To investigate the effects of the differentiation of regulatory T cells (Tregs) on radiation-induced lung injury and its mechanism.

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 samplet-test was used for comparison between the two groups.

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-γ secretion 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-γ increased (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.

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.

T-lymphocytes, regulatory; T-lymphocytes, helper-inducer; Models, animal; Th cell balance; Radiation-induced lung injury