的损伤情况,采用苏木精-伊红染色法检测小鼠肺组织的病理学改变;采用酶联免疫吸附测定法检测每组小鼠肺组织内转化生长因子β1(TGF-β1)、白细胞介素(IL)-17A、干扰素γ(IFN-γ)、IL-2和IL-4的水平变化。两组间比较采用独立样本t检验。结果流式细胞术检测结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内CD25+Foxp3+Treg百分比[(1.73±0.04)%、(2.13±0.15)%]均较空白对照组[(1.14±0.02)%、(1.70±0.06)%]明显升高,差异均有统计学意义(t=-26.680、-4.545,P=0.000、0.010),抑制Treg后,第4周和第8周时照射+CD25组小鼠肺组织内CD25+Foxp3+Treg百分比[(0.72±0.14)%、(0.27±0.02)%]均较单纯照射组明显降低,差异均有统计学意义(t=5.296、37.538,均P=0.000)。Western blot结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内NRP1蛋白表达水平均较空白对照组升高,差异均有统计学意义(t=-7.341、-9.127,均P=0.000)。免疫荧光法检测结果显示,照射后第4周和第8周,单纯照射组小鼠肺组织内CD25+NRP1+Treg的百分比均较空白对照组升高,而照射+CD25组CD25+NRP1+Treg百分比均较单纯照射组降低,且差异均有统计学意义(t=8.926、14.457,P=0.001、0.000)。观察小鼠皮肤损伤程度后发现,照射后第4周和第8周,单纯照射组小鼠皮肤损伤严重,而照射+CD25组小鼠照射后第4周时皮肤基本完好,第8周时出现脱毛脱皮。病理学结果显示,照射后第4周和第8周,与空白对照组相比,单纯照射组小鼠的肺组织结构破坏,肺泡壁增厚,细胞外基质增多,而照射+CD25组小鼠的肺组织结构完整,肺泡壁纤细。酶联免疫吸附测定结果显示,与空白对照组相比,照射后第4周,单纯照射组小鼠肺组织内IL-17A和IL-4的水平均升高,差异均有统计学意义(t=-8.492、-15.796,P=0.001、0.000),照射后第8周,TGF-β1和IL-17A水平升高,差异均有统计学意义(t=-11.072、-7.167,P=0.000、0.002),IL-2水平在第4周和第8周时均降低,IFN-γ水平在第4周时升高,差异有统计学意义(t=-27.393,P=0.000),第8周时下降;与单纯照射组相比,照射+CD25组小鼠TGF-β1和IL-17A水平在第4周和第8周时均降低(t=6.037、4.524、5.496、4.772,均P=0.000),IFN-γ水平升高(t=-7.006、-12.565,P=0.002、0.000),差异均有统计学意义,而IL-2和IL-4水平在第4周时均降低,第8周时均明显升高,差异均有统计学意义(t=2.866、-9.090、8.833、-7.191,均P=0.000)。结论放射性肺损伤小鼠的肺组织中出现Treg分化,并增强分泌TGF-β1促炎因子,同时干扰辅助T细胞(Th1、Th2型)细胞因子的平衡来促进放射性肺损伤的发生。

Objective To investigate the effects of the differentiation of regulatory T cells(Tregs)on radiation-induced lung injury and its mechanism.Methods A mouse model that inhibits Tregs was established.C57BL/6 mice were divided into the control group,simple irradiation group,irradiation+IgG group,and irradiation+CD25 group according to the random number table method.Each group comprised 12 mice.The mice in the simple irradiation group,irradiation+IgG group,and irradiation+CD25 group were given a single 20 Gy X-ray full chest irradiation.The mice in the irradiation+IgG group and irradiation+CD25 group were intraperitoneally injected with IgG antibody and CD25 antibody every week,respectively.Six mice were killed at 4 and 8 weeks after irradiation respectively.Flow cytometry was used to detect the percentage of CD25+Foxp3+Tregs in the lung tissue of each group of mice to identify whether the model was established successfully.Then,Western blot was used to detect the expression of neuropilin 1(NRP1)in the lungs of the mice in the control group and irradiation group.Immunofluorescence was used to detect the percentage of CD25+NRP1+Tregs.Photos were taken to observe the skin damage of each group of mice.Hematoxylin eosin staining was used to detect the pathological changes of lung tissue.The secretion of cytokines,namely,transforming growth factor-beta 1(TGF-β1),interleukin(IL)-17A,interferon-γ(IFN-γ),IL-2,and IL-4,in the lung tissue of each group was detected by enzyme-linked immunosorbent assay.Independent sample t-test was used for comparison between the two groups.Results The results of flow cytometry showed that the percentage of CD25+Foxp3+Tregs((1.73±0.04)%,(2.13±0.15)%)in the lung tissue of mice in the simple irradiation group was significantly higher than that in the control group((1.14±0.02)%,(1.70±0.06)%)(t=-26.680,-4.545;P=0.000,0.010)at 4 and 8 weeks.After the inhibition of Treg cells,the percentage of CD25+Foxp3+Tregs((0.72±0.14)%,(0.27±0.02)%)in the lung tissue of mice in the irradiation+CD25 group was significantly lower than that in the irradiation group alone(t=5.296,37.538;both P=0.000).The result of Western blot showed that the expression of NRP1 protein in the lung tissue of mice in the irradiation group increased significantly(t=-7.341,-9.127;both P=0.000).The results of immunofluorescence showed that the proportion of CD25+NRP1+Tregs in the lung tissue of mice in the irradiation group was higher than that in the control group at 4 and 8 weeks after irradiation.The proportion of CD25+NRP1+Tregs in the irradiation+CD25 group was lower than that in the irradiation group(t=8.926,14.457;P=0.001,0.000).The skin lesions observed were severe in the simple irradiation group.The skin in the irradiated+CD25 group was almost intact at 4 weeks,and hair removal and peeling still occurred at 8 weeks.Hematoxylin eosin staining showed that relative to that in the control group,the lung tissue structure of the mice in the irradiated group was destroyed,the alveolar wall was thickened,and the extracellular matrix was increased at 4 and 8 weeks after irradiation.The lung tissue of the irradiated+CD25 group was intact and the alveolar wall was slender.As indicated by the changes in inflammatory factors,relative to that in the control group,the secretion of IL-17A and IL-4 in the lung tissue of mice increased in the simple irradiation group(t=-8.492,-15.796;P=0.001,0.000)at 4 weeks.After 8 weeks of irradiation,the levels of TGF-β1 and IL-17A increased significantly(t=-11.072,-7.167;P=0.000,0.002),and the levels of IL-2 decreased at 4 and 8 weeks.IFN-γsecretion increased at 4 weeks(t=-27.393,P=0.000)and decreased at 8 weeks.Relative to that in the simple irradiation group,TGF-β1 and IL-17A decreased(t=6.037,4.524,5.496,4.772;all P=0.000),IFN-γincreased(t=-7.006,-12.565;P=0.002,0.000)at 4 and 8 weeks,and IL-2 and IL-4 decreased at 4 weeks and increased significantly at 8 weeks(t=2.866,-9.090,8.833,-7.191;all P=0.000)in the irradiation+CD25 group.Conclusion The differentiation of Tregs occurs in the lung tissue of mice with radiation-induced lung injury and promotes the development of radiation-induced lung injury by secreting TGF-β1 pro-inflammatory factor while interfering with the balance of helper T cell(Th)1/Th2 type cytokines.