| Pseudorabies virus（PRV）infection activates inflammatory responses to release robust pro-inflammatory cytokines,which is critical for controlling viral infection and clearance of PRV.However,the innate immune sensors and inflammasomes involved in production and secretion of pro-inflammatory cytokines,such as IL-1β,IL-18 and TNF-αduring PRV infection remain poorly characterized.To explore the mechanism of inflammatory response caused by PRV infection,we infected mouse peritoneal macrophages with PRV in vitro.Relative fluorescence quantitative PCR,western blotting and ELISA were used to detect the IL-1βat the levels of transcription,intracellular expression,and extracellular secretion,respectively.Immunoprecipitation and laser confocal were used to detect the formation of AIM2inflammasome and ASC oligomerization after PRV infection.In vivo,mice were intraperitoneally injected with PRV,the survival rate,serum levels of mature IL-1βand tissue transcription levels of inflammatory factors were measured.The results showed that PRV infection can induce the maturation and secretion of IL-1βand IL-18 in vivo and in vitro.In addition,PRV infection can cause ASC oligomerization and AIM2inflammasome formation.To investigate how PRV infection induces the transcription of pro-inflammatory cytokines,this study firstly used inhibitors of TLRs to pretreat the mouse peritoneal macrophages.The transcription,intracellular expression,and extracellular secretion of IL-1βwere detected.Secondly,different reporter detection systems were used to investigate the effect of overexpression of TLRs on NF-κB promoter activity and intracellular expression of pro-IL-1βafter mock-infection or PRV infection.Subsequently,si RNA targeting Tlrs and Myd88 were designed to explore the effect of silencing expression of these gene followed by reinfection with PRV on IL-1βtranscription and intracellular expression.Finally,the effects on PRV replication after the use of inhibitors or si RNA were measured respectively.The results showed that PRV infection increased the level of IL-1βthrough TLRs-NF-κB pathway,and shutting down this pathway could enhance PRV replication.In the exploration of the second signal,this study used different inhibitors and inflammasome gene knockout mice were used to explore the effect of inhibiting the expression of Caspase-1 and NLRP3 or in vivo inflammasome gene knockout on IL-1βsecretion,Caspase-1 activation,ASC oligomerization and PRV replication induced by PRV infection.UV-inactivated PRV were used to infect cells or exogenously transfect PRV genomic DNA to investigate whether PRV replication affected PRV-induced inflammatory response and which inflammasome participated in PRV-induced inflammation.The results showed that the replication of PRV affected the PRV-induced inflammatory response,and AIM2 inflammasome was involved in PRV-induced inflammatory responses.Finally,Gsdmd-/-and Gsdme-/-mice were used to explore the secretion process of mature IL-1β.The results showed that GSDMD was mainly involved in IL-1βsecretion during PRV infection.In this study,we reported that the transcription and expression levels of some proinflammatory cytokines,including IL-1β,IL-6 and TNF-α,are upregulated in primary peritoneal macrophages and in mice during PRV infection.Mechanistically,TLR2/3/4/5 sensed the PRV infection to enhance the transcription levels of pro-IL-1β,pro-IL-18 and GSDMD.Additionally,we found that PRV infection and transfection of its genomic DNA triggered AIM2 inflammasome activation,ASC oligomerization and Caspase-1 activation to enhance the secretion of IL-1βand IL-18,which was mainly dependent on GSDMD in vitro and in vivo.Taken together,our findings reveal that the activation of TLRs-NF-кB axis and AIM2 inflammasome as well as GSDMD is required for pro-inflammatory cytokines release,which resist the PRV replication and serve a critical role in host defense against PRV infection.Our findings provide novel clues to prevent and control the PRV infection. |
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