Study On Biological Characteristics Of Porcine CD83 And Its Soluble Protein Alleviates LPS-induced Abortion In Mice

CD83 is a transmembrane glycoprotein and belongs to the type ? immunoglobulin superfamily.CD83 was originally expressed on mature dendritic cells(DC),so it was used as a molecular marker for DC maturation.With the deepening of research,it was found that the expression of CD83 is not limited to mature DC,but also expressed in T cells,B cells and monocytes.There are two forms of CD83 molecules,expressing CD83 on the cell membrane,called membrane-bound CD83(Membrane-bound CD83,mCD83).and the extracellular domain of CD83 which is hydrolyzed off by protease is called soluble CD83.Both forms of CD83 have immunoregulatory functions,but they have opposite functions.The mCD83 promotes DC-mediated T cell activation and improves the immune response,while sCD83 exerts an immunosuppressive effect by inhibiting the differentiation of human monocytes into DC and DC-mediated T cell proliferation.And this immunosuppressive effect of sCD83 has a significant effect on the prevention and treatment of certain autoimmune diseases.Recent studies have confirmed that the pregnancy process of animals(including humans)is accompanied by an increase in serum sCD83,indicating that sCD83 may play an important regulatory role in regulating the immune tolerance of the maternal-fetal interface and maintaining normal pregnancy in animals.So far,studies on CD83 in humans and mice have been extensive and in-depth,while there have been few studies on CD83 in other animals.There is no research report on porcine CD83.To systematically study the biological characteristics of porcine CD83 and explore the application of sCD83 in animal breeding practice.This study started from the cloning and expression of porcine CD83,and analyzed the molecular and biochemical characteristics of porcine CD83 and its immunomodulatory function.The embryo protection and its molecular mechanism of porcine sCD83 was investigated by using LPS-induced abortion mice.1.To clone and express porcine CD83,the primers were first designed according to the predicted sequence of porcine CD83 gene in GenBank.The full-length CD83 and sCD83 genes were cloned from the spleen by RT-PCR.Sequences result showed that the porcine CD83 sequence has high homology with other mammalian species(>70%).Three N-glycosylation sites and five cysteine sites forming disulfide bonds are highly conserved across species.By constructing the expression vectors of full-length CD83 and sCD83 gene,the highly efficient expressions of CD83 were achieved in HEK293T cells.The expression of CD83 on the surface of mature DC cells(mDC)and the level of sCD83 in the mDC medium and serum were detected.Results proved that porcine CD83 has two forms(mCD83 and sCD83)in vivo.2.To analyze the glycosylation and biochemical characteristics of porcine CD83 and the production mechanism of sCD83.The N-glycosylation inhibitor(tunicamycin)was used during CD83 expression in HEK293T cells and analyzed the result of Western blot.The porcine CD83 was demonstrated to be a highly glycosylated protein.Through adopting site-directed mutagenesis strategy and using reducing loading buffer,the porcine CD83 was proved to be a homodimer mediated by an intermolecular covalent disulfide bond formed by the fifth cysteine.By using protein synthesis inhibitor CHX,the sCD83 is mainly produced by mCD83 digestion and hydrolysis3.To investigate the immunoregulatory functions of porcine mCD83 and sCD83,the lentiviral vector-mediated RNA interference strategy was used to down-regulate the expression of CD83 in DC cells,and the effect of CD83 down-regulation on DC-mediated T cell activation was analyzed.The results showed that down-regulating CD83 expression could significantly inhibit DC mediated T-cell activation and expression of related cytokines:IFN-?(P<0.05)?IL-2(P<0.01)?IL-4(P<0.05)and IL-10(P<0.01),indicating that porcine mCD83 can promote DC-mediated T cell activation.Administration of DCs with recombinant porcine sCD83 significantly inhibited DC-mediated T cell activation and expression of IFN-y(P<0.01)and IL-2(P<0.01),but did not significantly affect the expression of IL-4.This result indicates that porcine sCD83 can inhibit DC-mediated T cell activation,and its inhibition on cytokine productions is biased to Thl cytokines,suggesting that porcine sCD83 can regulate the balance of Thl/Th2 cytokines.The glycosylation of porcine CD83 was proved to plays an important role in its regulatory function.In addition,porcine sCD83 can inhibit the differentiation of monocytes into DC cells in vitro.4.To investigate whether porcine sCD83 has the biological function of regulating immune tolerance at the maternal-fetal interface.The LPS-induced abortion mice model was established to analyze the effects of porcine sCD83 on abortion rate of mice and its regulatory mechanism.Results showed that treating pregnant mice with recombinant porcine sCD83 could significantly reduce the abortion rate induced by LPS in mice(P<0.01),suggesting that porcine sCD83 could protect embryos.Further analysis showed that porcine sCD83 could inhibit the IFN-y expression(P<0.01)of Th1 type cytokines and promote the IL-10(P<0.01)and IL-4(P<0.05)expression of Th2 type cytokines.It was shown that porcine sCD83 can regulate the balance of Th1/Th2 cytokines and shift them toward Th2 cytokines which are conducive to embryo stability.Meanwhile,we found that treating pregnant mice with porcine sCD83 can increase the frequency of Treg cells in the uterus and inguinal lymph nodes(P<0.05),which can promote the maintenance of immune tolerance at the maternal-fetal interface.5.Embryonic-derived trophoblast cells are key cells for the implantation of the embryo into the mother's body and placenta formation.To analyze whether porcine sCD83 has the function in regulating trophoblast cells.Based on the determination that sCD83 can be combined with trophoblast cells.Transwell cell invasion experimental device was performed and indicated that porcine sCD83 significant promote the invasion of trophoblast cells(P<0.01).In order to explore its mechanism,we have confirmed that porcine sCD83 can promote the expression of matrix metalloproteinases(MMPs)in trophoblast cells,and inhibit the TNF-?(P<0.01)and IL-6(P<0.01)expression induced by LPS.These results indicated that porcine sCD83 promotes trophoblast cell invasion and it is involved in the promotion of MMPs expression and the inhibition of the expression of pro-inflammatory cytokines.In conclusion,this study systematically studied the biochemical characteristics,immunoregulatory functions of porcine CD83,and the embryo protection of recombinant porcine sCD83 in vivo and in vitro.Results showed that,like humans and mice,porcine CD83 is a dimeric glycoprotein that exists in two forms(mCD83 and sCD83)and have opposite immunomodulatory effects.Using the LPS induced mouse abortion model,we demonstrated that porcine sCD83 significantly inhibited LPS-mediated abortion.This embryo protection of the sCD83 is related to the promotion of Th2 cytokines expression,Treg cells frequency and trophoblast invasion.This study not only enriched the knowledge of the biological characteristics of porcine CD83,but provided an important scientific basis for the application of sCD83 in pig production to improve the fertility rate of pigs and other animals.This study provides new ideas for the prevention and treatment of some diseases related to immune tolerance disorder in animals.