Proteome-based Toll-like Receptor 2 Mediates The Inflammatory Response Induced By Japanese Encephalitis Virus

Japanese encephalitis virus（JEV）uses mosquitoes as the main carrier and pigs as the host.Over 60%of the world’s population lives in the coverage region of JEV.Each year,50000 to 170000 cases are reported to suffer the disease caused by JEV.The mortality rate in human is around 30%,and 50%survived patients have long-term neurological sequelae.JEV infection in pigs mainly occurs in the epidemic areas,infection with JEV can cause miscarriage and stillbirth in pigs.Infected piglets exhibit serious neurological diseases.Thus JEV poses a major risk to human and animal health.Epidemic Japanese encephalitis caused by JEV has spread in 24 countries,including Australia,Japan,India and most of the western Pacific and Southeast Asian countries.Based on the nucleotide sequences of JEV envelope protein,it can be divided into five genotypes,namely genotype I–genotype V.Genotype III was first detected in China,Genotype I has become the dominant type since it was isolated in Yunnan in 1977,then coexists with Genotype III.Understanding the distribution and prevalence of JEV genotypes have of great significance for the follow-up study,prevention and control of JEV.JEV can pass through the blood-brain barrier and cause damage to the central nervous system（CNS）.Microglia are residential macrophages of the the CNS.Microglia are the first line of defense against invasive pathogens,this cell type plays an important role in the inflammatory response via rapid activation and release of inflammatory cytokines to mediate inflammation.The recruitment of macrophage is a key step in the first line defense for prevention from viral induced damage.The innate response to the virus may enhance the inflammation and lead to pathological damage of the CNS.Toll-like receptor（TLR）can recognize the pathogen-associated molecular patterns（PAMP）and the active immunity and induce host defense.TLR family is one of the earliest determinants of immune activation.Dengue virus,Zika virus,Japanese encephalitis virus can be recognized by TLR and cause the activation of innate immunity.Previous studies showed that only TLR3,TLR4,TLR7,TLR8 and TLR9 are the key receptors for JEV,while the role of TLR2 in JEV induced inflammation is not clear.This study aims to reveal the mechanism underlying TLR2 mediated inflammatory response induced by JEV.Through investigation of JEV molecular epidemiology to understand the current popular JEV genotypes in China;to explore the role of TLR2 in mediating inflammatory response in JEV infected microglia and macrophages,and to verify it in mice.The main results of my study are as follows.Detection of JEV molecular epidemiology amplified 13 JEV E gene sequences,confirmed that the epidemic strain was genotype I.In total,74 phosphorylation sites and their kinases were predicted,of which 9 sites were mutated.In order to determine the molecular prevalence of JEV and the genotype of JEV strains in China,a JEV molecular epidemiological survey was conducted using 3105 mammals（pigs,foxes,raccoon dogs,yaks and goats）and 17300 mosquitoes in 11 provinces of China.JEV was detected in pigs in Heilongjiang（12/328,3.66%）,Jilin（17/642,2.65%）,Shandong（14/832,1.68%）,Guangxi（8/278,2.88%）and Inner Mongolia（9/952,0.94%）,goats in Tibet（1/51,1.96%）and mosquitoes in Yunnan（6/131,4.58%）.Thirteen gene sequences of JEV envelope（E）proteins were amplified from pigs in Heilongjiang（5/13）,Jilin（2/13）and Guangxi（6/13）for phylogenetic analysis,the glycosylation and phosphorylation sites of E protein’N154 were predicted.The results showed that the dominant JEV strain in China is Genotype I.JEV activates microglial inflammatory response through TLR2-PI3K-AKT pathway.In order to fully display changed pattern of protein expression in BV2infected by JEV,we took approaches of proteomics and phosphorylation proteomics to evaluate the extensive responses caused by JEV infected BV2 cells.The immune response at 6,12 and 24 hours past infection（hpi）confirmed the role of TLR2 in the inflammatory response of BV2 cells infected by JEV.In total,212 up-regulated proteins were detected at 6hpi,754 at 12h and 191 at 24h.According to GO and KEGG enrichment analyses,the up-regulated proteins were related to immune response.The results of parallel reaction monitoring（PRM）,Western blot and q PCR showed that the expression levels of TLR2,PI3K and AKT increased,and the phosphorylation levels of PI3K and AKT increased.My D88 and downstream proteins of My D88 did not be activated,these downstream proteins included IRAK1,IRAK4and TRAF6.By inhibiting expression of key proteins（TLR2,PI3K and AKT）,we confirmed that JEV activated the TLR2-PI3K-AKT pathway,leading to a strong inflammatory response.JEV activates the inflammatory response of macrophages through TLR2-PI3K-AKT pathway.JEV activates macrophage pyroptosis via TLR2.Results of proteomics and phosphorylation proteomics showed that proteins of cytokine-mediated signal pathway and TNF signal pathway were activated at 6 hpi.Innate immune response,apoptosis signal pathway and TLR-related pathway were all activated at 12 hpi.Defense reaction and enzyme inhibitor activity related signal pathways were activated after infection of raw264.7 cell line at 24 hpi.Comparison was carried out at the same time the difference of pathway activation between raw264.7 and BV2 cells after JEV infection.It was determined that JEV infected raw264.7 induced strong inflammatory response through TLR2-PI3K-AKT pathway.The programmed cell death pathway was activated at 12 hpi based on the proteomics.Both pyroptosis and apoptosis were found to be related to inflammatory reaction.Western blot results showed that pyroptosis key proteins NLRP3,cas1 and GSDMD were activated.TLR2 regulated the expression of NLRP3,and confirmed that NLRP3regulated the key proteins Cas1 and GSDMD of pyroptosis.Apoptosis related proteins such as Bax,cyc,cas9 and cas3 expression were not activated.TLR2 mediates inflammation when JEV infection occurred in mice.The activation of TLR2 was not conducive to the survival of mice.Results showed that TLR2-PI3K-AKT pathway is the key pathway of inflammatory response in vitro.In order to further explore the role of TLR2 protein in JEV-induced inflammation in mice,wild-type（WT）and TLR2 knockout(TLR2^-/-)mice were infected with JEV and the role of TLR2 in mediating inflammation was determined in mice.We examined the expression of inflammatory factors in macrophages,brains,blood,and testes in WT and TLR2^-/-mice.TLR2^-/-mice showed weaker inflammatory response.The detection of viral load showed no significant difference between WT mice and TLR2^-/-mice.PI3K and AKT were shown to be regulated by TLR2 in both WT and TLR2^-/-mouse macrophages and brains,consistent with their expression in BV2 and raw264.7in vitro.We monitored the survival rate of WT mice and TLR2^-/-mice after JEV infection.The survival time of WT mice was shorter than that of TLR2^-/-mice,indicating that the activation of TLR2 may not conducive to the survival rate of JEV infected mice.In conclusion,this study surveyed JEV molecular epidemiology of mammals and mosquitoes in 11 provinces of China.Genotype I JEV was found to be the dominant genotype in in the northeastern provinces and southwestern provinces of China.Genotype I JEV infection caused inflammatory reaction of both in microglia and macrophage through TLR2-PI3K-AKT pathway,and induced pyroptosis in macrophages through NLRP3.TLR2 was a key protein that mediates inflammation in mice.The activation of TLR2 in JEV infection shortened the survival time of mice.In this study,the inflammatory reaction mechanism caused by JEV was revealed,the role of TLR2 and related pathways was determined.These results have laid an important foundation for the prevention,control and treatment of JE in the future.