Studies On The Mechanism Of Radiation-induced Lung Injury By Single Cell Transcriptome Sequencing

Radiation-induced lung injury(RILI)is a complication of clinical chest tumor radiotherapy and bone marrow transplantation pretreatment,which is characterized by progressive destruction of lung tissue and deterioration of lung function,resulting in respiratory failure and death.RILI includes two stages,namely,early reversible radiation interstitial pneumonia(RIIP)and later radiation-induced pulmonary fibrosis(RIPF).There are obvious species and individual differences in the occurrence and development of RIPF,while the underlying mechanisms has not been clarified,so it is of great significance to study the mechanism of species difference of RILI furtherly so to find new targets for prevention and treatment.In this study,the fibrosis-prone and fibrosis-resistant mouse model was established,and the single-cell map of dynamic multicellular ecosystem of RILI was constructed by single-cell transcriptome and spatial transcriptome sequencing techniques to find the cell subsets related to RIPF species difference,and to explore its role in RILI.Materials and methods 1.To construct single-cell map of dynamic multicellular ecosystem of RILI by single cell transcriptional and spatial transcriptional sequencing:(1)Establishment and phenotypic study of RIPF animal model:male C57BL/6N mice and C3H/He N mice were exposed toγray at a dose of 20 Gy on the chest.Before irradiation and 4,14,24 weeks after irradiation,respiratory function,lung CT imaging,pulmonary histopathology,collagen content and cytokines of lung were detected to determine the difference of RILI process between the two kinds of mice.(2)Single cell transcriptome sequencing and data analysis:single cell suspension of lung was prepared for 10X single cell transcriptome sequencing and data analysis.Cell cluster analysis and subgroup identification,subgroup cell number change analysis,cell radiosensitivity analysis,signal pathway activity analysis and cell-cell interaction were analyzed.Sections of the lung of C57BL/6N mice and C3H/He N mice were prepared before and 14 weeks after irradiation.After tissue permeability and PCR reaction,visium spatial gene expression library was sequenced by standard short reading length sequencer,and the data were analyzed by Space Ranger analysis software.2.Studies on the role of Csf3r~+CD14~+Neu in RILI:flow cytometry and immunofluorescence were used for localization and quantitative verification of Csf3r~+CD14~+Neu.Csf3r~+CD14~+Neu and Csf3r~-CD14~+Neu were sorted by flow cytometry and co-cultured with fibroblasts and macrophages.The proliferation of fibroblasts and macrophages was detected by Edu,the migration of fibroblasts was detected by Transwell migration assay,the expression ofα-SMA was detected by immunofluorescence,and the contents of TGF-β,TNF-αand IL-10 in Neu and macrophage were detected by radioimmunoassay.Results 1.The differences of RILI progression between C57BL/6N mice and C3H/He N mice were as follows:(1)The imagine of chest by CT scanning showed that,24 weeks after irradiation,the lung volume and average lung density of C57BL/6N mice and C3H/He N mice were significantly higher than those of the control group,and the increase of C57BL/6N mice was more significant.(2)The results of respiratory function found that PIF,PEF,TV and MV of the two kinds of mice decreased compared with the control group,and the above indexes of C57BL/6N mice decreased more significantly than those of C3H/He N mice at 4,14 and 24 weeks after irradiation.(3)Pathological changes of lung showed that there were mainly interstitial inflammatory changes in the lung tissue of C3H/He N mice at 4,14 and 24 weeks after irradiation,while in the lung of C57BL/6N mice,cell hyperproliferation was found at 14 weeks after irradiation,and local fibrosis appeared at 24 weeks after irradiation.(4)The content of hydroxyproline and typeⅠcollagen in lung tissue of C57BL/6N mice increased significantly 24 weeks after irradiation,but there was no significant change in lung tissue of C3H/He N mice.(5)At 4,14 and 24 weeks after irradiation,the contents of TNF-α,TGF-β,CTGF,IL-1,IL-1RA and IL-4 in lung tissue increased in both kinds of mice,and the increase in C57BL/6N mice was more significant than those in C3H/He N mice.2.The results of single cell transcriptome sequencing analysis were as follows:Lung tissue cells were divided into 56 groups and 14 main cell types,mainly alveolar epithelial cells,macrophages,T lymphocytes,fibroblasts and granulocytes.The quantitative changes of cell subsets and the activity of signal pathway were different in the RILI process of the two kinds of mice,and the difference of Csf3r~+CD14~+Neu was the most significant in RIPF of the two kinds of mice.3.The results of spatial transcriptome analysis clarified the spatial distribution of epithelial cells,endothelial cells,macrophages,granulocytes and fibroblasts in lung tissue and their changes after irradiation.The spatial location and distribution map of lung tissue cells of C57BL/6N and C3H/He N mice before and after irradiation were constructed.4.The role of Csf3r~+CD14~+Neu in RILI:(1)The number and location of Csf3r~+CD14~+Neu changed in the process of RILI.14 and 24 weeks after irradiation,Csf3r~+CD14~+Neu in lung tissue increased significantly,and the increase of Csf3r~+CD14~+Neu in lung tissue of C57BL/6N mice was more significant than that of C3H/He N mice.Most of the increased Csf3r~+CD14~+Neu were located near the pleura and coexisted with LFB in fibrotic niches.(2)The contents of TGF-βin Csf3r~+CD14~+Neu and supernatant were significantly higher than those in Csf3r~-CD14~+Neu.(3)Csf3r~+CD14~+Neu had no effect on the proliferation of lung fibroblasts and M(?),but could promote the activation of LFB.Csf3r~-CD14~+Neu could promote the migration of LFB.(4)After cocultured with Csf3r~+CD14~+Neu,the content of TGF-βin RAW264.7 M(?)increased,while the content of IL-10 decreased.Conclusion:1.Based on sc RNA-seq and spatial transcriptome sequencing techniques,a landscape dynamic single cell map of RILI was constructed.Differences were found in cell subsets and related genes and pathways in lung of fibrosis-prone and fibrosis-resistant mice in different periods after irradiation.2.The increase of Csf3r~+CD14~+Neu was most significantly in the process of RILI,and its number in fibrosis-prone mice was higher than that in fibrosis-resistant mice.Csf3r~+CD14~+Neu highly expressed pro-fibrogenic cytokines,and promoted the activation of LFB and the expression of pro-fibrogenic cytokines in M(?),which might be a fibrogenic cell subset.