Mechanism Of SARM Inhibits TRIF On Airway Inflammation And Airway Hyperresponsiveness After RSV Infection

THE LEVEL OF SARM AND TRIF EXPRESSION POSTRSV INFECTIONObjective: Respiratory syncytial virus (RSV) is the most importantcause of lower respiratory tract infection in young children worldwide.TLR signaling play an important role in protecting from RSV infection. Inthe present study, RSV act as a trigger which activate TRIF dependentpathway and cause airway inflammation and AHR. To investigate the roleof SARM in airway inflammation after RSV infection, SARM and TRIFexpression after RSV infection.Methods: An overnight culture9HTEo cells in a6-well plate wasinfected with RSV at a multiplicity of infection of10for2hours. Theinfection was allowed to continue for12h,24h,36h,48h, or72h at37°Cunder5%CO2. Cells were collected for analysis at each time point.6-8week old female BALB/c mice, free of specific pathogens were incubated withRSV or UV-inactivated RSV. Total protein extracts from lung tissues or cellswere obtained using a total protein extraction kit. The protein concentrationwas determined using BCA assay reagent. The protein level was measuredby Western blot. Mice were divided into Control group, RSV+PBS groupand RSV+RES group, total protein extracts from lung tissues were obtainedusing a total protein extraction kit. The protein level was evaluated byWestern blot.Results: RSV was able to suppress SARM expression within36h ofinfection. SARM expression levels continued to gradually decrease withtime. In contrast, TRIF expression was increased in a time dependentmanner. RSV reduced SARM expression3days after infection. The levelsof SARM remained low, but the levels of TRIF increased at day5and day7post infection. Unlike live virus, UV-inactivated RSV was not able toreduce SARM expression in a time-dependent manner in vivo.Conclusion: Live but not UV-inactivated RSV suppressed SARMexpression in vivo and in viro. SARM interacts with TRIF and that onceSARM expression is induced by RES, the level of TRIF is reduced. MECHANISM OF SARM INHIBIT AIRWAYINFLAMMATION AND AIRWAYHYPPERRESPONSIVNESS AFTER RSV INFECTIONObjective: TLR signaling is important in RSV infection. In the firstpart we found that TRIF was induced and it’s inhibitor SARM wassuppressed. RES upregulated SARM expression after RSV infection.Previous studies has found that RES inhibited TRIF and reduced theinflammation and AHR caused by enhanced IFN-γ after RSV infection.This study is to investigate the relationship between airway inflammation,AHR and SARM after RSV infection. To investigate it’s mechanism.Methods: Mice were divided into Control group, RSV+PBS group,RSV+RES group, RSV+RES+siRNA negative group andRSV+RES+siRNA3-1group. CYP was administered, and5days later, micewere infected with RSV and were injected with RES one hourpost-inoculation. SARM siRNA was administrated to RSV-infected andRES-treated mice. Protein level was measured by Western blot. GFPexpression was detected by confocal microscopy. Lung function wasmeasured by whole-body plethysmography, lung histopathology wasexamined, and lymphocytes in bronchoalveolar lavage fluid were quantified. SARM and TRIF protein expression were detected in lung byWestern blot analyses. The expression of interferon-γ in BALF wasevaluated by ELISA.Results: Successful build in vivo model of SARM siRNA transfection.RSV increased TRIF expression by Western blot, while at the same timeinfection reduced SARM expression. In contrast, in mice treated with RESafter RSV infection, TRIF expression was inhibited and SARM expressionincreased. These results indicated that RES mediated inhibition of theTRIF-dependent pathway might rely on SARM expression. SARMexpression was elevated in the lungs of BALB/c mice treated with RESafter RSV infection compared to infection with RSV alone. siRNA wasused to knock down SARM mRNA level and reduce the protein expressionlevel in RES treated mice. The SARM siRNA construct effectivelysuppressed the level of SARM protein expression in the lungs of RSVinfected BALB/c mice treated with RES and the negative siRNA vector hadno effect. However, following SARM knock down TRIF expression wasincreased and the TRIF dependent pathway was induced. Mice infected withRSV had severe airway inflammation compared with uninfected controlmice. Treatment with RES reduced inflammation in mice infected withRSV. The effect of RES was reduced when SARM expression was knockeddown using siRNA. RES treated mice with SARM knocked down hadsimilar histologic findings as mice inoculated with RSV in the absence of RES. RSV infected mice had significantly more cells in the BALF thanuninfected mice. While treatment with RES reduced the cellularity of theBALF. SARM knockdown was sufficient to significantly increase the totalnumber of cells present in the BALF of RES treated mice compared to thenegative control siRNA treated group. There were qualitative differences incell types observed between treatment groups. The group treated with RESand siRNA3-1-treated RSV-infected mice had significantly increasedlymphocyte numbers (P<0.05) compared to negative control. RES andsiRNA negative-treated RSV-infected mice. Mice infected with live RSVhad significantly greater AHR than uninfected controls at methacholineconcentrations of between12.5and50.0mg/ml. Treatment with RESsignificantly reduced AHR caused by RSV. However, in mice with SARMknocked down after transfection, AHR was significantly higher than inmice transfected with the negative control siRNA vector and mice treatedwith RES after RSV infection. RSV infection significantly increased theinterferon-γ level in BALF compared to the uninfected control mice. REStreatment showed some protection and significantly reduced the level ofinterferon-γ compared to the RSV group. However, SARM knockdownabrogated the protective effect of RES treatment. The level of interferon-γwas significantly increased in the SARM knockdown group compared tothe negative control siRNA vector group. RSV-infected mice andRES-treated SARM siRNA3-1transfected RSV-infected mice had a similar RSV viral titer.Conclusions: SARM expression was reduced and TRIF expressionwas increased after infection with RSV. RES increased SARM expressionand decreased TRIF expression after RSV infection. SARM knockdown inRES-treated mice enhanced interferon-γ production, RSV-induced airwayinflammation, and AHR. RES decreased TRIF expression and prevented theRSV-mediated reduction of SARM expression. SARM attenuated airwayinflammation and AHR by the inhibition of the TRIF-dependent pathway,which suggested that SARM can be used as anti RSV therapeutic targets.