Study On The Mechanism Of Salmonella Typhimurium In Inducing Type Ⅰ Interferon Response In Murine Macrophages

Salmonella Typhimurium（S.Typhimurium）is a common food-borne enteric pathogen.During long-term coevolution with microorganisms,the host has evolved different pattern recognition receptors（PRRs）to recognize a variety of pathogenic microorganisms’conserved pathogen-associated molecular patterns（PAMPs）that induce strong innate immunity to respond to microbial infections.Lipopolysaccharide（LPS）from S.Typhimurium is a classical PAMPs that can be recognized by Toll like receptor 4（TLR4）,a membrane receptor in the PRRs family,and activates several innate immune responses,including type Ⅰ interferon response.Type Ⅰ IFN response is a powerful tool for the host to counteract pathogen infection.Different studies have confirmed that S.Typhimurium infection can induce type Ⅰ IFN response,which plays an important role in combating gastrointestinal S.Typhimurium infection.Therefore,unveiling the mechanism of S.Typhimurium infection-induced type Ⅰ IFN response could benefit the development of new strategies to control S.Typhimurium infection.In this study,S.Typhimurium and mouse macrophages were used as study materials and the transcriptome analysis and cell biology experiments were performed.We revealed a new mechanism of S.Typhimurium infection-induced type Ⅰ IFN response in murine macrophages.The main findings are as follows:（1）To detect the activation of the innate immunity of S.Typhimurium infected mouse peritoneal macrophages（PMs）,the gene expression changes in PMs after infection were analyzed by RNA-seq.An unbiased KEGG pathway enrichment analysis of differentially expressed gene data was performed and the results suggested that the cytoplasmic DNA sensing pathway may be activated during S.Typhimurium infection.Next,S.Typhimurium was used to infect WT or TLR4^-/-PMs,and q RT-PCR was performed to detect the expression of Interferon-stimulated genes（ISGs）.The result showed that the gene expression was substantially attenuated but was not totally diminished,indicating that S.Typhimurium infection-induced type Ⅰ IFN response is not completely dependent on TLR4;（2）To further explore whether the S.Typhimurium induced type Ⅰ IFN response is related to the cGAS-STING pathway,q RT-PCR was performed to detect the expression of ISGs in S.Typhimurium-infected WT,cGAS^-/-or STING^-/-PMs.The results showed that gene expression was significantly attenuated in cGAS^-/-or STING^-/-PMs,indicating that the S.Typhimurium infection-induced type Ⅰ IFN response partially dependent on the cGAS-STING pathway.To examine the role of cGAS-STING in host defense against S.Typhimurium infection,WT,cGAS^-/-or STING^-/-mice were intragastrically infected with S.Typhimurium,the survival was monitored and the bacterial loads in the spleen and liver were also examined.cGAS^-/-and STING^-/-mice exhibited attenuated survival rates and higher bacterial loads after infection.These results indicate that cGAS-STING can help the host defense against S.Typhimurium infection;（3）To explore the mechanism of S.Typhimurium infection-induced activation of cGAS-STING,the cytoplasm fraction of the S.Typhimurium-infected cells was isolated,and free mitochondrial DNA was detected by q PCR.After depleting part of the mitochondrial DNA with Et Br treatment,we observed that the S.Typhimurium infection-induced type Ⅰ IFN response was significantly downregulated.Pretreatment of reactive oxygen species（ROS）inhibitor,which could prevent the release of mitochondrial DNA,reduced S.Typhimurium infection-induced cytoplasmic mitochondrial DNA level and cellular type Ⅰ IFN response was significantly downregulated correspondingly.These results indicate that S.Typhimurium infection-induced mitochondrial DNA release can activate cGAS-STING-dependent type Ⅰ IFN response.Summary:This study demonstrated that S.Typhimurium infection caused mitochondrial damage,resulting in the release of mitochondrial DNA into the cytoplasm.Cytoplasmic mitochondrial DNA is recognized by cGAS,which in turn activates the cGAS-STING-dependent type Ⅰ IFN response.In addition,the innate immune response mediated by cGAS-STING constrains S.Typhimurium infection.