Mechanism Of Enhanced Respiratory Gram-positive Bacterial Secondary Infection Due To Virus-induced Expression Of IFN-I

Objective: Respiratory viruses are one of the main pathogens responsible for global epidemics,and secondary bacterial infections often accompany respiratory viral infections,with Gram-positive bacteria being the most common.Type I interferons(IFN-Ⅰ)are important cytokines induced by viral infections that can induce an antiviral state in almost all cell types.This study found a close correlation between increased susceptibility to secondary bacterial infections and type I interferon.The major immune cells on the surface of the respiratory mucosa are macrophages and neutrophils,which form the first line of defense against respiratory bacterial infections.Does the induction of high expression of type I interferon by viral infections affect the survival and function of macrophages and is it related to secondary bacterial infections? In order to further elucidate the potential mechanisms involved,we conducted an in-depth investigation to provide new data support for subsequent clinical prevention and treatment of bacterial secondary infections caused by viral infections and to improve patient outcomes.Methods:1.Wild-type female BALB/c mice were intranasally infected with respiratory syncytial virus(RSV),and lung tissue pathology and inflammatory cell infiltration were observed using HE staining.QRT-PCR and ELISA were used to detect the m RNA levels of IFN-Ⅰ in mouse lung tissue.After 24 hours of RSV and influenza virus(H1N1,H3N2,IBV)infection in A549 cells,ELISA was used to detect the expression levels of IFN-Ⅰ in the cell culture supernatant.2.The changes in cell membrane integrity were assessed using propidium iodide(PI)staining and flow cytometry after treating THP-1 cells with type I interferon.3.After treating THP-1 cells with IFN-Ⅰ,the changes in differentially expressed genes were analyzed through transcriptome sequencing.Bioinformatics analysis was conducted to select target genes and target proteins.The expression levels of necroptosis key proteins,RIPK3 and MLKL,were detected using Western blot.4.After pre-stimulation of THP-1 cells using IFN-β,THP-1 cells were stimulated with the G+ bacteriophage mimic Pam3CSK4 or heat-inactivated GAS(HK-GAS),and the expression levels of p-MLKL,p-RIPK3,the key protein of cell scorch death,GSDMD-N,and the key protein of apoptosis were detected by Western blot.5.After pre-stimulation of HL60 cells using IFN-β,HL60 cells were stimulated with Pam3CSK4 or heat-inactivated GAS(HK-GAS),and the expression levels of p-MLKL,p-RIPK3,GSDMD-N and apoptosis key proteins were detected by Western blot.6.Differential gene enrichment and protein-protein interaction(PPI)analysis were performed to identify proteins potentially interacting with MLKL and RIPK3.After treating THP-1 cells or HL60 cells with Pam3CSK4,the expression levels of p-p65,cellular inhibitor of apoptosis protein 2(CIAP2),and cellular FLICE-like inhibitory protein(CFLIP)were detected using Western blot.7.After pre-treating THP-1 or HL60 cells with IFN-β,they were stimulated with Pam3CSK4 or heat-killed group A Streptococcus(HK-GAS),and the expression of CIAP2 and CFLIP was detected using Western blot.Following si RNA knockdown of CIAP2 in THP-1 cells,the cells were stimulated with Pam3CSK4,and the expression levels of RIPK3,MLKL,and GSDMD were detected using Western blot.8.THP-1 cells were treated with the proteasome pathway inhibitor MG132 and stimulated with Pam3CSK4.The protein expression levels of ubiquitination(UB),RIPK3,and MLKL were detected using Western blot.In another experiment,293 T cells were co-transfected with p CDNA3.1-FLAG-RIPK3 or p ECMV-FLAG-MLKL,p CAGGS-HA-CIAP2,and HA-UB for 24 hours,along with a control group using empty vectors.Co-immunoprecipitation(Co-IP)was performed using anti-FLAG monoclonal antibody on the whole-cell lysates of each sample to detect the expression of RIPK3,MLKL,UB,and CIAP2 in the IP group and the whole-cell lysates.9.THP-1 cells were pre-treated with type I interferon and stimulated with Pam3CSK4.The expression levels of chemokines CXCL1,CXCL2,and the anti-inflammatory cytokine IL-10 were detected using q RT-PCR.After inducing THP-1 cells with IFN-α,GO enrichment analysis was performed to screen for differentially expressed genes that have a negative regulatory effect.The expression levels of p-p65,SOCS1,and IDO1 were validated using Western blot to assess the effects of type I interferon treatment on THP-1 cells.Results:1.RSV and influenza virus H3N2 can significantly induce high expression of type I interferon.In vivo experiments,it has been observed that after RSV infection,the lung tissue of mice maintains a high level of IFN-Ⅰ expression for up to 7 days post-infection.Furthermore,there is a positive correlation between the significant infiltration of inflammatory cells in the mouse lungs and the high expression of type I interferon.2.Type I interferon can damage the membrane integrity of THP-1 cells,while the individual stimulation of Pam3CSK4 or heat-killed group A Streptococcus(GAS)does not significantly disrupt the cell membrane of THP-1 cells.3.Transcriptome sequencing reveals that treatment of THP-1 cells with type I interferon induces the expression of cell death-related genes.These genes encompass key players in necroptosis(RIPK3 and MLKL),pyroptosis(GSDMD),and apoptosis(caspase 8 and caspase 3).Importantly,the upregulation of MLKL protein levels by type I interferon shows a timedependent pattern,with increased expression observed over time.4.Compared to the control group,treatment with type I interferon significantly upregulates the protein expression levels of phosphorylated RIPK3,phosphorylated MLKL,and the N-terminal active form of GSDMD in THP-1cells.However,type I interferon does not induce the activation of caspase 3.Neither Pam3CSK4 nor HK-GAS can significantly induce the activation of RIPK3,MLKL,and GSDMD.5.Compared to the control group,stimulation of HL60 cells with type I interferon also leads to a significant upregulation of phosphorylated RIPK3,phosphorylated MLKL,and the N-terminal active form of GSDMD.However,neither Pam3CSK4 nor HK-GAS can induce the activation of RIPK3,MLKL,and GSDMD in HL60 cells.6.Enrichment analysis of differentially expressed genes revealed that Pam3CSK4 significantly upregulates the expression of various pro-survival genes in THP-1 cells.Protein-protein interaction(PPI)analysis was conducted to investigate the potential interactions between the upregulated pro-survival genes and MLKL,RIPK3,and GSDMD.The analysis showed that CIAP2 and CFLIP have potential interactions with both MLKL and RIPK3,but no interaction was found with GSDMD.7.After treatment with type I interferon in THP-1 cells or HL60 cells,there is a significant downregulation of CIAP2 expression compared to the control group.However,there is no significant impact on the protein levels of CFLIP.Furthermore,in THP-1 cells,knocking down CIAP2 and stimulating with Pam3CSK4 result in a significant upregulation of RIPK3 and MLKL protein levels compared to the control group,but there is no increase in the protein expression of GSDMD.8.After stimulating THP-1 cells with Pam3CSK4,the addition of MG132 treatment leads to an increase in the protein expression of RIPK3 and MLKL compared to the control group.Moreover,when THP-1 cells are treated with different concentrations of MG132,the protein levels of RIPK3 and MLKL gradually increase in a dose-dependent manner.In addition,the coimmunoprecipitation(Co-IP)results show that overexpression of CIAP2 enhances the ubiquitination modification of RIPK3 and MLKL.9.Type I interferon can inhibit the expression of chemokines CXCL1 and CXCL2 in THP-1 cells after stimulation with Pam3CSK4.GO enrichment analysis reveals that type I interferon treatment of THP-1 cells can induce the upregulation of various genes encoding negative regulatory proteins.Additionally,it was found that SOCS1 and IDO1 are enriched in multiple GO molecular functions associated with negative regulation.Furthermore,it was observed that IFN-β can inhibit the phosphorylation of p65 during the induction of IDO1 expression.Conclusions:1.Respiratory Syncytial Virus(RSV),like other respiratory viruses,can significantly induce the expression of type I interferons(IFN-Ⅰ).The high expression of IFN-Ⅰ can be sustained throughout the recovery period from viral infection,which may be associated with the heightened inflammatory response in the host’s lungs.2.Type I interferons induce necroptosis and cell pyroptosis in macrophages and neutrophils,which may be important factors contributing to the diminished antimicrobial capability.3.Type I interferons can induce cell pyroptosis by causing the cleavage of Gasdermin D(GSDMD)into its active form,GSDMD-N.4.Type I interferons may enhance the phosphorylation of RIPK3 and MLKL and thus induce necroptosis by downregulating CIAP2 expression.5.Type I interferons inhibit the expression of chemokines CXCL1 and CXCL2,which are mediated by Pam3CSK4.This effect may be associated with the upregulation of SOCS1 and IDO1 by type I interferons,leading to the suppression of NF-κB signaling pathway activation.This mechanism could be another important factor contributing to bacterial secondary infections caused by type I interferons.