Study On The Mechanisms Of STING Regulating The Activation Of Alveolar Macrophages And Lung Fibroblasts Involved In The Silicosis Process

Objectives:Silicosis is a serious and fatal occupational pulmonary disease caused by long term inhalation of free silica dust.Recent studies have shown that the activation of STING signal pathway plays a crucial role in the progression of silicosis,but the specific regulatory mechanism remains unclear.In this study,STING protein was taken as the research target to explore its key role in regulating the progression of silicosis by affecting the activation of alveolar macrophages and lung fibroblasts.This will provide effective help to determine the potential pathogenesis of silicosis and explore new treatment strategies for silicosis.Methods:In this study,a mouse silicosis model was constructed by non-exposed bronchial perfusion of silica suspension.dsDNA content in mouse alveolar lavage fluid was determined using the Quant-iTTMPicoGreen? dsDNA kit.The expression level of STING in mouse lung tissues was detected by Western Blot and RT-PCR.Then,STING protein silencing model of mouse lung tissue was constructed by non-exposed bronchial perfusion of adeno-associated virus type 6 carrying related gene sequence.Mice were randomLy divided into four groups according to body weight:AAV-sh-NC+S aline,AAV-sh-NC+CS,AAV-sh-STING+Saline,and AAV-sh-STING+CS.dsDNA content in mouse alveolar lavage was determined using the Quant-iTTMPicoGreen? dsDNA kit.Western Blot was used to detect the expression levels of STING,IRF3,P-IRF3,IκBα,P-IκBα,Fn,Col-1 and a-SMA in mouse lung tissue.The secretion levels of TNF-α,IL-6,and TGF-β in mouse alveolar lavage fluid were determined by ELISA.T cells expressing IFN-γ(Th1),IL-17A(Th17),and Foxp3(Treg)in mouse hilar lymph nodes and spleen were measured by flow cytometry.H&E staining was used to observe the inflammatory cell infiltration in mouse lung tissue.The collagen deposition of mouse lung tissue was observed by Masson staining.The expressions of Fn and Col-1 in mouse lung tissues were observed by immunohistochemistry.Through the above experiments,the protective effect of targeted silencing STING protein on mouse lung tissue inflammation and fibrosis caused by exposure to silica dust was proved.In vitro,normal alveolar macrophage cell lines were divided into two groups:Saline group and CS group(50 μg/cm2)stimulated for 12,18,and 24 h.dsDNA content in cell supernatant was determined by Quant-iTTMPicoGreen? dsDNA kit.The expression of STING and its downstream proteins in alveolar macrophages was detected by Western Blot.The gene levels of STING,IRF3,and IFN-β in alveolar macrophages were detected by RT-PCR.The secretion levels of TNF-α,IL-6,TGF-β,and IFN-β in the supernatant were detected by ELISA.We then used lentiviruses to silence STING proteins in alveolar macrophages.The alveolar macrophage cell lines transfected with lentivirus were divided into the following four groups:sh-NC+Saline,sh-NC+CS,sh-STING+Saline,and shSTING+CS,and cultured for 12,18,and 24 h.The expression of STING and its downstream proteins in alveolar macrophages was detected by Western Blot.The gene levels of STING,IRF3,and IFN-β in alveolar macrophages were detected by RT-PCR.The secretion levels of TNF-α,IL-6,TGF-β,and IFN-β in the supernatant were determined by ELISA.Subsequently,we established a co-culture system between the cell supernatant(18 h)of alveolar macrophages of each group in the above experiments and lung fibroblasts(MLg[Mlg 2908]).The expression levels of STING,Fn,Col-1 and α-SMA in lung fibroblasts were detected by Western Blot and RT-PCR.The expression levels of IRF3,PIRF3,IκBα,and P-IκBα in lung fibroblasts were determined by Western Blot.The migration ability of lung fibroblasts was observed by cell migration assay.According to the above experimental results,the stimulation of silica dust could effectively activate the STING signal pathway of alveolar macrophages and secrete a series of pro-inflammatory and pro-fibrosis factors,which could effectively promote the proliferation and activation of lung fibroblasts to a large extent,and then produce the subsequent fibrosis effect.Moreover,targeted inhibition of the STING protein expression in alveolar macrophages could effectively inhibit the occurrence of these responses.In addition,normal lung fibroblast lines were divided into two groups:Saline group and TGF-β1 group(10 ng/mL)and treated for 48 h respectively.The expressions of STING,IRF3,P-IRF3,IκBα,P-IκBα,Fn,Col-1 and α-SMA in lung fibroblasts were detected by Western Blot.Subsequently,lentivirus transfection was used to silence the expression of STING protein in lung fibroblasts.Lung fibroblasts transfected with lentivirus were divided into four groups:sh-NC+Saline,sh-NC+TGF-β1(10 ng/mL),sh-STING+Saline,and sh-STING+TGF-β1(10 ng/mL).dsDNA content in lung fibroblast culture supernatant was determined using Quant-iTTMPicoGreen? dsDNA kit.The expression levels of STING,IRF3,P-IRF3,IκBα,P-IκBα,Fn,Col-1 and α-SMA in lung fibroblasts were detected by Western Blot.The gene expression levels of STING,Fn,Col-1 and α-SMA in lung fibroblasts were detected by RT-PCR.The migration ability of lung fibroblasts was observed by cell migration assay.The above experimental results indicated that STING signal pathway was involved in the activation of lung fibroblasts induced by TGF-β1 and the production of pro-fibrotic protein.Results:1.Silica dust exposure induced the release of dsDNA and the expression of STING protein in mouse lung tissue.Quantitative detection results of dsDNA showed that compared with mice in Saline group,dsDNA content in alveolar lavage fluid of mice in CS group was significantly increased,and the difference was statistically significant(P<0.05).Western Blot and RT-PCR results showed that STING protein and gene expression levels in lung tissue of mice in Saline group were significantly increased,with statistical significance(P<0.05).dsDNA content showed a strong positive correlation with STING protein expression.These results indicated that STING signal pathway might play a key role in the regulation of pulmonary inflammation and fibrosis induced by silica dust.2.Silencing STING protein in mouse lung tissue could block the activation of STING signal pathway caused by silica dust exposure.dsDNA test results showed that compared with AAV-sh-NC+S aline group,dsDNA content in alveolar lavage fluid of mice in AAV-sh-NC+CS group was significantly increased,with statistical significance(P<0.05).Compared with AAV-sh-NC+CS group,dsDNA content in alveolar lavage fluid of mice in AAV-sh-STING+CS group was significantly decreased,and the difference was statistically significant(P<0.05).Western Blot results showed that compared with AAV-sh-NC+Saline group,the expression levels of STING,P-IRF3,and P-IκBα in the lung tissue of mice in AAV-sh-NC+CS group were significantly increased.The difference was statistically significant(P<0.05),but there was no significant difference in the expressions of total protein IRF3 and IκBα.Compared with AAV-shNC+CS group,the results showed that the expression levels of STING,P-IRF3,and PIκBα in lung tissue of mice in AAV-sh-STING+CS group were significantly decreased,with statistical significance(P<0.05),while the expressions of total proteins IRF3 and IκBα were not changed.ELISA results showed that compared with mice in AAV-shNC+S aline group,the secretion of TNF-α,IL-6,and TGF-β in alveolar lavage fluid of mice in AAV-sh-NC+CS group was significantly increased,with statistical significance(P<0.05).Compared with AAV-sh-NC+CS group,the secretion of TNF-α,IL-6,and TGF-β in AAV-sh-STING+CS group was significantly reduced,with statistical significance(P<0.05).The above results indicated that STING protein silencing in lung tissue of mice could effectively inhibit the activation of STING signal pathway caused by silica dust exposure.3.STING protein was involved in the regulation of T cell differentiation in mouse hilar lymph nodes and spleen tissue.No matter in hilar lymph nodes or spleen tissue,the proportion of Th1,Th17 and Treg in mice of AAV-sh-NC+Saline group significantly increased in silicosis inflammation stage(7,14 days)and fibrosis stage(56 days),compared with that of AAV-sh-NC+Saline group.Compared with AAV-sh-NC+CS group,the proportion of Th1,Th17 and Treg in AAV-shSTING+CS group was significantly decreased,the difference was statistically significant(P<0.05).4.Silencing STING protein in mouse lung tissue could improve lung tissue inflammation and fibrosis caused by silica dust exposure.Giemsa staining showed that silica dust caused the increase of inflammatory cell infiltration in mouse lung tissue,and STING protein silencing in mouse lung tissue could greatly alleviate the increase.The results of H&E staining showed that silica dust caused increased inflammation of mouse lung tissue,and silencing STING in mouse lung tissue could greatly relieve the inflammatory lesions.Masson staining results showed that in the early stage of silica dust exposure(days 7 and 14),silica dust did not cause significant collagen deposition in lung tissue;however,in the evening stage of silica dust exposure(days 56),serious collagen deposition occurred in the lung tissue of mice in the silicosis group,and the silencing of STING protein in the lung tissue of mice could effectively reduce collagen deposition.Western Blot results showed that there were no differences in the expressions of Fn,Col-1 and α-SMA in the lung tissues of all groups at 7 and 14 days(inflammatory stage).However,at day 56(fibrosis stage),we found that silica dust exposure significantly induced the expressions of Fn,Col-1 and α-SMA and STING protein silencing could inhibit this increase,with statistical significance(P<0.05).The immunohistochemical experiments of Fn and Col-1 also showed the same results.These results indicated that targeted silencing STING protein of mouse lung tissue had a positive protective effect on lung tissue inflammation and fibrosis caused by silica dust exposure.5.Silica dust exposure could induce the release of dsDNA and the activation of the STING signal pathway of the alveolar macrophages.Compared with Saline group,dsDNA content in the supernatant of alveolar macrophages in CS group was significantly increased,with statistical significance(P<0.05).Western Blot and RT-PCR results showed that compared with Saline group,the expression levels of STING,P-IRF3,P-IκBα,and IFN-β of alveolar macrophages in CS group were significantly increased,with statistical significance(P<0.05).ELISA results showed that the secretion levels of TNF-α,IL-6,TGF-β,and IFN-β of alveolar macrophages in CS group were increased,and the differences were statistically significant(P<0.05).dsDNA level was strongly positively correlated with the overexpression of STING,P-IRF3,PIκBα,TNF-α,IL-6,and IFN-β.These findings suggested that silica dust exposure might induce the release of dsDNA to activate the STING signal pathway of alveolar macrophages,thus producing a variety of pro-inflammatory and pro-fibrotic factors leading to inflammation and fibrosis.6.Silencing STING protein in alveolar macrophages could effectively block the activation of STING signal pathway of alveolar macrophages caused by silica dust exposure.Western Blot results showed that,compared with the sh-NC+Saline group,the protein expressions of STING,P-IRF3,P-IκBα,and TGF-β in alveolar macrophages of the shNC+CS group were significantly increased,while the expression levels of various proteins in the alveolar macrophages of the sh-STING+CS group were significantly decreased.The difference was statistically significant(P<0.05).There was no difference in the expression of total protein IRF3 and IκBα.RT-PCR results showed that the gene expression of STING,IRF3,and IFN-β in alveolar macrophages of each group was consistent with the protein expression.ELISA results showed that compared with the sh-NC+Saline group,the secretion levels of TNF-α,IL-6,TGF-β,and IFN-β in the supernatant of alveolar macrophages in the sh-NC+CS group were significantly increased,while the secretion levels of various cytokines in the sh-STING+CS group were significantly decreased.The difference was statistically significant(P<0.05).7.Silencing STING protein in alveolar macrophages could effectively inhibit the activation of pulmonary fibroblasts and the generation of fibrosis effect mediated by pulmonary fibroblasts.Western Blot and RT-PCR results indicated that compared with sh-NC+Saline group,the protein and gene expression levels of Fn,Col-1 and α-SMA in pulmonary fibroblasts treated with alveolar macrophage supernatant in the sh-NC+CS group were significantly increased.The protein and gene expression levels of Fn,Col-1 and α-SMA in pulmonary fibroblasts treated with alveolar macrophage superneant in sh-STING+CS group were significantly decreased,with statistical significance(P<0.05).The results of cell migration experiment showed that the migration ability of pulmonary fibroblasts treated with the supernatant of alveolar macrophages in the sh-NC+CS group was significantly enhanced compared with the control group,while the migration ability of pulmonary fibroblasts treated with the supernatant of alveolar macrophages in the sh-STING+CS group was significantly weakened.These results indicated that many cytokines secreted by silica dust treated alveolar macrophages could lead to the activation of lung fibrocytes and the generation of fibrosis effect mediated by pulmonary fibroblasts,which were accompanied by the involvement of STING signal pathway of alveolar macrophages.8.The STING driven alveolar macrophage factor induced by silica dust could effectively activate the STING signal pathway of lung fibroblasts.Western Blot results showed that compared with sh-NC+Saline group,the protein expression levels of STING,P-IRF3,and P-IκBα in pulmonary fibroblasts treated with alveolar macrophage supernatant in the sh-NC+CS group were significantly increased.The protein expression levels of STING,P-IRF3,and P-IκBα in pulmonary fibroblasts treated with alveolar macrophage supernatant in sh-STING+CS group were significantly decreased,with statistical significance(P<0.05).STING was positively correlated with the overexpression of P-IRF3,P-IκBα,α-SMA,Fn and Col-1.9.TGF-β1 activated the STING signal pathway and led to a series of fibrosis effects of lung fibroblasts.Western Blot results showed that the protein expression levels of STING,P-IRF3,P-IκBα,Fn,Col-1 and α-SMAin lung fibroblasts treated with TGF-β1 were significantly increased,with statistical significance(P<0.05).Moreover,the expression of STING was strongly correlated with the expression of Fn,Col-1 and α-SMA.These results strongly suggested that TGF-β1 might mediate the pro-fibrosis response by activating the STING signal pathway of lung fibroblasts.10.The silencing of STING protein in lung fibroblasts could effectively inhibit the activation of STING signal pathway and the generation of lung fibroblasts-mediated fibrosis effect caused by TGF-β1.Western Blot results showed that compared with sh-NC+Saline group,the protein expression levels of STING,P-IRF3,P-IκBα,Fn,Col-1 and α-SMA in lung fibroblasts of sh-NC+TGF-β1(10 ng/mL)group were significantly increased.The difference was statistically significant(P<0.05).Compared with sh-NC+TGF-β1(10 ng/mL)group,the protein expression levels of STING,P-IRF3,P-IκBα,Fn,Col-1 and α-SMA in lung fibroblasts in sh-STING+TGF-β1(10 ng/mL)group were significantly decreased.The difference was statistically significant(P<0.05).The gene expressions of STING,IRF3,Fn,Col-1 and α-SMA in lung fibroblasts were detected by RT-PCR,and the results were consistent with those of the proteins.Cell migration experiment showed that compared with sh-NC+Saline group,the migration ability of lung fibroblasts in sh-NC+TGF-β1(10 ng/mL)group were significantly enhanced.Compared with sh-NC+TGF-β1(10 ng/mL)group,the migration ability of lung fibroblasts in sh-STING+TGF-β1(10 ng/mL)group were significantly decreased.dsDNA assay results of Quant-iTTMPicoGreen? showed that dsDNA content in lung fibroblasts supernatant of sh-NC+Saline group was significantly increased compared with that of sh-NC+Saline group.Compared with sh-NC+TGF-β1(10 ng/mL)group,dsDNA content in lung fibroblast supernatant of sh-STING+TGF-β1(10 ng/mL)group was significantly reduced.The difference was statistically significant(P<0.05).dsDNA content and STING had strong positive correlation with Fn,Col-1 and αSMA overexpression.These results suggested that STING was also a key factor in the fibrogenic effect of lung fibroblasts.Conclusion:1.STING signal pathway could regulate the activation of alveolar macrophages to promote the occurrence and development of inflammation and fibrosis caused by silica dust by secreting various proinflammatory and pro-fibrotic factors.2.The STING signal pathway and the pro-inflammatory and pro-fibrotic factors secreted by alveolar macrophages mediated by STING signal pathway could regulate the activation of lung fibroblasts,resulting in subsequent fibrosis effects.3.STING signal pathway might participate in the occurrence and development of inflammation and fibrosis in silicosis mice by regulating the activation of alveolar macrophages,lung fibroblasts and T lymphocytes differentiation.