| We examined the dynamic effects of PCV2 on the secretion of IL-4 and IL-12 in piglet lymphocytes, and the signal transduction pathway. To gain insight into the mechanism of immunosuppression caused by porcine circovius type 2 (PCV2) infections in piglet to provide a new strategy for prevention of PCVD.Eight conventionally weaned piglets with no antibodies and antigen against PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) were chosen as the experimental animals. The suspensions of the single cells isolated from spleens were divided into four groups following:control group, nuclear factor kappa B (NF-κB) inhibition group (inhibition group), PCV2 group, and NF-κB inhibitor-PCV2 group (inhibitor-PCV2 group). Cells were inoculated, and then supernatants and lymphocytes were respectively harvested after 0,6,12,24 and 48 h. The observation of the PCV2-infected cells and the nuclear translocations of nuclear factor kappa B (NF-κB) were conducted using Indirect Immunofluorescence Assay (IFA). Flow cytometry was used for quantitative assessment of the numbers of infected cells. The concentrations of IL-4, IL-10 and IL-12 secreted in the supernatants were measured by ELISA. The proteins concentrations of NF-κB/p65 in nucleus, myeloid differentiation factor 88 (MyD88) phosphorylated IκB (p-IκB) and NF-κB/p65 in cytoplasm were detected by Western blot. Electrophoretic mobility shift assay (EMSA) detected NF-κB DNA binding activity. Analysis of the levels between IL-4, IL-10 and IL-12 with the MyD88-NF-κB signaling pathway. The results demonstrated that the PCV2 antigens were seen in lymphocytes at 6 h and most in cytoplasm by IFA; Using flow cytometry, the positive rate was 1.19% at 6 h. At 48 h, the positive rate was up to 17.48%. The levels of IL-4 in the PCV2 group was significantly decreased campared with the control group and the inhibition-PCV2 group (P<0.05) after 12 h,24 h and 48 h infection. The levels of IL-10 had no difference in the inhibitor-PCV2 group campared with PCV2 group. After 12 h PCV2 infected, the levels ofIL-12 was higher than PCV2 group compared with control group (P<0.05). In the same time points, there was no significant changes between PVC2 group and inhibition-PCV2 group. The levels of IL-12 in PVC2 group was higher than the control group, and it is a time-dependent rise in 6 h,12 h and 24 h (P<0.05). The levels of IL-12 was decreased or significantly decreased in inhibition-PCV2 group compared with PCV2 group (P<0.05, P<0.01). The expression of MyD88 was significant increased compared with control group after infected with PCV2 (P<0.05, P<0.01). There was no significantly difference between PCV2 group and inhibition-PCV2 group. The levels of p-IκB in infected PCV2 cells were increased at 24 h (P<0.05) and significantly increased at 48 h (P<0.01). The levels of p-IκB in inhibited-PCV2 group was significantly decreased compared with PCV2 group after 6 h (P<0.01). The nuclear translocations of NF-κB/p65 were detected by IFA. After 6 h, the results showed that NF-κB/p65 can detected in the nucleus of lymphocytes, and the signals were increased in a time-dependent way. After 12 h, Western Blot results showed that levels of cytoplasmic NF-κB/p65 in PCV2 group was decreased compared with control group (P<0.05). There were no significant changes of the cytoplasmic p65 expression among control group, inhibition group and inhibition-PCV2 group. The levels of nuclear NF-κB/p65 was increased in PCV2 group compare with control group after 6 h,12 h,24 h and 48 h (P<0.01). The levels of nuclear NF-κB/p65 was significantly decreased in inhibited-PCV2 group compared with control group at each time points (P<0.01, P<0.05). The EMS A results showed that after 12 h and 48 h PCV2 infected NF-κB DNA-binding was significantly increased than control group (P<0.01). The NF-κB DNA-binding was still increasing at 24 h (P<0.05). The NF-κB DNA-binding in inhibition-PCV2 groups were significantly decreased compared with PCV2 groups (P<0.01). The results demonstrated that PCV2 could infect piglet lymphocytes in vitro and the expression of IL-4 and IL-12 in the PCV2-infected piglet lymphocytes was regulated through the activation of MyD88-NF-κB signaling pathway. |
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