Mechanism Of Radiation-induced Antigen Presentation Disorder In Treg-mediated Radiation-induced Pulmonary Fibrosis

Objectives:This study was based on radiation-induced abnormal activation of Treg to mediate radiation-induced pulmonary fibrosis,to investigate the effects of radiation on antigen-presenting cells and the mechanism of Teg-specific activation of Treg,and to clarify the regulation of radiation-induced antigen presentation dysfunction.T cells mediate EMT in lung epithelial cells and are involved in radiation pulmonary fibrosis.This study provides new ideas and experimental basis for the prevention and treatment of radiation-induced pulmonary fibrosis.Methods:Mouse primary regulatory T lymphocytes(Treg),conventional T lymphocytes(Tcon)and dendritic cells(DC)were sorted by immunomagnetic beads sorting.mouse immature dendritic cells were derived from in vitro cytokine induction experiments.Flow cytometry was used to determine the purity and cell markers.The source of cells and animals is 60CO-γ rays.In vivo animal experiments using lead brick partial shielding technology to construct changes in Treg and DC detected by mouse radioactive pulmonary fibrosis model.In vitro experiments were mouse primary cell co-culture techniques,and knockdown was performed by TSC1-specific siRNA and TSC1 plasmid construction.And overexpressing cell models to verify the mechanism by which TSC1 regulates DCs and specifically activates regulatory T cells.Results:The results of flow cytometry showed that the purity of mice primary cells sorted by immunomagnetic beads was 90%,and the sorting effect was good,which satisfied the subsequent experiments.Cytokine-induced bone marrow-derived dendritic cells(BMDC)meet the experimental requirements,and the positive cell rate of CD80,CD86 and MHC-Ⅱ after irradiation is 60%,showing a significant activation state.4Gy The percentage of Treg cells induced by co-culture with Tcon cells after irradiation with DC cells at 6Gy dose was 15%and 25%,respectively,which could significantly induce Treg cell differentiation.In vivo animal experiments showed that DC cells in the body showed significant activation after irradiation.qRT-PCR,Westernblot,immunofluorescence experiments showed that the expression of TSC1 decreased after DC irradiation,and TEC1 could be induced to induce Treg cell differentiation after knocking down TSC1.At the same time,overexpression and irradiation experiments showed that TSC1 normal expression and high expression could not induce Treg cell differentiation.Flow cytometry detection of CD80,CD86 and MHC-II after BMDC irradiation showed that DC secretion of cytokines after irradiation showed that IL-1β,IL,and MHC-Ⅱ were knocked down in TSC1 compared with the control group.The over expression plus irradiation group exhibited a significant activation state compared to the overexpression group.IL-2,IL-4,IL-6,IL-12,IFN-γ,TNF-α had a very significant increase in the content after irradiation compared with the control group,among which IL-1β,IL-6 The secretion was significantly increased after knocking down TSC1.There was no statistically significant change in the amount of secretion after overexpression of TSC1.The difference in the amount of overexpression and irradiation in the irradiated group compared with the control group,the irradiation group and the overexpression group was statistically significant..Flow cytometry results showed that IL-1β failed to induce Treg cell differentiation at different concentration gradients(positive cell expression rate was 4%).IL-6 induced an increase in Treg differentiation with increasing concentration,and the difference was statistically significant compared with the control group.We used flow cytometry to detect the expression of ZO-1 and N-cadherin in MLE-12 cells.The results showed that compared with the control group,the experimental group induced EMT in MLE-12 cells with the increase of DC addition.The ability to enhance EMT was most significant with a DC to TCON ratio of 1:1(positive expression rate was 3%),and Western blot results showed that the epithelial markers E-cadherin and Zo-1 were co-cultured.The increase of the proportion showed a significant downward trend,and the interstitial markers N-cadherin and Twist showed a significant upward trend with the increase of the co-culture ratio Immunofluorescence experiments showed that the expression of epithelial marker E-cadherin was significantly decreased,and the expression of the interstitial marker N-cadherin was significantly enhanced.Conclusion:1.radiation activated DC cell antigen presentation function.Treg is specifically activated by TSC1.2.TSC1 specifically regulates the expression of MHC-II and IL-6 in activated DCs,and further promotes EMT and participates in the pathogenesis of RPF.