Study On Mechanism Of IFN-Ⅰ Response Induced By Streptococcus Pneumoniae Hydrogen Peroxide-mediated Mitochondrial Dysfunction In Lung Cells

Objective:To investigate whether Streptococcus pneumoniae hydrogen peroxide causes mitochondrial damage and oxidative damage of mtDNA in alveolar epithelial cells,and whether this aberrant mtDNA was released into cytoplasm to further induce type I IFN cascade response in host cells.Background:Streptococcus pneumoniae（S.pn）is one of the important pathogenic bacteria in human nasopharynx,which can cause pneumonia,otitis media,meningitis,bacteremia and so on.S.pn spxB gene encodes pyruvate oxidase,which can decompose pyruvate to produce a large amount of hydrogen peroxide.The deletion of the spxB gene results in a significant decrease in the amount of hydrogen peroxide during the growth of S.pn.S.pn H₂O₂ can induce mitochondrial damage and ultimately mediate apoptosis.Studies have reported that damaged mitochondria release a large number of mitochondrial damage-related molecular patterns（mtDAMPs）,including mtDNA,ATP and so on,which activate the host immune response.However,the mechanism of S.pn H₂O₂-induced damage to alveolar epithelial cells and subsequent activation of host immune response is not fully understood.We speculate that S.pn H₂O₂ may cause oxidative damage of mtDNA in alveolar epithelial cells,reduce the content of mtDNA,and then lead to the release of mtDNA into the cytoplasm to induce the expression of IFNβ.Methods and Results:1.In pneumonia mice model,the expression of IFNβin serum and lung tissues of C57BL/6 mice was detected by ELISA and real time-PCR.Compared with D39ΔspxB infection group and D39+Cat infection group,D39 infection could induce the expression of IFNβin serum and lung homogenate of C57BL/6 mice.In vitro,the expression of IFN-I（IFNаand IFNβ）in A549 cells and culture supernatant was detected by ELISA and real time-PCR.Compared with D39ΔspxB infection group and D39+Cat infection group,D39 infection induced IFN-I cascade response in A549cells.At the same time,exogenous hydrogen peroxide stimulation is capable of inducing IFN-I cascade response in A549 cells.2.In vitro,Western Blot detected the expression of STING protein in A549 cells under different infection conditions.Compared with D39ΔspxB infection and D39+Cat infection,D39 infection could up-regulate the STING protein in A549 cells.The expression of Ifnβwas detected by real time-PCR when Sting was knocked down.Neither D39 infection nor H₂O₂stimulation could induce the expression of Ifnβin A549 cells.3.In vivo,compared with D39ΔspxB infection and D39+Cat infection,D39 infection group caused significant histopathological damage,accumulation of PINK1 and decrease of Pgc1-a expression in lung tissue of C57BL/6 mice.In vitro,compared with D39ΔspxB infection and D39+Cat infection,D39 infection could cause the decrease of mitochondrial membrane potential and mitochondrial ultrastructural changes in A549cells,including mitochondrial ultrastructural changes,mitochondrial swelling,shrinkage and destruction of cristae structure.4.The co-localization of DNA oxidative damage marker 8-OHdG and mitochondria was detected by immunofluorescence assay.Compared with D39ΔspxB infection and D39+Cat infection,D39 infection could up-regulate the expression of 8-OHdG in mitochondria of A549 cells.mtDNA content in lung cells was detected by real time-PCR.Compared with D39ΔspxB infection and D39+Cat infection,D39 infection could reduce the copy number and transcription level of mtDNA in lung tissue and A549 cells and then lead to the release of mtDNA into cytoplasm.Next,the mtDNA in cytoplasm was isolated and transfected into A549 cells.The expression of IFNβwas detected by real time-PCR.The result showed that mtDNA released into cytoplasm could up-regulate the expression of IFNβin A549 cells.Meanwhile,mtDNA-deficient A549 cells were constructed in vitro.The expression of IFNβwas detected by real time-PCR at different time points after treatment with H₂O₂ and infection with D39,respectively.Compared with wild type A549 cells,H₂O₂ and D39 stimulation could not promotes the expression of IFNβin mtDNA-deficient A549 cells mtDNA.Conclusions:This study confirms that H₂O₂ secreted by S.pn can induce mitochondrial damage in alveolar epithelial cells,oxidative damage of mtDNA and leakage into cytoplasm,and then activate STING signaling pathway to induce type I IFN cascade response in host cells.This study is the first to elucidate the mechanism of type I IFN cascade response in alveolar epithelial cells induced by S.pn H₂O₂.It also demonstrates that mtDNA play an important role in mediating host immune response.This will help us to further understand the interaction between S.pn and innate immune response,and provide new ideas for finding new strategies to prevent and treat pneumococcal disease.