IRPS Modulate The Maturation And Function Of MoDCs Affected By PRV In Vitro

In this study, Monocyte-derived DC (MoDC) generated with GM-CSF and IL-4 were established in vitro. And the very virulent Pseudorabies Virus isolated from vaccinated Swine, named PRV CH/HN-12/6, was characterised. The modulation of maturation and founction by IRPS and PRV were analyzed respectively. And we also researched that IRPS modulated the maturation and function of MoDCs affected by PRV in vitro. All the results could help us learn the pathogenic mechanism of PRV and the immunomodulation mechanism of IRPS.1. Morphological, phenotypic and functional properties characterization of porcine monocytes-derived dendritic cells.To establish an optimal method for maturation induction of dendritic cells (DCs) in vitro, porcine monocyte-derived DCs (MoDCs) were induced in the presence of GM-CSF and IL-4. On Day 5, MoDCs were induced with LPS for 48 h, and harvested to detect morphological, phenotypic and functional properties characterization of MoDCs with LPS treatment or not. The cells were observed via light microscopy and scanning electron microscopy. FITC-dextran endocytic activity and the surface molecules of MoDCs were measured by FCM, and T lymphocyte proliferation following MoDCs stimulation by CCK-8 assay. The res μLts indicated that MoDCs had the typical morphology, and the surface molec μLar CD1a、CD80、CD86、 SLAIⅡ、D172a expression were changed with LPS treatment from 53.8%,42.4%,23.4%,54.2%, 48.2% to 55.2%,53.6%,43.6%,65.4%,61.1% differently. The ability of unstimulated DC to uptake FITC-dextran was higher than that of LPS-treated DC. And MLR assays revealed that LPS-treated DCs had a strong ability to stim μLate the naive T cells.Taken together, these dates demonstrated that the culture of porcine MoDCs induced from monocytes in vitro was established successf μLly.2. IRPS affect the maturation and function of dendritic cells in vitro.IRPS is known to have a variety of immunomodulatory activity. However, little is known about their immunomodulatory effects on porcine monocytes-derived dendritic cells (DC). DCs are professional antigen presenting cells, which play an important role in initiating Tcell responses against microbial pathogens. In this study, the regulatory effects of IRPS on maturation and function of cultured porcine MoDC were investigated in vitro. The cells were observed via light microscopy and scanning electron microscopy. FITC-dextran endocytic activity and the surface molecules of MoDCs were measured by FCM, and T lymphocyte proliferation following MoDCs stimulation byCCK-8 assay. The results indicated that MoDCs displayed a more mature morphology with long protrusions, and the surface moleculLar CD1a、CD80、CD86、SLAⅡ、 CD172a expression were significantly up-regulated by IRPS-treatment compared to LPS-treatment. The ability of unstimulated DC to uptake FITC-dextran was higher than that of IRPS- or LPS-treated DC. And MLR assays revealed that IRPS-treated DCs had a strong ability to stimulate the naive T cells.Taken together, these dates demonstrated that IRPS induce porcine MoDCs maturation in phenocytes and functions.3. Isolation, identification and pathogenicity of the very virulent Pseudorabies Virus isolated from vaccinated Swine.In this part, a pseudorabies virus was isolated and identified by PCR from dead piglets in many herds had suffered from a disease that was clinically similar to pseudorabies in Henan and Shandong province of China. The isolated PRV could induce typical cytopathogenic effect in vero cells and TCID50/mL was 1.45×108 which was detected in BHK-21 cells at the 8th passage. Experimental infection in newborn piglets was used to analysis the pathogenicity of PRV. The test group infected with this isolate developed typical nervous system symptom at 3 days post infection (dpi) and died at 4 dpi. The control group was normal. The gE gene of the virus was sequenced and and showed a nucleotide homology of 98.9% with GDSH-07 strain sequences published in GenBank, and phylogenetic analysis revealed that the sequences of this isolate clustered to the same region of the tree. The isolation was named PRV CH-HN.4. Modulations of phenotype, function and cytokine expression of porcine monecytes derived dendritic cells by PRV.In this part, phenotypic and functional property changes of porcine monecytes-derived dendritic cells (MoDCs) after PRV infection have been detailed. The surface phenotypes of MoDCs expression exposure to PRV were investigated. Exposure of PRV to MoDCs res μLted in a down-regulated expression of CD1a、CD172a、CD80、CD86、SLAII molecules in imDCs. These molecules were down-regulated significantly by PRV except for CD1a in imDCs. And this infection dramatically downregulated cell surface molecule expressions, such as CDla、CD 172a、 CD80、CD86、SLAII in mDCs exposure to PRV,especially CD86 and SLAII. MLR assays revealed an impaired capacity of PRV-infected DCs to activate allogeneic naive T cells. Antibody microarray demonstrated that exposure of Mo-imDCs to PRV resulted in a significantly increased secretion of IL-1β, TNF-a, IL-10 but not IL-12. And PRV inhibited the expression of IL-1β, TNF-a, IL-12 of DCs even upon response to LPS, but enhanced IL-10.5. IRPS modulate the maturation and function of MoDCs affected by PRV in vitroIn this part, the immunological function of IRPS was observed in MoDC exposure to PRV. MoDC were cultured for five days with medium which had GM-CSF and IL-4. On Day 5, MoDCs were stimulated with LPS or IRPS for 48 h, and exposure to PRV for 24 h, and then harvested to detect phenotypic and functional properties characterization of MoDCs. FITC-dextran endocytic activity, the surface molecules of MoDCs and cell apoptosis were measured by FCM, and T lymphocyte proliferation stimulation by CCK-8 assay, and cytokines expression by antibody microarray. The results indicated that IRPS could inhibit the down-regulation of MoDCs moleculars exposure to PRV, and inhibit the decreased ability of stimulating T cells compared to LPS-treated MoDCs.Antibody microarray demonstrated that IRPS inhibited the decline of IL-lβ and TNF-α expression by MoDCs exposure to PRV, and inhibited the expression of IL-10 induced by PRV, and enhanced IL-12 expression. Apoptosis was detected with Annexin V-FITC/PI double stains by FCM and the results indicated that PRV infection induced MoDCs apoptosis and IRPS could enhance the level of apoptotic cells. Conclusions:1. Monocyte-derived DC (MoDC) were generated with GM-CSF and IL-4 in vitro.2. IRPS induce porcine MoDCs maturation in phenocytes and functions.3. Identification and pathogenicity of the very virulent Pseudorabies Virus isolated from vaccinated Swine and named PRV CH-HN.4. PRV infection inhibited the maturation of imDCs and down-regulated the phenotypes, function and cytokine expression of mDCs derived from porcine monecytes.5. IRPS inhibited the effection on maturation and function of MoDCs by PRV in vitro, and enhanced the apoptosis induced by PRV infection.