Expression Pattern Of The Interferon Regulatory Factor Family Members In Influenza Virus Induced Local And Systemic Inflammatory Responses

Influenza virus(IV)is the pathogen of flu both in human and animal.It can be divided into three types: influenza A virus(IAV),influenza B virus(IBV)and influenza C virus(ICV).Of these,IAV is reported to cause the majority of global outbreaks as its antigenicity is easy to mutate and may induce immune damage such as acute lung injury(ALI).During influenza virus infection,a large number of infiltrated neutrophils in the lungs of the host are the key immune cells causing ALI.The cytokine storm caused by influenza virus infection is the main driver of ALI,in which type I interferon(IFN-I)is considered to be an important factor.Type I interferon is considered to be a key cytokine in influenza virus-induced acute lung injury(ALI),It is known that all mammalian nucleated cells can produce IFN-I.Type I interferon expression is regulated by the transcription of interferon regulators.Interferon regulatory factor(IRF)is a family of transcription factors including IRF1-9,of which IRF3 and IRF7 are vital transcriptional regulators that initiate viral type I interferon transcription.In the TLR7 signaling pathway,IRF7 is a major transcription factor that regulates type I IFN production,where influenza virus is a natural ligand of TLR7.After infecting the human body,it not only causes a large amount of type I interferon production,but also generates cytokine storms to induce acute lung injury,yet the molecular mechanism is unclear.Since all members of the IRF family can bind to sites that are rich in GAAA sequences on the promoter of the target gene,the changes in the expression profile of the IRF family members in the virus-induced inflammatory response may reveal an entry into the mechanism of influenza virus-induced immune damage point.In this research,qRT-PCR method was used to study the expression profile of IRF family in influenza virus-induced infection,different organ tissues of mice,and pDC-like CAL-1 cells at mRNA level,while the Western Blot method was applied to detect the phosphorylation of IRF3,IRF5 and IRF7 in CAL-1 cells under different time quantum of H1N1 IAV treatment.The results were as follows:1.mRNA levels of IRF family members in nasopharyngeal specimens of patients with influenza virus infectionIn order to study whether there were any change in the expression levels of IRF family members in local tissues infected with the influenza virus,we used qRT-PCR to detect the mRNA levels of IRF family members in all nasopharyngeal samples.The results showed that the mRNA levels of IRF family members in all samples changed to varying degrees.IRF7 and IRF9 mRNA levels in the samples with either IAV or IBV infection were significantly increased compared with that of controls,while in IBV infection,the IRF2 levels were also elevated which accompanied a decrease in IRF4 and IRF5 levels.The results indicate that there are significant different in the expression profiles of the IRF family genes during IAV or IBV infection,in which only two IRFs responded to IAV infection and five IRFs responded to IBV infection.2.The mRNA expression profile of IRF family in the lungs and other organs of mice infected with H1N1 IAV and the regulatory effect of GAAA ODN on the expression of IRFsIn order to fully understand the influence of the influenza virus infection on the expression profile of IRF family genes in vivo,mice were infected with H1N1 IAV FM1 with or without the treatment of GAAA ODN M1 or PBS by intraperitoneal injection on day 2 and day 4 post infection(p.i.).On day 5 p.i.,the WBCs,lungs and other organs of the mice were isolated for detecting the mRNA levels of IRF family members by qRT-PCR.The results are showed below :1)After the infection,the expression profiles of IRF family genes in the lungs and WBCs of the mice were similar,in which the levels of IRF5 and IRF7 increased significantly but not other members.In the intestines,hearts and kidneys of the mice,the expression levels of multiple IRF family members increased.The expression levels of IRF2,IRF4,IRF5,IRF7 and IRF9 in intestines,IRF1,IRF2,IRF3,IRF4,IRF5 and IRF7 in hearts,and IRF1,IRF6,IRF7 and IRF9 in kidneys were significantly increased after the infection.Unexpectedly,there was almost no change in the expression level of any IRF family member in the spleens and livers of the mice.The results indicate that the response of IRF family members to the influenza virus infection can be either local or systemic,but the expression profiles are different in different organs.2)The levels of some IRF family members in the examined organs of the mice were significantly reduced by GAAA ODN M1 treatment.However,the expression levels of IRF7 and IRF9 in lungs,IRF4 in intestines and hearts,IRF1,IRF7 and IRF9 in kidneys,and IRF7 and IRF8 in livers were not decreased.In addition,the levels of IRF3,IRF6 and IRF8 in kidneys,IRF9 in livers and IRF4 in stomaches were increased.These results indicate the treatment with GAAA ODN can affect the expression profile of IRF family genes induced by influenza virus infection.3.Influence of influenza virus infection on IRF family expression profile in human pDC-like CAL-1 cellsIn order to determine the effect of direct infection of influenza virus on the expression profile of IRF family genes in target cells,considering the fact that pDC was a dedicated cell of IFN-I production and influenza virus can induce a high levels of IFN-I,we used H1N1 IAV to infect human pDC-like CAL-1 cells in culture for detecting the mRNA levels of IRF family members in the cells dynamically by qRT-PCR.The results showed that the mRNA levels of all IRF family members increased to varying degrees after infection.By comparison,the expression levels of IRF6 and IRF7 were the highest.This result indicates that all members of the IRF family are involved in the response to influenza virus infection.4.Effect of influenza virus infection on the expression of TLR3/7,RIG-I and their downstream signal molecules in CAL-1 cellsTLR3/7 and RIG-I signaling pathways can induce the generation of a large amount of IFN-I and inflammatory cytokines/chemokine when responding to viral infection.Our results showed that H1N1 IAV infection could significantly up-regulate the mRNA levels of TLR3/7 and RIG-I.In comparison,the level of TLR7 mRNA increased earlier than that of TLR3 and RIG-I,in which the level reached to 50 times of that in control group at 6 h post-infection,while at this time,TLR3 and RIG-I were not expressed.The level of TLR7 was fluctuating in 72 h post-infection,the highest level was up to about 200 times of that in control group,which appeared at 12 h,36 h,60 h and 72 h post-infection,and kept about 50 times of that in control group in other times post-infection.The level of TLR3 mRNA was up-regulated in the middle stage,the highest level reached to about 600 times of that in control group,but was at baseline levels in the early and late stages of the infection.The level of RIG-I mRNA increased from 12 h to 72 h after the infection,the highest level appeared at 24 h and 36 h post-infection and reached to over 200 times of that in control group,after that,kept at 10 to 50 times of that in control group.Based on the analysis of the expression levels of cytokines/chemokine induced by influenza virus infection,we found that the levels of IFN-? and IFN-? mRNA increased significantly,the highest levels of them reached to 400 times or 500 times of that in control group.The level of IFN-? increased earlier than that of IFN-?.The level of IFN-? was slightly up-regulated with only 10 times increased,and TNF-? was at low level during the infection.The mRNA level of CXCL10,a chemokine that could recruit neutrophils to the inflammation site,was significantly up-regulated at 6 h post-infection to about 100 times of that in control group,the highest levels appeared at 24 h and 36 h post-infection with 2500 times of the level compared to the control,and after that began to decline to baseline level.These results indicate that influenza virus can activate TLR3/7 and RIG-I to induce the production of IFN-I/II and CXCL10.The produced IFN-?/? may be not only directly induced by influenza virus infection,but also regulated through IFN-I/II positive feedback loop.5.Possible mechanism of changes in the expression profile of IRF family members caused by influenza virusIn order to explore the possible mechanism of changes in the expression profile of IRF family members caused by influenza virus infection,CAL-1 cells were infected with H1N1 IAV,which was treated with rIFN-?2b,chloroquine or GAAA ODN,respectively.The results are summarized below:1)When the rIFN-?2b and the virus act on the cells at the same time,the expression levels of most IRF family members were significantly inhibited except for IRF9 which showed a much lesser inhibition.When rIFN-?2b is given 24 h before or after the IAV infection,the levels of most IRF family members decreased significantly to the baseline level but IRF4,IRF6,IRF7 and IRF9 still maintained somewhat higher levels.These results suggest IFN-I may have different regulatory role on the expression of IRF family members during influenza virus infection.2)When the chloroquine and the virus act on cells at the same time for 24 h,the expression levels of all IRF family members were significantly inhibited,suggesting that influenza virus relies on endosomal TLR signals to induce the expression of IRF family memebrs.3)The GAAA ODN added to the culture of H1N1 IAV infected CAL-1 cells displayed an inhibitory effect in a dose-dependent manner on the expression of most IRF family members except for IRF6 which showed an obvious enhancement in a high dose.It is suggested that the behavior of IRF6 in cytoplasm is different from that of other IRF family members.Overall,influenza virus infection relies on endosomal TLR signals and IFN-I positive feedback loop to regulate the expression of IRF family members.The regulatory mechanism of IRF6 seems to be different compared with that of other IRF family members.6.Effect of influenza virus infection on the phosphorylation of IRF3,IRF5 and IRF7 in CAL-1 cellsThe expression of IFN-/ and CXCL10 was up-regulated by influenza virus infection,and IRF3 and IRF7 were the main transcription factors of IFN-/.IRF5 not only regulates the production of IFN-I,but also regulates the transcription of CXCL10.Therefore,we used Western Blotting and immunocoprecipitation to detect proteins of the IRF3,IRF7 and IRF5 expressed in CAL-1 cells infected by H1N1 IAV and their phosphorylation.The results showed that the expression levels of the IRF3 and IRF5 at all time points and IRF7 at 6 h post infection in CAL-1 cells were significantly enhanced.While the phosphoralations of IRF3 and IRF5 at 1 h and 3 h,and IRF7 at 1 h post infection in the cells were significantly increased.GAAA ODN A1 only inhibited the phosphorylation of IRF5,but promoted the phosphorylation of IRF3 and IRF7.This result shows that different IRF family members have different responses to influenza virus and GAAA ODN treatment.Conclusions: 1.All members of the IRF family are involved in the response to influenza virus infection to varying degrees.the expression profile of the IRF family is related to the pathogenicity of influenza virus.2.When more members of the IRF family members involved during the response to influenza virus infection may be more conducive to control the intensity of inflammation.3.Influenza virus mainly relies on endosomal TLR signals and the positive feedback loop of IFN-I to cause either direct or indirect differential expression of all IRF family members locally or systemically.4.In the in vitro study with influenza virus-infected CAL-1 cells,we noticed that IRF6 was somewhat different from other IRF family members.IRF6 may play a negative regulatory role in the IRF family,so as to balance the IRF family expression profile.5.It is inferred that there may be a mutual restriction mechanism among members of the IRF family,so as to ensure a moderate immune response,unless the overreaction of some member breaks through the restriction.In summary,the expression profile of the IRF family had a certain relationship with the influenza virus,indicating that regulating the expression profile of the IRF family may provide an experimental basis for the prevention and treatment of influenza-induced acute lung injury.