Preliminary Analysis Of Expression Changes Of Inhibitory Receptor And Cytokine In Chicken Infected Accurately By IBDV

PD-1and PD-L1belong to the B7-CD28superfamily, which was discovered inthe study of the hybridoma cell apoptosis regulation. Programmed death1(PD-1)and its ligands, PD-L1and PD-L2, deliver inhibitory signals that regulate thebalance between T cell activation and tolerance. Blockade of the PD-1/PD-L1pathway is able to restore functional capabilities to dysfunction or exhausted CD8+Tin the viral persistent infection. The PD-1:PD-L pathway delivers inhibitory signalsthat regulate both central and peripheral immune response, in control of cytokine andantibody production, proliferation and activation of CTL and tolerance ofautoantigen. The study about T cell immunity suppressive receptor PD-1just start inanimal immunosuppressive disease or virus persistent infection, especially in thepoultry disease.In this subject, expression changes and the relationship about immune status ofT lymphocyte immune inhibitory receptor PD-1and its ligands PD-Ls infected byIBDV were studied. The specific primers were designed according to the sequencesof PD-l, PD-L1, PD-L2, IL-2and IFN-γ from GenBank to amplify the objectivegenes for corresponding plasmids construction, and then they were used asquantitative template to construct the standard curve and melting curve for detectionsensitivity, specificity and repeatability. The relative expression level of chickenPD-l, PD-L1, PD-L2, IL-2, IL-6and IFN-γ mRNA in PBMCs from chicken weredetected on the3rd,5th,7th and10th day after IBDV infection by a SYBRS Greenreal-time fluorescent quantitative PCR assay, which established in this research. Atthe same time, the changes in expression of the important cytokines IL-2, IL-6andIFN-γ which associated with the immune regulation were determined at the sametime. After analysis of IBDV infection, The virus replication in vivo and therelationship between expression changes of PD-1, PD-L1and PD-L2on chickeninfected by IBDV.The established assay worked well with a good coefficient correlation(R2>0.99)while the template concentration was from1×101-1×109copies/μL, the sensitivity was sufficient to detect at least101copies of the samples. Real-time RT-PCRanalysis showed that the expression of PD-1increased significantly (p<0.05) in the7th day after infection, the expression of PD-L1and PD-L2increased significantly(P<0.01, P<0.05) in the3rd day after infection and the IBDV loads in bursal tissueare highest in the5th days after infection. The expression of IFN-γincreasedsignificantly (p <0.05) in the3rd、5th、7th day after infection, the expression of IFN-γ decreased significantly (p <0.05) after the expression of PD-1increased. Theexpression of IL-2and IL-6increased significantly (p <0.05) in the3rd day afterinfection. During the infection period, the expression of IL-2and IL-6decreasedsignificantly (p<0.05) in the5th,7th,10th day.The established assay was able to detect the expression of the six genes inPBMC. Results revealing the change of chicken PD-l, PD-L1, PD-L2, IL-2, IL-6and IFN-γ genes after IBDV infection, which showed that PD-l, PD-L1, PD-L2expression increased positively correlated with IBDV loads, and PD-L1, PD-L2increasing expression precede PD-1. After infection, the expression of IL-2、IL-6andIFN-γ were significantly increased with the expression of PD-l, PD-L1and PD-L2elevated, while the expression of IL-2, IL-6and IFN-γ were decreased. Theseresults suggest that expression of T cell immunity suppressive receptor PD-1and itsligands of PD-L1, PD-L2can be increased by infecting IBDV. Meanwhile theexpression of IL-2, IL-6and IFN-γ gene decreas in organism which appearsimmunosuppressive condition.