Analysis Of Endothelial-derived IL-8 Induced By PCV2 Regulating NF-?B Signal Pathway Related To The Maturation Of Monocyte Derived Dendritic Cell

Porcine circovirus type 2?PCV2?can cause porcine immunosuppressive diseases.Vascular endothelial cells?VEC?,the target cell of PCV2,play an important role in immune response and inflammatory regulation.Previous study showed PCV2-infected iliac artery endothelial cells?PIECs?could upregulate the secretion of endothelial IL-8 which inhibited the function and maturation of monocyte-derived dendritic cells?MoDCs?.Co-culture system of PIEC and MoDC by transwell was used to analyze endothelial-derived IL-8 induced by PCV2 regulating NF-?B signal pathway related to the maturation of MoDC from gene and protein levels with gene chip technology,western blot,and laser confocal microscopy.Probe technology improved the effect of endothelial-derived IL-8 induced by PCV2 on the adhesion of MoDC.All will provide the experimental basis for elucidating the pathogenic mechanism of PCV2.According to the different treatment methods of the iliac artery endothelial cells?PIEC?,control group?PIEC-DC?,PCV2-infected group?PCV2-PIEC-DC?,IL-8-silenced group(IL-8^low-PIEC-DC),IL-8-overexpressed group(IL-8^hi-PIEC-DC),IL-8-overexpressed group infected with PCV2(PCV2-IL-8^hi-PIEC-DC),and IL-8-antibody group?Ab-PIEC-DC?were prepared.The adhesion function of MoDCs was detected by molecular probe,and adhesion rate was calculated by fluorescence microscope.The results showed that two groups displayed similar tread.Compared to the PCV2-PIEC-DC group and the IL-8hi-PIEC-DC group,the adhesion rate of the PIEC-DC group was significantly upregulated,but was significantly lower than that of the IL-8 low-PIEC.The adhesion rate of PIEC-DC group and Ab-PIEC-DC group was higher than that of PIEC-DC group.These results suggested that PCV2-induced endothelial-derived IL-8 inhibit adhesion function of MoDC.Control group?DC?,co-culture control group?PIEC-DC?,PCV2 infection group?PCV2-PIEC-DC?,IL-8-silencedgroup(IL-8^low-PIEC-DC),IL-8-overexpressedgroup(IL-8^over-PIEC-DC)were set up.The gene expression profiles of MoDC in different treated group were detected by Agilent microarray,and two times more than the differentially expressed genes were analyzed by GO and Pathway analysis.The differential genes of MoDC in different groups were mainly related to immune response,antigen presentation,inflammatory response,angiogenesis,cell adhesion and other functions,and differential genes mainly focused on NF-?B signaling pathway related to MoDC maturation.The expressions of NF-?B p65 and I?B?of MoDC in different treatment groups were analyzed by Western Blot,and the nuclear transporation of NF-?B p65 in MoDC was observed with the confocal laser scanning.Compared with the control group?DC?,the expression of NF-?B p65 was significantly decreased,and the expression of I?B?was significantly increased in PIEC-DC.Compared with the PIEC-DC,the expression of NF-?B p65 was significantly decreased and the expression of I?B?was significantly increased in IL-8^hi-PIEC-DC and PCV2-PIEC-DC.The expression of NF-?B p65 was significantly increased and the expression of I?B?was significantly decreased in IL-8^low-PIEC-DC compared with PIEC-DC.All these indicated that endothelial-derived IL-8 inhibited the activation of NF-?B signaling pathway and nuclear transfer of NF-?B p65 in MoDC,which suppressed the maturation of MoDC.In addition,PCV2-PIEC-DC,PCV2-Ab-PIEC-DC and its BAY11-7082 pretreatment PCV2-Ab-PIEC-BAY-DC,IL-8^hi-PIEC-DC,IL-8^hi-Ab-PIEC-DC and its BAY 11-7082 pretreatment IL-8^hi-Ab-PIEC-BAY-DC were established.The expressions of IL-12 and GM-CSF related to the maturation of MoDC were detected by real-time RT-PCR.The results showed that the expression levels of IL-12 and GM-CSF in PCV2-Ab-PIEC-DC and IL-8hi-Ab-PIEC-DC were significantly higher than those in PCV2-PIEC-DC and IL-8^hi-PIEC-DC.The expression levels of IL-12 and GM-CSF in PCV2-Ab-PIEC-BAY-DC and IL-8^hi-Ab-PIEC-BAY-DC were significantly lower than those in PCV2-Ab-PIEC-DC and IL-8^hi-Ab-PIEC-DC,which indicated the low expressions of IL-12 and GM-CSF caused by the secretion of endothelial-derived IL-8 induced by PCV2 were closely related with NF-?B signaling pathway.In summary,the endothelial IL-8 induced by PCV2 infection suppressed the maturation of MoDC by inhibiting the activation and degradation of I?B?in MoDC and the activation of NF-?B p65,impeding nuclear transfer of NF-?B p65 and the expression of related cytokines IL-12 and GM-CSF,which inhibit the maturation of MoDC.