The Anti-APEC Innate Immune Response Mediated By DuNLRP3

Avian Pathogenic Escherichia coli(APEC)is an important Extraintestinal Pathogen E.col(ExPEC),which can cause local or systemic colibacillosis in chickens,ducks and other poultry.APEC is a common pathogen in duck production.It can infect ducks of any age.After infection,the incidence and mortality of ducks are high.The typical anatomical manifestations of APEC are fibrous pericarditis,perihepatitis,balloon inflammation,peritonitis and panoculitis.It has caused enormous economic losses to poultry breeding industry.Patterns recognition receptors are an important part of innate immunity,which can recognize pathogen associated molecular pattern(PAMP)and damage-associated molecular pattern(DAMP),activate downstream signal transduction pathways,promote the expression of immune-related genes and induce innate immune response.Nucleotide-binding oligomerization domain-like receptors(NLRs)can recognize many pathogenic components,tissue damage and cell stress in cytoplasm.NLRP3,a member of the P subfamily in NLRs,is well known for its ability to form a cytoplasmic complex,inflammatory,with ASC and caspase-1.Many studies have shown that NLRP3 plays an important role in promoting the expression of inflammatory cytokines IL-1?,IL-18 and TNF-?,regulating the signal pathway of NF-?B and promoting apoptosis.In mammals such as humans and mice,NLRs can recognize many components of bacteria in the cytoplasm,such as bacterial flagella,RNA,lipopolysaccharide,lipoteichoic acid and mural acyl dipeptides on peptidoglycan.However,some studies have pointed out that there are some differences of PRRs between the ducks and other species.Therefore,in order to determine whether there are NLRP3 receptors in ducks and whether this receptors participate in the host's innate immune response to bacteria,the full-length CDs sequences of duck NLRP3(duNLRP3)receptors were cloned;the virulence gene,growth curve and pathogenicity to Cherry Valley duck of strain APEC O1K1 were identified;the expression of innate immune related genes in vivo and in vitro after APEC infection was detected by relative fluorescence quantitative qRT-PCR;the role of duNLRP3 in innate immunity caused by APEC infection was clarified;In addition,we also identified the sub-localization of duNLRP3 in cells as well as its induction for the apoptosis and NF-?B.It mainly includes the followingfive parts:1.Cloning,identification and bioinformatics analysis of duNLRP3In this study,full-length CDs of duNLRP3 were obtained with the healthy duck spleen DNA as template.We found that the full-length CDs sequences of duNLRP3 was 2805 bp,encoding 934 amino acids(GenBank No.MH373356.1).Through software analysis,we found that duNLRP3 protein is an unstable hydrophilic protein without signal peptide and transmembrane domain.Du NLRP3 proteins contained NAHT domains,C-terminal LRR repetitive domains,and the N-terminal PYD domain.Homology and phylogenetic tree analysis showed that duNLRP3 proteins was located on Bird branching,and had close homology with Gallus,but far homology with Mammals.2.Pathogenicity of APEC on Cherry Valley DuckFirstly,we identified that APEC O1K1 isolates used in this experiment mainly contain aatA,tsh,fimC,mat,ibeB,vat,yijp,ompA,neuC,cva/cvi,iss,iroN,fyuA and irp2 virulence genes.These virulent genes may be responsible for the strong pathogenicity of the isolate to ducks.In addition,the growth curve of the strain was studied by the method of colony plate counting.It was found that the logarithmic growth period of the strain was 2-6 h,the stable period was 6-14 h,and the decay period was 14 h later.APEC could reach its peak at 2 dpi(days post infection)and could replicate rapidly and effectively in liver,spleen and brain.In addition,the infected ducks showed typical clinical symptoms and pathological change from 2dpi.These results indicate that APEC can cause severe infection in ducks,including the extensive tissue damage and dysfunction.3.Innate immune response caused by the APEC infectionTo clarify the innate immune response of host induced by APEC infection,we studied the expression of innate immune-related genes in liver,spleen,brain and DEFs cells after the bacterial infection.We found that the expression of TLR4 was significantly up-regulated at 1and 3 dpi.In spleen,TLR2,4,5 and 15 were down-regulated at 1 and 3 dpi.In the brain,TLRs were up-regulated significantly at 1 and 2 dpi.This phenomenon indicates that TLRs play an important role in regulating innate immunity in the process of bacterial infection,especially in the early stage of bacterial infection.In addition,after APEC infection,AvBD2 and 16 in duck liver,spleen and brain were up-regulated significantly,while AvBD4,5,7 and9 were down-regulated mainly.The expression of IL-1? in liver,spleen and brain was up-regulated at 1-3 dpi.As a major pro-inflammatory factor,IL-6 is up-regulated in liver and spleen and maintains the background level in brain.The expression of IL-8,another proinflammatory factor,was up-regulated steadily and significantly in all three tissues,especially in the brain.IL-8 was up-regulated 12-14 times steadily at 1-3 dpi.As one of the anti-inflammatory factors,the expression of IL-10 in liver,spleen and brain also increased in varying degrees.These results suggest that APEC infection causes inflammation in the host.In addition,the expression of MHC-I in liver,spleen and brain was up-regulated at 1-3 dpi,but MHC-II was down-regulated significantly.In addition,the in vitro and in vivo results are basically consistent,indicating that APEC infection can induce host to produce a large number of TLRs(such as TLR2),AvBDs(such as AvBD2),ILs(such as IL-6 and IL-8)and MHCs(such as MHC-I).Overexpression of these factors(especially IL-6 and IL-8)may lead to excessive inflammation in the body,which may cause some damage to host cells.4.Anti-APEC innate immunity mediated by duNLRP3We studied the tissue distribution of duNLRP3 in healthy duck tissues.We found that the expression of duNLRP3 was highest in pancreas,but lower in esophagus and ileum.These results indicated that duNLRP3 was widely distributed in healthy duck tissues and varied greatly.Du NLRP3 was up-regulated in the liver,down-regulated in the spleen at 1-3 dpi;but up-regulated in the brain at 2 and 3 dpi.These results suggest that duNLRP3 is involved in the innate immune response induced by APEC infection.In addition,through lentivirus-mediated duNLRP3 expression and si-RNA knockdown of duNLRP3 expression in DEFs cells,we found that overexpression of duNLRP3 could significantly inhibit the proliferation of APEC in vivo and in vitro.At the same time,after overexpression of duNLRP3,the expression of IL-1? in liver,spleen and brain was significantly up-regulated at 2 and 3 dpi;and the up-regulated multiples of IL-18 and TNF-? in brain were significantly lower than those in liver and spleen.The same results were also found in DEFs cells infected with NLRP3 lentiviral vector.Especially after APEC infection,the expressions of IL-1?,IL-18 and TNF-?were significantly up-regulated.When duNLRP3 was knocked down,the expression of IL-1?and IL-18 in DEFs cells induced by APEC infection was significantly decreased,but the inhibition of TNF-a was not significant.These results suggest that overexpression ofduNLRP3 can significantly induce the production of inflammatory factors.5.Preliminary study on duNLRP3 mediated signaling pathway of anti-APEC infectionWe found that duNLRP3 was mainly expressed in the cytoplasm and also in the nucleus.In addition,we co-transfected pc-duNLRP3 with pGL3-NF-?B-luc,pGL3-IFN-?-luc and pGL3-IRF-7-luc,respectively,and studied the activation of duNLRP3 on the promoters of NF-?B,IFN-? and IRF-7 by double luciferase reporter gene detection.We found that over-expression of duNLRP3 alone did not significantly increase the promoter activity of NF-?B and IRF-7,but slightly decreased the promoter activity of IFN-?.However,under APEC or LPS stimulation,over-expression of duNLRP3 could significantly enhance the activity of NF-?B,but had no significant effect on the activation of IFN-? and IRF-7promoters,and the promoter activity induced by LPS stimulation was higher than that induced by APEC infection.In addition,the transfection experiment of pSi-NLRP3 also proved that duNLRP3 could significantly activate NF-?B stimulated by APEC or LPS.These results suggest that duNLRP3 can significantly induce the activation of NF-?B under APEC infection or LPS stimulation.At the same time,overexpression of duNLRP3 significantly promoted the apoptosis of DEFs cells,and this effect was more significant after APEC infection.