PIL6-TLR4 Transgenic Sheep Identification And Study Of The Immune Response To Salmonella

Lipopolysaccharide(LPS),also called endotoxin that is released after destruction of the bacterial cell wall,is a major component of the outer membrane of Gram-negative bacteria.There are many kinds of Gram-negative bacteria such as Escherichia coli,V.cholera,Salmonella and Brucella.They often cause many infectious diseases in digestive tract,reproductive system and blood of humans or other animals,which leads to great losses in animal husbandry and threatens human health.In the infected host,Toll-like receptors can recognize pathogen-associated molecular patterns(PAMP),which results in an immediate host response to infection with pathogens and initiates the innate immune response.TLR4(Toll-like receptors 4),which recognizes LPS and initiates a series of intracellular responses,invokes a vigorous cytokine response among immune cells against Gram-negative bacteria.TLR4 activates the My D88 and TRIF-dependent pathways through nuclear factor-κB(NF-κB)–associated signaling events.TLR4 also acts as a regulator in the immune system.Following the activation of TLR4,adaptive immune responses are improved.Eukaryotic expression vector 3S-p CMV-TLR4 was constructed and the recombinant plasmids were transfected into sheep fibroblasts.Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs by others in our lab.In this study,effects of TLR4 overexpression on inflammation response were addressed in vitro.39 sheep were detected using Southern blot analysis and four positive transgenic sheep carrying the exogenous TLR4 were obtained.The peripheral blood mononuclear cells of sheep were isolated and infected with Salmonella(MOI = 100)to investigate the effect of transgenic and wild type sheep on the intracellular parasitism of Salmonella.The expression of TLR4 m RNA and protein was determined respectively by q PCR and ELISA.Several important cytokines(TNF-α,IL-6,IL-8,IL-10,IFN-γ)that have different physiological functions in inflammatory responses were detected.In the meanwhile,several oxidative stress factors(NOS,NO,GSH,GSSG,ROS,MDA)that can be act as indicators of oxidative damages were measured.DAPI and PI staining were performed on the infected cells to compare the death of peripheral blood mononuclear cells between transgenic sheep and wild type sheep.The number of intracellular Salmonella in transgenic sheep was significantly lower than that in wild type sheep at 24 h and 48 h(p <0.05).The expression peak of TLR4 m RNA in transgenic sheep was at 4h.The m RNA expression of IL-6,TNF-α,IL-8 and IFN-γ in transgenic sheep was significantly higher than that in non-transgenic sheep at 4 h,12 h and 24 h(p <0.05).The expression of anti-inflammatory factors IL-10 was up-regulated at the later stage of inflammatory response,and inhibited the expression of pro-inflammatory cytokines and inflammatory cytokines.With the presence of Salmonella,transgenic sheep induce earlier and higher expression of inflammatory cytokines than that in non-transgenic sheep,and also trigger the earlier and higher expression of anti-inflammatory cytokines,which is a coexistence of high oxidative level and high reduction level,protecting the cells from damage.There were no significant differences between the peripheral blood mononuclear cell death rate of transgenic sheep and wild type sheep by DAPI and PI staining and HCA(high content analysis)statistical analysis.The Salmonella infection of sheep peripheral blood mononuclear cells showed that the expression of TLR4 was up-regulated in transgenic sheep,and the expression of oxidative stress factor was up-regulated,which improved the killing capability for Salmonella.At the cellular level,the transgenic sheep could effectively inhibit the intracellular parasitism of Salmonella.