Study On The Effects And Mechanisms Of Fibroblast Regulating The Plasticity Of Type 2 Innate Lymphocyte(ILC2) Via Notch3 In The Progression Of Silicosis

Objective:Silicosis is an occupational lung disease characterized by silicon nodules and diffuse pulmonary fibrosis,resulting from exposure to inhalable silica dust.The pathogenesis of silicosis is unknown and its pathological process is irreversible,which is a major global public health problem seriously threatening the physical and mental health of workers.Exploring a new mechanism can provide a scientific basis for preventing the occurrence and development of silicosis fibrosis.Innate lymphoid cells（ILCs）are a recently discovered tissue-resident cells enriched on the mucosal barrier surface of the body,and an increasing number of studies have revealed their important role in chronic lung diseases.In recent years,immune dysregulation in the local microenvironment of lung tissue is thought to accelerate the progression of fibrosis.In silicosis fibrosis,it is not clear whether ILCs are involved in the progression of fibrosis and how they regulate lung immunity.Therefore,this study aims to explore the mechanism of lung immune regulation from a new perspective with the change of immunological characterization of ILCs as the starting point,in order to provide new clues for preventing the occurrence and development of silicosis fibrosis.Methods:In this study,an experimental silicosis mouse model was constructed by instillating crystalline silica suspension into the trachea without exposure.Flow cytometry was used to describe the changes in immunological characterization of ILCs in silicosis model mice,and to explore the mechanism of the regulation of ILCs on pulmonary immunity in silicosis fibrosis.Flow cytometry was used to detect the changes in the proportion of ILC1and ILC2 in silicosis model mice at different time points during the course of fibrosis,and to explore the influencing factors,so as to explore the immune regulatory mechanism of ILCs in silicosis fibrosis.Lung ILC2 of CD45.1/2 mice was purified by Magnetic activated cell sorting（MACS）.The role of ILC2-ILC1 remodeling in silicosis fibrosis was verified by adoptive transfer and flow cytometry.The expression of IL-18Rαand IL-12Rβ2 in ILC2 remodeling in silicosis fibrosis was detected by flow cytometry in ST2^{e GFP/+}reporter mice.Using Il18^–/–mice,flow cytometry was used to verify the role of IL-18 in regulating ILC2-ILC1 remodeling in silicosis fibrosis.Immunofluorescence staining was performed on lung tissue sections of silicosis model mice to explore the spatial relationship between fibroblasts and ILC2 in fibrotic lung tissue.Mouse lung fibroblast line MLg（Mlg 2908）and primary mouse lung fibroblast cells were stimulated by matrix gel with different hardness.Immunofluorescence staining and Western blot were used to investigate the effect of activation of fibroblasts on the secretion of IL-18.Lung ILC2 extracted from MACS were cultured using conditioned medium of fibroblasts stimulated by matrix gel of different hardness.Flow cytometry was then used to detect the phenotypic changes of ILC2 in order to explore the regulatory role of activated fibroblasts in the remodeling of ILC2.The expression of Notch3 in fibroblasts of mice with silicosis fibrosis was detected by flow cytometry.Mouse lung fibroblast line MLg and primary mouse lung fibroblast cells were stimulated with matrix gel of different hardness in vitro,and the expression of Notch3 in fibroblast cells was detected by immunofluorescence staining and Western blot.Fibroblast specific Notch3 knockout mice were constructed,and the Notch3 gene was effectively knocked out by administering Tamoxifen（TAM）.Primary mouse lung fibroblasts were extracted and the knockout effect of Notch3 was verified by Western blot assay.Primary mouse lung fibroblasts with Notch3 knockout were stimulated with hard gel,and the effect of Notch3 knockout on the expression of pro-IL-18 was explored by immunofluorescence staining.The supernatant of cell culture was collected for protein purification,and the effect of Notch3 knockout on IL-18 secretion was detected by Western blot assay.Fibroblast specific Notch3 knockout mice were constructed and given TAM at different time points in the progression of fibrosis to achieve the time-specific knockout of fibroblast Notch3.Flow cytometry was used to detect the effect of fibroblast Notch3 knockout at different stages of fibrosis on the remodeling of ILC2.Macrophage-specific Notch3 knockout mice were constructed and given TAM at 3-4 weeks of silicosis modeling to complete the Notch3knockout in the initial stage of macrophage fibrosis.Flow cytometry was used to detect the effect of macrophage-specific Notch3 knockout on ILC2 remodeling.HE staining,Masson staining and q PCR were used to detect the effect of Notch3 gene knockout on the progression of silicosis fibrosis at different stages.Results:1.Pulmonary ILCs enhance immunological activity in silicosis fibrosis.Flow cytometry was used to detect the immunological characterization of lung ILCs in silicosis mice.The results showed that the proportion of lung ILCs and the number of lung cells increased significantly in silicosis fibrosis.The expression of lung ILCs cytokine receptor was changed,especially the expression level of IL-18Rαwas significantly increased.The expression level of lung ILCs resident related molecules was significantly increased.The expression of functional signaling molecules of lung ILCs and the secretion levels of cytokines were significantly changed,including decreased secretion of IL-13 and increased secretion of IFN-γ.（P<0.05）2.The proportion of pulmonary ILC1 in silicosis fibrosis increased and the proportion of ILC2 decreased.Flow cytometry was performed to detect the proportion of ILC1 and ILC2 in the lung in the condition of silicosis fibrosis.It was found that with the progression of fibrosis,the expression levels of phenotypic markers and nuclear transcription factors on cell surface significantly increased the proportion of ILC1 and decreased the proportion of ILC2,and the relative ratio of ILC1 to ILC2 gradually increased.Silicosis was modeled in Rag1^–/–mice,and flow cytometry showed that the proportion of ILC1 increased and the proportion of ILC2decreased with the progression of fibrosis.（P<0.05）3.The proportion of ILC1 and ILC2 in silicosis fibrosis is related to IL-18Rα.Flow cytometry was performed to detect IL-18Rαexpression levels of ILC1 and ILC2 in the lungs of silicosis model mice,and it was found that the change of the proportion of ILC1and ILC2 was correlated with the increase of IL-18Rαexpression level of ILC2.Further examination of silicosis model mice showed that there was no number of deaths of ILC2 in the lung,and almost no ILC2 in the lymph nodes.Fluorescently labeled CD45 antibody was injected into the caudal vein,and after 5 minutes of stable circulation in vivo,the samples were taken for flow detection.It was found that there were few lung ILC1 and ILC2 from circulation in mice with silicosis fibrosis,and there was no difference between them and the control group.（P<0.05）4.IL-18 mediates the remodeling of lung ILC2 to ILC1 in silicosis fibrosis.Using MACS technique and adoptive transfer experiment,flow cytometry demonstrated that lung ILC2 was remodeled into ILC1 during silicosis fibrosis,and almost all of the converted ILC1 expressed IL-18Rα.Silicosis modeling was performed on ST2^{e GFP/+}fluorescein mice.Flow cytometry was used to detect the correlation of IL-18Rαexpression during ILC2 remodeling,and the correlation of IL-12Rβ2 expression was excluded due to the absence of IL-12Rβ2 expression on ILC2.Flow cytometry of Il18^–/–mice modelled with silicosis showed that Il18 deletion resulted in an increase in the proportion of ILC2 and a decrease in the proportion of ILC1 in the lung.（P<0.05）5.Pulmonary ILC2 is commonly found around activated fibroblasts in silicosis fibrosis.Liposome treatment was given to silicosis model mice that had been constructed for 7 weeks to effectively reduce the pulmonary interstitial macrophages and alveolar macrophages.Flow cytometry showed that it could not effectively block the remodeling of lung ILC2 to ILC1 in the progression of silicosis fibrosis.Using the immunofluorescence staining technique of lung tissue,we found that lung ILC2 is commonly seen around activated fibroblasts in silicosis fibrosis.（P<0.05）6.Activated fibroblasts secreted more IL-18 mediating the remodeling of ILC2 to ILC1.Mouse lung fibroblast lines MLg and primary mouse lung fibroblast cells were stimulated by matrix gel with different hardness in vitro,andα-SMA expression was increased in the fibroblast cells cultured with hard gel by immunofluorescence staining and Western blots assay.The expression of pro-IL-18 and the secretion of IL-18 were detected.It was found that the expression of pro-IL-18 and the secretion of IL-18 were significantly increased in the fibroblasts activated by hard gel.Primary mouse lung fibroblasts were stimulated with matrix gel of different hardness in vitro,and the conditioned medium was collected to culture lung ILC2 extracted by MACS.ILC2 was collected for flow cytometry,and cells expressing the marker of ILC1 were found in lung ILC2.The transwell chamber was used to co-culture fibroblasts treated with matrix gel of different hardness and lung ILC2 at different time lengths.Flow cytometry was used to detect the phenotypic changes of ILC2.It was found that cells expressing the marker of ILC1 appeared in lung ILC2,and the expression level changed with the co-culture proportion and time length.（P<0.05）7.Notch3 signaling in activated fibroblasts promotes IL-18 secretion.Flow cytometry showed that the expression level of Notch3 in mouse lung fibroblasts increased significantly in silicosis fibrosis.Mouse lung fibroblast line MLg and primary mouse lung fibroblasts were stimulated by matrix gel with different hardness in vitro.Immunofluorescence staining showed that the expression of Notch3 was significantly increased in activated fibroblasts.The fibroblast specific Notch3 knockout mice were effectively knocked out by intraperitoneal injection of TAM,and the primary fibroblasts were extracted.After stimulation by hard glue in vitro,immunofluorescence staining and Western blot detection,it was found that fibroblast specific knockout Notch3 could effectively reduce the expression of pro-IL-18 and secretion of IL-18.（P<0.05）8.Fibroblast Notch3 signaling regulates the remodeling of lung ILC2 in silicosis fibrosis.Fibroblast specific Notch3 knockout mice were treated with TAM at different time points to complete the Notch3 knockout at the initial stage of fibrosis（3-4 weeks after silicosis modeling）and the advanced stage of fibrosis（7-8 weeks after silicosis modeling）.Flow cytometry showed that fibroblast specific knockout of Notch3 could effectively block the remodeling of ILC2-ILC1 in fibrosis progression.（P<0.05）9.Specific knockout of Notch3 in fibroblast can delay silicosis fibrosis.Fibroblast specific Notch3 knockout mice were treated with TAM at different time points to knock out fibroblast Notch3 at the initial stage of fibrosis（3-4 weeks for silicosis modeling）and at the advanced stage of fibrosis（7-8 weeks for silicosis modeling）.HE and Masson staining and q PCR detection showed that specific knockout of fibroblast Notch3 in the early stage of fibrosis could reduce inflammatory infiltration,collagen deposition,and the transcription levels of fibrosis indicators Col1 and Fn in mice.（P<0.05）Conclusion:1.The number and immunological activity of pulmonary ILCs in silicosis fibrosis are increased,with the proportion of ILC1 increased and the proportion of ILC2 decreased associating with IL-18Rα.2.The secretion of IL-18 by fibroblasts regulates the remodeling of lung ILC2 to ILC1during the fibrosis of silicosis.3.Fibroblast Notch3 signals regulate lung ILC2 remodeling and affect the progression of silicosis fibrosis.