Study On The Synergistic Damage Effects And Related Mechanism Of TNF-α And IFN-β On Respiratory Epithelial Cells

ObjectiveIn this study,human lung bronchial epithelium BEAS-2B cells were used to investigate the role of TNF-α and IFN-β in respiratory epithelial cell injury and the related molecular mechanism.Method1.IFN-β,IFN-γ,and TNF-α were used alone or in combination to stimulate BEAS-2B cells to simulate the effect of pro-inflammatory cytokines on human respiratory epithelial cells.The cytotoxic effects of IFN-β,IFN-γ and TNF-α on respiratory epithelial cells were analyzed by measuring cell viability,lactate dehydrogenase release,and SYTOX Green-DAPI staining,and the phenotype changes of cells at different concentrations and times were also observed.2.The death modes of human lung bronchial epithelial cells BEAS-2B induced by TNF-α and IFN-β were initially screened by ZVAD-FMK,GSK-872,NSA and other inhibitors of key molecules in apoptosis and necroptosis pathways.Cell viability assay,lactate dehydrogenase release assay,SYTOX Green-DAPI staining and Annexin V FITC-PI staining were used to determine the inhibitory effects of various inhibitors on TNF-α and IFN-β induced BEAS-2B cell death.Western Blot analysis was performed to detect the activation of key molecules in the pathway after TNF-αand IFN-β stimulation of BEAS-2B cells.3.The CRISPR-CAS9 technique was used to knockdown caspase-8 or/and caspase-3 in human lung bronchial epithelial cells BEAS-2B,and Western Blot was used to detect the expression of caspase-8 or/and caspase-3.The effect of caspase-8or/and caspase-3 knockdown on TNF-α and IFN-β induced BEAS-2B cell death was investigated by cell viability assay,and the changes of related molecular pathways were detected by Western Blot.Result1.The results of cell viability assay showed that IFN-β,IFN-γ and TNF-α could inhibit the proliferation of human lung bronchial epithelial cells BEAS-2B to some extent,and the effect of IFN-β was most significant among them.When BEAS-2B cells were treated with different combinations of TNF-α,IFN-β and IFN-γ,and the TNF-α/IFN-β combination significantly decreased cell viability,but there was no significant difference between the TNF-α/IFN-β/IFN-γ and TNF-α/IFN-β.These results indicated that TNF-α and IFN-β could synergistically inhibit cell proliferation in a time-dependent manner.2.Compared with the control group,the death morphology of BEAS-2B cells treated with TNF-α and IFN-β was more obvious,and the percentage of SYTOX Green positive cells and LDH level in cell medium supernatant were significantly increased.These results suggest that TNF-α combined with IFN-β induced BEAS-2B cell death.In addition,the results of cell viability assay showed that the survival rates of human bronchial epithelial cells 16 HBE and human lung adenocarcinoma cells H1975 were also significantly decreased after TNF-α and IFN-β combined treatment.3.The results of cell viability assay showed that the caspase inhibitor ZVAD-FMK and MLKL inhibitor NSA could partially inhibit TNF-α/IFN-β induced cell death.Meanwhile,the Annexin V-FITC-PI staining was double positive in TNF-α/IFN-β treated BEAS-2B cells.The results of immunofluorescence staining of activated caspase-3 and Western Blot showed that caspase-8 and caspase-3,the key executer molecules of apoptosis pathway,were activated in IFN-β and TNF-α/IFN-βtreatment groups.Meanwhile,PARP1 cleavage increased in a time-dependent manner.In addition,the combination of TNF-α and IFN-β activated caspase-8 and caspase-3more effectively than IFN-β alone,and ZVAD-FMK inhibited their activation.4.The results of cell viability assay and SYTOX Green-DAPI fluorescence staining showed that NSA could partially inhibit TNF-α/IFN-β induced cell death when caspase-8 activity was inhibited by ZVAD-FMK.The results of Western Blot analysis showed that although IFN-β and TNF-α/IFN-β increased the expression of MLKL,but did not induce phosphorylated MLKL.Further detection of RIPK3 expression showed that RIPK3 was not expressed in BEAS-2B cells.5.Bubble-like membrane swelling was observed in TNF-α/IFN-β treated BEAS-2B cells,and IL-1β release was detected in the medium supernatant.In addition,caspase-3 and caspase-8 inhibitors partially improved the survival rate of TNF-α/IFN-β treated cells and reduced LDH release.After caspase-8 or/and caspase-3was/were knockdown in BEAS-2B cells,TNF-α/IFN-β induced cell death was inhibited and the activation cleavage of pyroptosis-related molecules GSDMD and GSDME were reduced.Conclusion1.IFN-β,IFN-γ and TNF-α could partially inhibit the proliferation of human lung bronchial epithelial cells BEAS-2B when used alone.The survival rate of human lung bronchial epithelial cells could be effectively reduced by low concentration of IFN-β,and the addition of TNF-α could enhance the effect of IFN-β.TNF-α and IFN-β synergistically damage human lung bronchial epithelial cells BEAS-2B in a time-dependent manner.In addition,TNF-α and IFN-β synergistically inhibited the proliferation of other lung tissue-derived cells.2.TNF-α and IFN-β could synergistically activate caspase-8 and caspase-3,and at the same time increase lysed PARP1,leading to apoptosis of BEAS-2B cells.Although TNF-α and IFN-β promoted the expression of MLKL,necroptosis could not be induced due to the non-expression of RIPK3 in BEAS-2B cells.3.TNF-α and IFN-β lead to BEAS-2B cell pyroptosis by activating caspase-8and caspase-3 and further inducing GSDMD and GSDME activation cleavage.Inhibition of caspase-8 and caspase-3 activity or reduction of their expression could alleviate the toxic effects of TNF-α and IFN-β on BEAS-2B cells.In addition,ZVAD-FMK and NSA could synergistically inhibit this cytotoxic effect of TNF-α and IFN-β.