Effects And Mechanisms Of Stimulator Of Interferon Gene STING In Staphylococcus Aureus Respiratory Infection

STING（Stimulator of interferon genes）is known as an important adaptor protein or direct sensor in the detection of CDNs.While it is activated,STING serves as a scaffold for TBK1 and the transcription factor IRF3.IRF3 is phosphorylated by TBK1,after which it translocates into the nucleus to stimulate the transcription of type I interferons.Presently,research on STING has predominantly focused on its role in type I interferons signaling,which participates in antiviral responses and autoimmune disorders.However,STING is a double-edged sword in host antibacterial immunity.The implication of STING during pulmonary microbial infection remains unknown to date.The severity of S.aureus pulmonary infection could be varied from asymptomatic infection to necrotizing pneumonia.Methicillin-resistant Staphylococcus aureus（MRSA）infections have become a global threat to public health.Exploration on the molecular mechanisms of the respiratory immune response and find the intervention target,which contributes to the prevention and control of S.aureus pneumonia.Herein,we further investigated the effect and mechanism of STING in host defense against S.aureus pulmonary infection.To assess whether STING influences susceptibility in pulmonary host defense against S.aureus infection,the S.aureus pneumonia model was constructed using WT and STING^-/-mice.We found that STING^-/-mice were more susceptible to S.aureus infection than WT mice at 24 hpi,which was manifested by increased mortality,more bacteria burden in BALF and lungs,severe destruction of lung architecture,increased inflammatory cells infiltration,and inflammatory mediators secretion.These results demonstrated that STING plays an indispensable role in protection against S.aureus-induced pneumonia.Based on the above results,it is challenging to delve into the mechanisms underlying the STING-mediated protection to S.aureus infection.Infection and inflammation are always intertwined and are mutually the cause or consequence.Therefore,to further explore the underlying causes,we analyzed the initial interplay between S.aureus and the host at 6 hpi.Intriguingly,we observed a higher S.aureus burden in lung tissues of STING^-/-mice at the acute stage of infection.Meanwhile,there was no difference in inflammatory mediators secretion between WT and STING^-/-mice.These results suggested that STING contributes to host defense against S.aureus lung infection by potentially promoting early bacterial clearance.Subsequently,the question arose as to how STING limits early pulmonary colonization of S.aureus.Host cell death is an acute immune response against microbial intrusion.Bacteria can mediate host cell death to achieve immune escape.We further investigate whether STING prevents S.aureus pathogenesis by mediating cell death.Experiments were carried out as follows.We first examined cell death of the lung tissue by TUNEL and LDH assay.Our results revealed that STING^-/-mice presented more TUNEL-positive cells compared to WT lung sections at 6 hpi.The increased cell death was confirmed by more LDH release in STING^-/-BALF.We further quantified the number of immune cells in BALF using flow cytometry.Compared with WT mice,STING^-/-mice had equivalent numbers of neutrophils in BALF,but a significantly decreased number of macrophages accompanied by increased dead macrophages in BALF.Conclusively,these results indicated that STING suppresses macrophage death during the early stage of S.aureus infection.We further investigate the modality of STING-mediated cell death.Recently,S.aureus toxins such as Agr,Hla,Luk AB,and Psms are known to deplete alveolar macrophages through activating necroptosis,a mode of cell death,which is a major mechanism of S.aureus induced lung damage.Therefore,we analyzed whether STING-mediated cell death is necroptosis.Our results revealed that the RIPK3 and phosphorylation of MLKL definitely increased in the lungs of STING^-/-mice during early infection compared to the control mice.Due to STING mainly located in the recruited inflammatory cell,BMDMs isolated from WT and STING^-/-mice were used to examine the effect of STING on cell death.Consistent with in vivo results,increased cell death,and higher levels of RIPK3 and p-MLKL were also presented in infected STING^-/-BMDMs compared to infected WT macrophages.The increased cell death in STING^-/-BMDMs can be inhibited by the necroptosis inhibitor,Nec-1.Thus,our in vivo and in vitro findings show that STING inhibits necroptosis activation during S.aureus infection.We then utilized MLKL^-/-mice to evaluate the effect of necroptosis on S.aureus infection.The results showed that MLKL^-/-mice exhibited decreased bacterial burden in lung tissues at 6 hpi.The level of LDH release and the numbers of dead macrophages were also decreased in MLKL^-/-mice compared to WT mice.At 24hpi,there was significantly improved host defense against S.aureus infection in MLKL^-/-mice as compared with WT mice.More importantly,blocking necroptosis by MLKL inhibitor GW806742X rescued host defense defect against S.aureus pneumonia in STING^-/-mice.Taken together,STING promotes pathogen control via necroptosis suppression during the early stage of pulmonary S.aureus infection.The above results show that blocking necroptosis in STING^-/-mice possesses an intensified immune protection.There remains a significant gap between WT and STING^-/-mice,suggesting that STING may contribute to host defense against S.aureus pneumonia via additional mechanisms.Extracellular Traps is a novel extracellular protective defense mechanism against various pathogens,which is related to the development of lung disease.We further investigate whether STING prevents S.aureus infection by mediating ETs formation.Experiments presented in the current study demonstrated that DNase I treatment effectively reduced cf DNA in the BALF utilizing SYTOX green straining.Importantly,DNase I treatment facilitated pathogen colonization without increased inflammatory cell infiltration and pro-inflammatory factors secretion.Consistent with this,STING^-/-mice presented decreased cf DNA level,Cit H3 expression,and ETs structure compared to WT mice.To further verify this finding,BMDMs or BMDNs from WT and STING^-/-mice were employed.We first demonstrated that a significant reduction of extracellular DNA was seen in STING^-/-BMDMs or BMDNs compared with WT cells,as well as decreased the expression of ET‐representative protein Cit H3 in BMDMs.In this study,the colocalization of DNA with Cit H3,MPO,and NE in S.aureus-triggered ETs was significantly reduced in STING^-/-BMDMs compared to WT BMDMs.Meanwhile,STING^-/-BMDMs exhibited significantly increased extracellular bacteria burden compared to WT BMDMs pretreatment with or without Cyt D,such differences also disappeared after DNaseⅠtreatment.Hence,our results indicated that STING promotes ETs formation to defense against S.aureus infection.We set out to explore the underlying molecular mechanism by which STING regulates the formation of ETs.The vital role of NADPH oxidase-dependent ROS during NETs formation has been reported previously.We further investigate whether STING-mediated ETs formation depends on ROS production.Consequently,the interaction between ROS generation from NADPH oxidase and S.aureus-induced p38MAPK or ERK1/2 activation were detected.Thus,NADPH oxidase inhibitor DPI was performed to explore the role of ROS in S.aureus-induced ETs in macrophages.Indeed,our results showed that DPI treatment definitely reduced the formation of S.aureus-induced METs,mainly characterized by decreased cf DNA level and the colocalization between DNA and relevant proteins（Cit H3,MPO,and NE）.ROS could function as an important second messenger to transmit diverse signals such as ERK1/2 and p38MAPK signaling pathway.In our study,DPI treatment could suppress the activation of ERK1/2,but not the p38 MAPK signaling pathway.To clarify the role of STING in ROS-ERK mediated ETs formation,we detected the expression of p-ERK and ROS production.Compared with control cells,STING^-/-BMDMs exhibited less production of ROS and decreased level of p-ERK.Importantly,inhibiting the ROS-ERK signal with inhibitors reduced the ETs formation and the differences disappeared between WT and STING^-/-BMDMs after S.aureus infection.Hence,our study emphasized the importance of STING in promoting S.aureus-induced ETs formation via the ROS-ERK signaling pathway.Collectively,our study demonstrates that STING limits early pulmonary colonization of S.aureus by inhibiting necroptosis activation in macrophages.Besides,STING-mediated ETs improved host defense during S.aureus infection by enhancing the extracellular killing capacity via the ROS-ERK signaling pathway.