Mechanism Of PCFs Regulation RSV-induced Early Antiviral Responses And Respiratory Inflammatory Responses

PARTⅠ PCFS REGULATE RSV-INDUCED ANTIVIRAL RESPONSES VIA IFN-ALPHA/BETA-STAT1 SIGNALING PATHWAYObjectives RSV infection is the major cause of respiratory tract infection in infants worldwide for which no vaccine or antiviral treatment is available.Previously we found that PCFs degeneration significantly decreased viral titers following RSV infection,suggesting that PCFs were involved in the antiviral effect in RSV infection.Antiviral responses,characterized by IFN-α/β induction,play important roles in defense against viral infections.Upon viral invasion,the virus components are recognized by PRRs,which induces the production of IFN-α/β.IFN-α/β exerts its antiviral activity via induction of ISGs by activating STAT1.Therefore,we hypothesized that PCFs degeneration may increase the induction of IFN-α/β uopn RSV infection,and enhance the antiviral responses against RSV.Methods Lungs and BALF of 12 h,24h and 48 h post-RSV infection were collected from Intact and KPCF mice.The expression of the following important molecules of “PRRs-IFN-α/β-STAT1-ISGs” pathway was detected: RIG-1,TLR3,TLR4;IFN-α,IFN-β;STAT1,p STAT1;IP-10 and Mx1.Results PCFs degeneration showed significantly increases of RIG-1 and TLR3 m RNA expression in lung tissues,IFN-α and IFN-β secretion in BALF,STAT1 m RNA expression and p STAT1 protein expression in lung tissues,as well as the IP-10 secretion in BALF and Mx1 m RNA expression in lung tissues in 12-48 h post-RSV infection,which all peaked at 12 h.Conclusion PCFs degeneration promoted RSV-induced antiviral responses via IFN-α/β-STAT1 signaling pathway.PART Ⅱ VIP INVOLVED IN PCFS-REGULATED ANTIVIRAL RESPONSES INDUCED BY RSV VIA IFN-ALPHA/BETA-STAT1 SIGNALING PATHWAYObjectives PCFs exerts various biological effects by secreting neuropeptides such as SP,CGRP and VIP.Several studies have shown that neuropeptides affected viral replication.We hypothesized that PCFs degeneration may increase certain neuropeptide to promote IFN-α/β-STAT1 antiviral responses following RSV infection.Methods 1.BALF of 12 h,24h and 48 h post-RSV infection were collected from both groups,and the secretion level of SP,CGRP and VIP were detected by ELISA.2.VIPhyb(a pan VIP receptors antagonist)were administrated in KPCF mice,VIP or VPAC1 agonist were administrated in Intact mice respectively,mice were euthanasized at 12 h post-RSV infection,RIG-1,TLR3,IFN-α,IFN-β,STAT1,p STAT1,IP-10,Mx1 levels were detected.Plaque assay was performed on day 3 post infection to assess virus titers.Results 1.PCFs degeneration increased VIP secretion.2.After VIPhyb treatment in KPCF mice,RIG-1 and TLR3 m RNA expression in lung tissues,IFN-α and IFN-β secretion in BALF,STAT1 m RNA expression and p STAT1 protein expression in lung tissues,as well as IP-10 secretion in BALF and Mx1 m RNA expression in lung tissues were all significantly decreased in 12 h following RSV infection,and virus titers were increased.3.After VIP and VPAC1 agonist treatment in Intact mice,RIG-1 and TLR3 m RNA expression in lung tissues,IFN-α and IFN-β secretion in BALF,STAT1 m RNA expression and p STAT1 protein expression in lung tissues,as well as IP-10 secretion in BALF and Mx1 m RNA expression in lung tissues were all significantly increased in 12 h following RSV infection,and virus titers were decreased.Conclusions PCFs degeneration elevated the induction of VIP,which promoted RSV-induced antiviral responses via IFN-α/β-STAT1 signalling and decreased virus titers.PART Ⅲ PCFS AFFECT RSV-INDUCED IFNGR1 EXPRESSION VIA IFN-ALPHA AND MODULATE IFN-GAMMA MEDIATED AIRWAY INFLAMMATORY RESPONSESObjectives Previous study showed that IFN-γ plays important roles in the induction of airway inflammatory responses post-RSV infection,while PCFs degeneration alleviated RSV-induced airway inflammation and AHR,the IFN-γ was still at high levels.IFN-γ exerts its effects by binding to its receptor IFNGR1.We hypothesized that PCFs degeneration may downregulate RSV-induced IFNGR1 expression to antagonize IFN-γ-mediated airway inflammatory responses,and the increased IFN-α/β-STAT1 antiviral responses may be related to the downregulation of IFNGR1.Methods 1.Intact and KPCF mice were infected with RSV,HE staining of lung and ELISA of SP in BALF were performed on day 5.2.Immunohistochemistry and Western Blot were used to detect the expression of IFNGR1 in both groups post-RSV infection.3.Intact mice were given IFNGR1 Mab to neutralize IFNGR1,then airway inflammatory responses and IFN-γ levels in BALF were detect on day 5 post-RSV infection.4.The expression of STAT1 and p STAT1 was detected on day 5,and were also detected on days 1-5 post infection.5.Recombinant mouse IFN-α(r IFN-α)was intranasally administrated in Intact mice,then IFNGR1 expression was detected on day 5 post-RSV infection.Results 1.Both inflammatory cells infiltration in lung tissues and SP secretion in BALF were significantly decreased in KPCF mice post-RSV infection.2.IFNGR1 expression was increased in Intact mice,while PCFs degeneration decreased IFNGR1 expression post-RSV infection.3.After IFNGR1 neutralization in Intact mice,the total number of inflammatory cells in BALF,especially lymphocytes,were reduced post-RSV infection,AHR was also decreased.However,the level of IFN-γ in BALF was not significantly different from those of RSV group.4.The expression of STAT1 and p STAT1 were all increased significantly on day 5 post-RSV infection in KPCF mice,and the expression of STAT1 and p STAT1 increased continuously on 1-5 days after infection.5.After treatment with recombinant IFN-α,the expression of IFNGR1 was decreased in Intact mice post-RSV infection.Conclusion PCFs affect IFNGR1 expression by inducing IFN-α to regulate IFN-γ-mediated airway inflammation and AHR.