Effects Of IRF7-IFN-α Signaling Pathway On Acute Lung Injury Induced By Influenza Virus And Its Underlying Mechanisms

Influenza is a highly contagious respiratory disease,which poses a great threat to human health and causes worldwide morbidity and mortality.The excessive inflammatory responses induced by influenza virus are the main cause of acute lung injury(ALI),which could develop into acute respiratory distress syndrome(ARDS)in severe cases.In response to influenza virus invasion,the host mobilizes the innate immune system to produce IFN-α to defend against them.However,IFN-α is considered as a major contributor to influenza virus induced ALI.Considering the interferon regulatory factor 7(IRF7)is a transcription factor that mainly regulates IFN-α expression,we speculate that IRF7-IFN-α signaling pathway may be involved in the development of influenza virus induced ALI and attenuation of IRF7 activity could decrease IFN-α production to alleviate influenza virus induced ALI.As reported,the immune regulatory oligodeoxynucleotide(r ODN)is widely used in the treatment of autoimmune diseases such as systemic lupus erythematosus(SLE)and arthritis(RA).Therefore,we designed the IRF7-r ODNs,based on the binding sites of IRF7 on the genes of IFN-α,and explored the effects of IRF7-r ODNs on influenza virus induced ALI.IFN-α exists several subtypes in human and mice and they all bind to the same IFNAR receptor to induce the expression of several interferon stimulated genes(ISGs)through the JAK/STAT signaling pathway.However,the diverse IFN-α subtypes activate different downstream signaling pathways and induce different downstream genes.The discrepancies may be related to the receptor binding affinity or the protein structure caused by the different amino acids at specific positions.So,we hypothesized that the different inflammatory responses and disease severities induced by different influenza virus may be related to the diverse IFN-α subtype induction,and the specific IFN-α subtype induction in local infectious sites may be a clinical indicator to reflect the disease severity.In this paper,we observed the the activation of IRF7 and the m RNA levels of IRF7-IFN-α signaling pathway related molecules in epithelial cells and trachea and lung tissues infected with influenza virus,designed IRF7-r ODNs,based on the mouse and human IRF7 binding sites,and detected the effects of IRF7-r ODNs on influenza virus induced ALI.Moreover,we also detected the diverse IFN-α subtype induction in human nasopharyngeal epithelial cells or mouse lung tissues infected with different influenza virus.1.The activation of IRF7-IFN-α signaling pathway in local infectious sites in vitroTo detect the activation of IRF7-IFN-α signaling pathway,we established the influenza virus infected epithelial cells and mouse trachea and lung tissue model to observe the activation and expression of IRF7 and the m RNA levels of IRF7-IFN-α signaling pathway related molecules.The results showed that: 1)The mouse trachea and lung tissues are constitutively expressed IRF7.2)The IRF7 phosphorylation was increased in trachea and lung tissues infected with H1N1 influenza virus.3)The m RNA levels of TLR7,IRF3,IRF7,IFNA,IFNB and CXCL10 were significantly increased in epithelial cells infected with H1N1 influenza virus.4)The m RNA expression of Tlr7,Irf3,Irf7,Ifna,Ifnb and Cxcl10 were obviously increased in trachea and lung tissues infected with H1N1 influenza virus.The results suggested that influenza virus infection could rapidly activate IRF7 and increase the m RNA levels of IRF7-IFN-α related signaling pathway molecules.2.Effects of IRF7-r ODN on ALI induced by influenza virusTo study the role of IRF7 and IRF7-IFN-α signaling pathway in influenza virus induced ALI,we designed the IRF7 targeting IRF7-r ODNs and detected the role of IRF7-r ODNs in ALI induced by influenza virus.2.1 Establishment of influenza virus induced acute lung injury mouse modelTo establish influenza virus induced acute lung injury mouse model,the mice were intranasally inoculated with H1N1 influenza virus.After that,we observed the appearance of the infected mice,recorded the body weights and survivals and detected pathological changes of lungs and the percentage of neutrophils in BALF.The results showed that: 1)On day 2 post-infection,the mice began to display signs of inappetence,hunching and ruffled fur.The body weights of H1N1 influenza virus infected mice were decreased twenty percentage to original body weights on day 5 post-infection.Death of mice began on day 6 post-infection,and they were all dead within 10 days postinfection.2)Lung index and wet-to dry ratio were significantly increased on day 3 postinfection.3)Interstitial and alveolar oedema and diffuse alveolar damage,evaluated by pathological scores,began to occur on day 1 post-infection,and then worsened afterwards.4)In the BALF,the percentage of neutrophils was increased to nearly 40% on day 1 post-infection,and reached a peak on day 3 post-infection.The results suggested that the influenza virus infection could induce ALI.2.2 Effects of IRF7-r ODNs on acute lung injury induced by influenza virusTo observe whether the IRF7-r ODNs could alleviate influenza virus induced ALI,the mice were intranasally inoculated with H1N1 influenza virus and treated with the IRF7-r ODNs.After that,we recorded the survivals,observed pathological changes of the lungs and detected neutrophil percentage in BALF.The results showed that: 1)The IRF7-r ODN M1 could significantly increase the survival of the H1N1 influenza virus infected mice.2)IRF7-r ODN M1 could alleviate the pathological damage of lung tissues in mice infected with H1N1 influenza virus.3)IRF7-r ODN M1 could reduce the percentage of neutrophils in BALF.The results suggested that the IRF7-r ODN M1 could alleviate ALI induced by influenza virus.2.3 Effects of IRF7-r ODNs on IRF7-IFN-α signaling pathwayTo detect the effects of IRF7-r ODNs on IRF7-IFN-α signaling pathway,we isolated the lung tissues from the H1N1 influenza virus infected mice treated with IRF7-r ODNs,detected IRF7 protein expression and Ifna and Cxcl10 m RNA levels in lungs and verified the binding of IRF7-r ODNs with IRF7.The results showed that: 1)The IRF7-r ODN M1 could decrease the IRF7 protein expression in lungs of the mice.2)IRF7-r ODN M1 could decrease the m RNA levels of Ifna and Cxcl10 in lungs of the mice.3)IRF7-r ODN M1 could bind to IRF7.The results suggested that: the IRF7-r ODN M1 could reduce the downstream cytokine IFN-α and CXCL10 expression,by binding to IRF7 to interfere IRF7 activity to alleviate ALI induced by influenza virus.2.4 Effects of IRF7-r ODNs on IRF7 activity in RAW264.7 cells infected with influenza virusTo test the interfering role of IRF7-r ODNs on IRF7 activity,we cultured the H1N1 influenza virus infected RAW264.7 cells treated with IRF7-r ODNs and detected the IRF7 expression and nuclear translocation.The results showed that 1)H1N1 influenza virus infection could significantly increase the m RNA level of Irf7 in RAW264.7 cells infected with H1N1 influenza virus.2)IRF7-r ODN M1 could decrease the m RNA level of Irf7 in RAW264.7 cells infected with H1N1 influenza virus.3)IRF7-r ODN M1 could inhibit IRF7 translocation in RAW264.7 cells infected with H1N1 influenza virus.The results suggested that the IRF7-r ODN M1 could interfere IRF7 activity by interfering IRF7 translocation.3.The relationship between the severity of influenza virus infection and IFN-αsubtype inductionIFN-α exists several subtypes in human and mice.They all bind to the same IFNAR receptor,whereas they activate the distinct downstream signaling pathways.The relationship between the severity of influenza virus infection and IFN-α subtype induction are still unclear.We collected the nasopharyngeal epithelial cells infected with influenza A virus(IAV)or influenza B virus(IBV)and the lung tissues infected with H1N1 or H9N2 to detect the relationship between the immune responses and the diverse IFN-α subtype induction.3.1 The diverse IFN-α subtype induction in nasopharyngeal epithelial cells infected with different influenza virusesTo detect the IFN-α subtype induction in nasopharyngeal epithelial cells infected with different influenza viruses,we collected the nasopharyngeal epithelial cells by nasopharyngeal swabs,classified them into the IAV or IBV infected cells by direct fluorescent antigen assays and detected the m RNA levels of IFNA and CXCL10.We also used the consensus primers to amplify all IFN-α subtypes in PCR method to construct a c DNA library to analyze the percentage of IFN-α subtypes.The results showed that: 1)The m RNA levels of IFNA and CXCL10 were higher in IAV infected nasopharyngeal epithelial cells.2)The percentage of IFN-α14 was increased in nasopharyngeal epithelial cells infected with IAV.3)The percentage of IFN-α5 and IFN-α21 was increased in nasopharyngeal epithelial cells infected with IBV.The results suggested the influenza virus induced different inflammatory responses and disease severity may be correlated to the diverse IFN-α subtype induction.3.2 The diverse IFN-α subtype induction in lungs of the mice infected with different influenza virusesTo explore the diverse IFN-α subtype induction in lungs of the mice infected with different influenza viruses,the mice were intranasally inoculated with H1N1 or H9N2 influenza virus.After that,we recorded the survivals,observed the pathological changes of lung tissues and detected the percentage of neutrophils in BALF and the m RNA levels of Ifna and Cxcl10 in lungs.We also used the consensus primers to amplify all IFN-α subtypes in PCR method to construct a c DNA library to analyze the IFN-α subtype percentage.The results showed that: 1)The mice infected with H1N1 influenza virus were all dead,whereas the mice infected with H9N2 were all alive.The pathological damage in lungs of the H1N1 influenza virus infected mice was much more serious than that in the H9N2 influenza virus infected mice.The percentage of neutrophils was higher in H1N1 influenza virus infected mice.The m RNA levels of Ifna and Cxcl10 were higher in lungs of the H1N1 influenza virus infected mice.2)The percentage of IFN-α1 and IFN-α9 was higher in lungs of the H1N1 influenza virus infected mice,whereas the percentage of IFN-α2 and IFN-α12 was higher in lungs of the H9N2 influenza virus infected mice.The results suggested that the different inflammatory responses and disease severity were correlated to the diverse IFN-α subtype induction.In conclusion,this study demonstrated that influenza virus infection could rapidly activate IRF7-IFN-α related signaling pathway in trachea and lung tissues in vitro and the IRF7-r ODN M1 could alleviate ALI induced by influenza virus,by interfering IRF7 activity.Furthermore,it was demonstrated that the different inflammatory responses and disease severity were correlated to the diverse IFN-α subtype induction.The study provides experimental basis for therapeutic drugs of influenza virus infection and new ideas for clinical diagnosis and treatment.