The Mechanism Of Host Inflammatory Response Induced By Pseudorabies Virus Through Activation Of TLR2 Pathway

Pseudorabies virus（PRV）is an alpha herpesvirus that can cause neuroinflammation in a variety of mammals such as ruminants,rodents and carnivores.Since pigs are its natural host,PRV has caused huge economic losses to the pig industry worldwide.Toll-like receptors,as intrinsic pattern recognition receptors（PRRs）on the cell surface,play an important role in host resistance to pathogens,and TLR2 has been shown to play an important role in resistance to infection by some herpes viruses such as human herpes simplex virus（HSV）and varicella-zoster virus（VZV）.Previous studies in our laboratory have demonstrated that PRV infection of mouse macrophages induces the production and secretion of various inflammatory factors such as IL-1β,IL-6,and TNF-α.However,it is still unclear how TLR2 and its downstream molecular pathways function in the process of PRV infection and activation of pro-inflammatory cytokine production.This experiment was conducted in vitro and in vivo using wild-type mice and TLR2^-/-mice after infection with PRV,aiming to investigate the role played by TLR2 and its downstream signals in PRV infection.1、The study of inflammatory response induced by PRV in vivo after infecting susceptible hostWT mice were infected intraperitoneally with 5×10⁵ TCID₅₀ of PRV JS-2012,and the weight changes and symptom changes of the infected mice were recorded for3 days.Lung and spleen tissues were photographed and HE stained after 1 and 3 days of mouse attack,and sections were taken to observe the inflammatory pathological changes in lung and spleen tissues.To investigate whether TLR2/MyD88 plays a role in the inflammation induced by PRV infection in mice and in pathogenicity,lung,brainstem and trigeminal nerve were taken for grinding after 1 and 3 days of attack.q PCR was performed to detect the expression of inflammatory factors,TLR2,MyD88and the viral content in these tissues;after intraperitoneal infection of WT and TLR2-deficient mice with 5×10⁵TCID₅₀ of PRV,their survival was recorded.The results showed that the mice became progressively sicker and their body weight decreased gradually after the attack,and there was a significant difference from the original body weight by the third day;three days after the attack,severe inflammatory pathological changes occurred in the lung and spleen tissues of the mice compared with the non-attacked group:interstitial hyperplasia,inflammatory cell infiltration,splenic nodule atrophy,etc.;the expression of IL-1βand IL-6 in the lung and brainstem of the mice three days after the attack was significantly In the lung and brainstem,the expression of TLR2 was significantly higher on the first day after attack compared with the uninfected group,while in the brainstem,the expression of TLR2 was significantly higher on the first and third day after attack,but no significant changes were observed in the trigeminal nerve;three days after attack,the expression of MyD88 was significantly higher in the brainstem,but not in the lung or trigeminal nerve.Three days after virus infection,the expression of MyD88 was significantly higher in the brainstem,but not in the lung and trigeminal nerve;compared with the non-attacked group,only the viral content in the brainstem was significantly higher on the third day,while there was no significant change in the viral content in the lung and trigeminal nerve;ten days after virus infection,the survival rate of TLR2^-/-mice was80%higher compared with that of WT mice,while the survival rate of WT mice was20%.These results suggest that PRV infection in mice can cause severe inflammation in the lung,spleen and brainstem and that the virus is mainly concentrated in the brainstem.TLR2/MyD88 was also associated with the inflammation and pathogenicity of PRV in mice.2、The role of TLR2/MyD88-NF-κB in the PRV-induced host inflammatory responsePeritoneal macrophages from WT mice were infected with PRV,and the m RNA expression of TLR2 and MyD88 was detected by RT-PCR,which showed that the expression of TLR2 and MyD88 was significantly higher after PRV infection.To investigate how TLR2/MyD88 plays a role in PRV-induced cytokine production,WT and TLR2^-/-mouse macrophages were infected with PRV,and cytokine m RNA expression was detected by RT-PCR.The results showed that TLR2^-/-mice showed significantly lower m RNA expression of numerous cytokines,including IL-1βand IFN-β,compared with WT mice.The production of IL-1β,IL-6,and IFN-βwas further examined by ELISA,and the results showed that the production levels of these three factors were significantly decreased after deletion of TLR2 gene.Similarly,the production of IL-1β,IL-6,and IFN-βwas significantly reduced in the si-MyD88group compared with the si-Con group when these three factors were examined by ELISA after silencing MyD88.Further,mouse macrophages infected with PRV with WT and TLR2^-/-and after silencing MyD88 were examined by Western blot for changes in related proteins.The results showed that P-65,IRF3 proteins were hardly phosphorylated after deletion of TLR2 gene or silencing of MyD88.Taken together,our results show that there is no activation of NF-κB pathway and phosphorylation of IRF3,and correspondingly no production of cytokines such as IL-1β,IL-6,and IFN-β,after PRV infection of macrophages in the absence of TLR2/MyD88 gene,indicating that PRV infection of macrophages induced inflammatory response is dependent on the activation of TLR2/MyD88.3、The mechanisms of PRV-induced inflammation mediated by MAPKs and AKT signaling pathwaysTo determine the role of MAPK and AKT signaling pathways in inducing inflammatory activation in PRV-infected macrophages,in a PRV-infected WT mouse macrophage model,cells were pretreated with PRV using NF-κB pathway inhibitor（BAY11-7082）,AKT pathway inhibitor（Wortmannin）,JNK pathway inhibitor（SP600125）,ERK pathway inhibitor（FR180204）and P38 pathway inhibitor（SB203580）were pretreated cells for 1 h and then infected with PRV.The phosphorylation of P-65,AKT,JNK,ERK and P38 proteins was first detected by Western blot,and the results showed that the activation of the relevant pathways was significantly inhibited after using each inhibitor.Next,the m RNA expression of IL-1βand IFN-βand their production by ELISA were detected by RT-PCR,and the results showed that the m RNA expression as well as protein production of IL-1βand IFN-βwere significantly higher in the AKT inhibitor group and significantly lower in the JNK,ERK,and P38 inhibitor groups compared with the group not treated with inhibitors.Then,the mouse macrophages were infected with PRV in WT and TLR2^-/-and after silencing MyD88,and the changes of related proteins were detected by Western blot.The results showed that there would be little phosphorylation of AKT and JNK proteins in the TLR2^-/-mouse group and si-MyD88 group compared to the WT mouse group and si-Con group.Further,WT mouse macrophages pretreated with PRV infection by three inhibitors BAY11-7082,Wortmannin,and SP600125 for 1 h.The expression of m RNA of TLR2 and MyD88 was detected by RT-PCR,while the related protein levels were detected by Western blot.The results showed no significant changes in the expression of m RNA and protein production of TLR2 and no significant changes in the expression of m RNA of MyD88 compared with the group not treated with inhibitors,but at the protein level,MyD88 was significantly elevated in the AKT inhibitor group and significantly decreased in both NF-κB and JNK inhibitor groups.Taken together,our results show that the MAPK pathway and AKT pathway play positive and negative regulatory roles in the induction of IL-1βand IFN-βproduction by PRV-infected macrophages,respectively,and that the activation of AKT and JNK pathways is equally dependent on TLR2/MyD88,meanwhile the activation of AKT,NF-κB and JNK pathways can also promote or inhibit the production of MyD88 protein.