The Mechanism Of S1PR1 Regulating IL-1? Expression Induced By Newcastle Disease Virus

Newcastle Disease(ND),caused by Newcastle disease virus(NDV)is an acute,febrile,septic and highly contagious disease.NDV is one of the important pathogens mainly harmful to domestic poultry and related industry.Virulent NDV strain infection induces numerous IL-1? and causes strong inflammatory response,which lead to death.IL-1?,as the central mediator of inflammation,can increase the synthesis and release of IL-6,IL-8and vascular cell adhesion molecules,activate lymphocytes and promote the leukocytes and eosinophils infiltrate to inflammation site.Appropriate IL-1? level can help host to clear virus and repair tissue injury,but excessive IL-1? will develop inflammatory response,causing high morbidity and mortality.Previous studies mainly focused on inflammatory responses diversity induced by different virulence NDVs,but the effects of IL-1? on NDV-induced inflammatory responses and the molecular mechanisms by which NDV induces IL-1? expression are rarely reported.S1PR1 is an important target in inflammatory diseases treatment.It has been confirmed in mammals that S1PR1 regulates inflammatory cell infiltration and inflammatory cytokine expression induced by viral infection.However,the role of S1PR1 signaling pathway in viral-induced inflammation remains unclear in poultry.Previous studies in our laboratory have found that inhibition or over-expression of S1PR1 can significantly regulate IL-1? expression,suggesting that S1PR1 may alter the degree of inflammatory response induced by NDV infection by regulating pro-inflammatory cytokine expression.However,the mechanism that how S1PR1 regulate IL-1? is still unknown.Based on the above problems and previous researches,this study started with NDV in vivo/in vitro infection,selected appropriate cells,S1PR1 signaling pathway molecule and IL-1?-mediated inflammatory response as the research object,using the method ofover-expression and gene function interference or inhibition to analyze the relationship between NDV/NDV-component,S1PR1 signaling pathway and IL-1?-mediated inflammatory response from different levels and angles,which will reveal the signaling pathway of S1PR1 regulating IL-1? expression and its impact on NDV pathogenic.This research mainly includes the following contents:1.The effect of IL-1? on pathogenesis induced by NDVIn this study,SPF chickens were infected with different virulence NDV strains,the clinical symptoms,body temperature and deaths of the animals after infection were observed,and the organs were collected at 3 day post infection(dpi),the NDV replication and chicken inflammatory cytokines in organs were tested.We found that the body temperatures were significantly increased after NDV virulent strain infection,the clinical symptoms were obvious,and the mortality was 100%,multiple tissues and organs showed inflammatory injury,and numerous IL-1?,IL-6,IL-8 and TNF-? were detected.The IL-1?protein level is higher in tissues with stronger inflammatory reaction;The clinical symptoms of chickens infected with La Sota were mild,and the body temperature returns to normal after 1 dpi,no death was found.IL-1? neutralize antibody was applied to treat virulent NDV strain GM infected chickens.The IL-1? tissue distribution,body temperature were detected and mortality was record.Results shown that in NDV infection,IL-1? neutralizing antibody can effectively reduce IL-1? level in lung and glandular stomach,relieve the increase of body temperature,and reduce the mortality rate from 100% to 71.4%.2.NDV induces IL-1? expression via NLRP3/caspase-1 inflammasome and MAPK signaling pathwayTo identify host molecules involved in NDV-induced IL-1? expression,we examined NLRP3,caspase-1,ERK,p38,and JNK/MAPK activities by using gene over-expression,silencing or inhibitor.The results showed that virulent NDV strain GM could induce the activation of NLRP3/caspase-1 inflammasome in SPF chicken and avian cells;over-expression of NLRP3 increased NLRP3 expression,and GM-induced IL-1?expression increased to 169.3 pg/m L,which significantly higher than the p CA transfection and GM infected group(113.8 pg/m L);after stimulated with Si-NLRP3,NLRP3 expressionlevels decreased,GM-induced IL-1? expression levels decreased from 152.8 pg/m L to 87.1pg/m L.After inhibition of caspase-1,the expression of IL-1? induced by GM decreased from 127.5 pg/m L to 58.9 pg/m L;after inhibition of ERK/MAPK,NDV-induced IL-1? did not decrease significantly;IL-1? decreased from 113.0 pg/m L to 64.9 pg/m L after p38/MAPK inhibited;after inhibition of JNK/MAPK,GM-induced IL-1? decreased from124.0 pg/m L to 90.5 pg/m L,indicating that NDV can activate NLRP3/ caspase-1inflammasome,p38/MAPK and JNK/MAPK and promote IL-1? expression.3.NDV genomic RNA induces IL-1? expression via NLRP3/caspase-1In order to explore the NDV components that involved in the induction of IL-1?expression,DF1 cells were infected with UV-inactivated GM and GM.It was found that UV-GM could not induce IL-1? expression in cells.Subsequently,we constructed the eukaryotic expression plasmids of GM-V and GM-W genes,obtained genomic RNA of GM virus,transfected them into cells separately,the results shown that both GM-V and GM-W could be effectively expressed in cells.The expression of IL-1? was inhibited by GM-V transfection and the IL-1? expression slightly increased with no statistical difference when compared with N1 F group after GM-W transfection.The expression level of IL-1? induced by GM RNA was 93.5 pg/m L,which indicate that GM RNA can induce IL-1? expression.Based on the third chapter,we further examined the activation of NLRP3,caspase-1,p38 and JNK/MAPK after GM RNA transfection,and found that GM RNA could activate NLRP3/caspase-1 inflammasome,but the activated of p38 and JNK/MAPK were weak.Over-expression of NLRP3 increased IL-1? stimulated by GM RNA from 85.3 pg/m L to108.5 pg/m L,and IL-1? decreased from 86.5 pg/m L to 42.7 pg/m L after inhibition of NLRP3;After inhibiting caspase-1 activity,IL-1? expression induced by GM RNA decreased from 87.9 pg/m L to 64.6 pg/m L,indicating that NLRP3/caspase-1 is involved in GM RNA-induced IL-1? expression.4.S1PR1 regulates NDV-induced IL-1? expression via NLRP3/caspase-1In order to clarify the molecular mechanism of S1PR1 regulating NDV-induced IL-1?expression,we examined NDV-induced IL-1? expression changes after S1PR1 agonist SEW2871 treatment and found: SEW2871 stimulation could increased NDV-induced IL-1?expression from 121.5 pg/m L to 135.5 pg/m L;NDV-induced IL-1? expression was reducedfrom 115.3 pg/m L to 75.8 pg/m L after using the W146 inhibitor.Based on the study in Chapter 3,we further examined the activation of NLRP3,caspase-1,p38 and JNK/MAPK activated by S1PR1,and found that the activity of NLRP3 and caspase-1 was significantly increased after stimulating with S1PR1 agonist SEW2871,no significant increase in the activation of p38 and JNK/MAPK were found.After stimulated with W146,the activities of NLRP3 and caspase-1 were significantly decreased,and no significantly decreased of p38 and JNK/MAPK were found,indicating that S1PR1 can activate NLRP3/caspase-1inflammasome and further regulate IL-1? expression.Based on the clinical characteristics of NDV,combined with the latest research progress,and based on our previous research,we studied the molecular mechanism of S1PR1 involvement in NDV-induced IL-1? expression.The results mainly proved that IL-1? neutralizing antibody can reduce morbidity and death caused by GM infection;NDV promotes IL-1? production through NLRP3/caspase-1 inflammasome,p38 and JNK/MAPK signaling molecules;GM genomic RNA induces IL-1? expression,and this process depend on activation of NLRP3/caspase-1 inflammasome;further studies have demonstrated that S1PR1 regulates IL-1? expression via NLRP3/caspase-1 inflammasome.This studys form a new perspective that S1PR1 regulates the pathogenesis of NDV infection and provides scientific theoretical guidance for the prevention and control of ND.