The Roles And Mechanisms Of Regulatory T Cells(Tregs) In Radiation-induced Pulmonary Fibrosis

Thoracic radiotherapy is one of the common therapeutic measurement for treating lung cancer, esophageal cancer, breast cancer, and lymphomas. However, its use is hampered by the moderate sensitivity of the lungs to irradiation, with resultant complications of radiation-induced pneumonitis and lung fibrosis. Symptoms of radiation pneumonitis, such as low-degrade fever, mild cough, usually occur early stage(within 3 months) after irradiation. Pulmonary fibrosis would appear late stage(after 6 months) after irradiation, and is diagnosed as excessive fibroblast proliferation, collagen and extracellular matrix deposition. Patients suffered from lung fibrosis have severe chronic respiratory failure and ultimately died. Fibrosis with high prevalence, low predictability, hard treatment, and even higher mortality than the primary cancer, seriously limits the clinical application of radiotherapy. Therefore, revealing the mechanism of the development of radiation-induced pulmonary fibrosis, as well as providing important theoretical basis for the prevention or alleviation of radiation-induced pulmonary fibrosis, are of important significance. Our previous study showed that during in the process of radiation-induced pulmonary fibrosis, regulatory T lymphocytes(regulatory T cells, Tregs) within the lung increased, leading to the immune balance toward Treg migration. Tregs,as subsets of CD4+ T cells, play an immune suppressive role in the maintenance of immune tolerance and homeostasis. Evidence showed that Tregs is more tolerant to irradiation than other T cells. Some evidence also reported that Tregs are involved in idiopathic pulmonary fibrosis(IPF), and a variety of fiber proliferative diseases. This study for Treg in the role of radiation pulmonary fibrosis, provide new clues for the treatment. Fibroblast as the key effector cells in pulmonary fibrosis, secrete the collagen with excessive deposition, which is the major contributor of pulmonary fibrosis. Existing research shows that in pathological conditions, in addition to the primary focal hyperplasia, differentiation of bone marrow-derived circulating fiber cells(circulating fibrocyte) and transitions of the epithelial cells to alveolar epithelial mesenchymal(Epithelia mesenchymal transitions, EMT) are also important source of fibroblasts, which involved VIII in the various fiber proliferative disease onset. Fibrocyte cells as bone marrow precursor cells that can secrete collagen and present in antigen. Epithelial-mesenchymal transitions(EMT) are involved in important biological process related to epithelial cell polarity disappearance, cytoskeletal rearrangement, and mesenchymal cells related characteristics obtaining. Research has shown that fibrocyte differentiation and EMT are regulated by the variety of immune factors, and associated with the immune balance. Tregs are of significance to maintain the immune balance, therefore potential to regulate fibrocyte differentiation and the process of EMT. This paper aims to study the changes, specific roles and potential mechanisms of Tregs in radiation-induced lung fibrosis, providing the effective targets in prevention or mitigation of radiation-induced pulmonary fibrosis. The related experimental work according to the main content is as follows: 1. In this study, C57BL/6 mice were thoracically irradiated with 20 Gy 60 Co γ-ray to establish the mouse model of radiation-induced pulmonary fibrosis. The results showed series of changes that weight decreasing, white blood cells(WBC) plummeting, and pathologic changes of lung tissues emerging in irradiated mice. HE staining revealed that alveolar exudate appearred, bronchial epithelial cell shedded 2 weeks after irradiation. After exposure to irradiation 1 month to 6 months, alveolar structure changed, and alveolar fused and collapsed, fibroblasts around peribronchial increased, the symptom of fibrosis was gradually severed. Masson trichrome staining also revealed pulmonary interstitial fibers and collagen deposition after irradiation 1 month until 6 months. Moreover, the total collagen content in the lung gradually increased from 1 month after exposure to 6 months, showing evident time dependent increase. The results via method of fluorescence activated cell sorting(FACS) showed that the percent of CD4+CD25+ Tregs to T cells in peripheral blood was reduced in 1 week after irradiation, then recovered to normal level 14 days post irradiation. Tregs in lung tissues showed continuous rise from 14 days to 6 months after irradiation. 2. To clarify the specific role of Tregs in radiation-induced pulmonary fibrosis, Treg depletion model was also established. We generated Treg-depletion mice by intraperitoneal injection of anti-CD25 antibody. The results via method of FACS showed that Tregs in peripheral blood was effectively inhibitted, Tregs in lung tissues was reduced and comparable to normal levels. We further investigated the regulation of Tregs on radiation-induced pulmonary fibrosis. After Treg depletion, irradiated mice showed intact alveolar structure, weakened alveolar fusion, and reduced lung collagen distribution compared with irradiated mice without Treg depletion via methods of HE and Masson stanining, showing that the symptoms of fibrosis were alleviate, and that may be regulated by Treg depletion. Those findings suggest that Tregs may promote the development of pulmonary fibrosis. 3. Tregs influencied in CD4 + T cells and Th1 / Th2 balance to participate in the pathogenesis of radiation pulmonary fibrosis. The results of CD4 + T cells and Th17 cells in the lungs examined via FACS revealed that the percent and number of CD4 + T cells and Th17 cells reduced in the early irradiation stage, but increased in the late irradiation stage, showing the similar tendency of CD4 + T cells and Th17 cells in radiation-induced lung fibrosis. After Treg depletion, the percent of number of CD4+ T cells and Th17 cells in T cells, increased compared with normal and irradiated mice without injection with CD25 antibody. The levels of Th1 / Th2 type cytokines that were related to Tregs were assayed through using a suspension microarray Luminex. The results showed that Th1-type cytokines IFN-γ, IL-12, IL-2, IL-1α and Th2 cytokines IL-6, IL-10, IL-4 and IL-5 expression increased, suggesting that the balance of Th1 / Th2 cytokines was in disorder after exposure to irradiation. After Treg depletion, Th1-type cytokines increased, but Th2-type cytokines decreased in mice with Treg depletion after irradiation compared with non-irradiation group mice and other 2 irradiated group mice. Hence, Th1 / Th2 balance was upset in favor to Th1-type cytokines. These findings suggest that Tregs may promote lung fibrosis through decreasing Th17 cell response or shifting Th1/Th2 balance, providing an important mechanism of Tregs regulating radiation-induced pulmonary fibrosis. 4. Tregs regulated fibrocyte differentiation to participate in the pathogenesis of radiation pulmonary fibrosis. We used FACS to detect the number of fibrocyte cells. The results in animal models showed a reduction in the number of fibrocyte within 2 weeks after irradiation, but a consistent increase from 14 days to 6 months after irradiation, which was similar to the change of Tregs in response to irradiation. After Treg depletion, the number of fibrocyte in lung tissues of mice was decreased with Treg depletion after irradiation compared with irradiated mice without Treg depletion, though fibrocyte number in irradiated mice with Treg depletion was comparable to non-irradiation group mice. Moreover, we examined the chemokines CCL2 and CCL3 content via Luminex, the results described that these two chemokines content were significantly increased in mice after irradiation, and consistent with the change of fibrocyte. Then CCL2 and CCL3 cytokine expression were reduced in irradiated mice with Treg depletion, but still higher than non-irradiated mice. The results showed that Tregs may accelerate radiation-induced pulmonary fibrosis through stimulating fibrocyte gathered in the lungs, and this effect may be mediated by CCL2 and CCL3 chemokines. To test this inference, we isolated mouse spleen Tregs and mononuclear cells and co-cultured these cells for 14 days, then examined the percent of CD45 + COLⅠ + fibrocyte in mononuclear cells by FACS. It has been reported that mononuclear cells can differentiate into fibrocyte in vitro. The results showed that the percent of CD45 + COLⅠ + fibrocyte of mononuclear cells in co-culture system was increased than mononuclear cells cultured alone, suggesting that Tregs can promote fibrocyte differentiation. Also, the CCL2 and CCL3 chemokine content increased in the supernatant of co-culture cells compared with monoclear cells cultured alone. These findings prove the mechanism that fibrocyte gathering and differentiation in the lungs via CCL2/CCL3 by Tregs may be mechanism in radiation-induced pulmonary fibrosis. 5. Tregs regulated epithelial cells EMT through β-catenin to participate in the pathogenesis of radiation pulmonary fibrosis. We examined the occurrence of epithelial mesenchymal transition(EMT) by immunofluorescence and the expression of EMT-related β-catenin by Immunohistochemistry in epithelial cells of lung tissue in animal models. The results showed that the alveolar epithelial cells obtained stromal cells markers N- cadherin(N-cadherin) and reduced epithelia cells markers pro-sufactant protein C(SPC) in the late stage of pulmonary fibrosis after irradiation, indicating that EMT enhanced in the process of radiation-induced pulmonary fibrosis. Inhibition of Treg after irradiation led to decreased N- cadherin expression as well as reduced SPC loss in lung epithelial cells, suggesting weaker EMT. Furthermore, the β-catenin expression in epithelial cell cytoplasm and nucleus increased from 1 month to 6 months after irradiation. Then abnormal β-catenin expression in lung epithelial cells of mice reduced in response to Treg depletion after irradiation compared with irradiated mice without Treg depletin, but increased than normal mice without irradiation. Thus, Tregs may promote EMT in the development of radiation-induced pulmonary fibrosis via β-catenin in epithelial cells. To explain this inference, we co-cultured Tregs that were isolated from mice spleen with irradiated mice lung epithelial cells(MLE12) in vitro. The percent of E-cadherin + Fibronectin + MLE12 cell measured by FACS showed that the occurrence of EMT from rate of 9.3% in culture of irradiated MLE12 to rate of 13.2%(p<0.05) in co-culture of irradiated MLE12 with Tregs. In addition, β-catenin expression measured by western-blot in irradiated MLE 12 cells that was co-cultured with Tregs was also increased than irradiated MLE12 cells cultured alone. The β-catenin si RNA to silence MLE12 cells effectively down-regulated the percent of E-cadherin + Fibronectin + MLE12 cells(14.3%) in β-catenin deficient MLE 12 cells that was co-cultured with Tregs than that(16.2%, p<0.05) normal MLE 12 cells that was irradiated and co-cultured with Tregs, suggesting that Tregs can promote the occurrence of EMT that is mediated in part by the β-catenin. These findings prove that β-catenin-mediated EMT might be another mechanism that Tregs promote the development of radiation-induced pulmonary fibrosis. In conclusion, we successfully established a mouse model of radiation-induced pulmonary fibrosis. Tregs were involved in the development of radiation-induced pulmonary fibrosis. And, Tregs may accelerate the process of radiation-induced pulmonary fibrosis by reducing Th17 cell response, modulating Th1/Th2 balance, accelerating fibrocyte accumulating and differentiation through CCL2/CCL3, as well as promoting β-catenin-mediated EMT in lung tissues. Therefore, This research provides an important reference for the use of the inhibition of Treg measures to alleviate and treat radiation-induced pulmonary fibrosis. Key words: Tregs; Th1/Th2; fibrocyte; EMT; β-catenin; Radiaton-induced pulmonary fibrosis.