| Porcine reproductive and respiratory syndrome(PRRS) was caused by PRRSV. It is characterized by the productive failure and stillbirth in sows, and respiratory problems in piglet. PRRS has become one of the most severe disease globally. In China, the first prevalence of PRRSV was reported in 1996. PRRS caused significant economic loss to swine industry of China.At present, vaccination is still a conventional method for the prevention of PRRS.Currently available vaccines against the disease include modified live vaccine(MLV) and inactivated vaccine(IV). MLV is well recognized for its protective efficacy but there is risk of virus gene recombination and virulence return. IV, is well known for its safety, but it cannot trigger effective cellular immune and only confers limited protection. An effective adjuvant is desiderated to increase the protection and safety of inactivated PRRS vaccine.Dendritic cells(DCs) are potent antigen-presenting cells that play an important role in inducing primary antigen-specific immune responses. Many pattern recognition receptors(PRRs) expressed on the surface of DCs. DCs could capture and deal with antigen, then submit the antigen complex to prime naive T cells, induce T cells differentiate and bridge the innate and acquired immunity. Toll-like receptors(TLRs) were a kind of important PRRs expressing on the surface of DCs, which could specifically identify pathogen-associated molecular patterns(PAMP), activate the downstream signal transduction pathway, cause the production of inflammatory cytokines, interferon I, chemotactic factor and antibacterial peptide, and then activate neutrophile granulocyte and macrophage to kill the pathogen directly, which plays an important role in innate immune defence and acquired immune defence.Proteomics has an important significance for the research of life activity, disease mechanism and finding the early molecular marker of disease. The development andapplication of proteomics technology is becoming more and more widely, the method is also constantly updated. In 2004, the American biological application system company launched a new proteomics research technology isobaric tags for relative and absolute quantitation(iTRAQ). This technology has the characteristics of quantitative sensitive, quickly responsive,tag completely, high repeatability, high throughput, which has been applied in microbes,plants, animals, and nerve tissue, etc.1. MoDCs were incubated with different combinations of TLR3/7 ligands and inactivated PRRSV.TLR3 ligand poly(I:C) and TLR7 ligand imiquimod were differently combined to incubated Mo DCs and to vaccinate mice. The mRNA levels of Th1-type cytokines and Th2-type cytokines were examined by real time PCR, the concentrations of both Th1- and Th2-type cytokines in cell supernatant were detected by ELISA. Results showed that poly(I:C)-imiquimod-inactivated PRRSV antigen group showed the highest mRNA and protein levels of both the Th1- and Th2-type cytokines(P<0.05).To clarify the immune response mechanism of Mo DCs treated with TLR3 and 7 ligands along with inactivated PRRSV antigen, the mRNA levels of TRIF, MyD88 and NF-κB P65,the cytoplasmic protein levels of TRIF, MyD88, phosph-IκBα and the nucleous protein levels of NF-κB P65 in Mo DCs treated with different combinations for different times were analyzed. Results showed that compared with inactivated PRRSV antigen, the mRNA levels of TRIF, MyD88 and NF-κB P65 were stimulated highest as early as 8 h in poly(I:C)-imiquimod-inactivated PRRSV antigen group(P<0.05). The cytoplasm protein levels of TRIF, MyD88 and phospho-IκBα were obviously increased in poly(I:C)-imiquimod-inactivated PRRSV antigen group at 8 h and 12 h compared with other groups.The nucleous protein levels of NF-κB P65 were gradually increased from 1 h to 12 h in poly(I: C)-imiquimod-inactivated PRRSV antigen group, but the protein levels of NF-κB P65 and IκBα were not obviously changed in other groups. Mo DCs were pretreated with a specific NF-κB inhibitor BAY11-7082, the mRNA levels of cytokines were decreased to a basal level in poly(I: C)-imiquimod-inactivated PRRSV antigen group. Above all, we predicted that TRIF/MyD88-NF-κB signaling pathway was activated in Mo DCs treated with poly(I: C),imiquimod along with inactivated PRRSV antigen, and NF-κB play a key role for thesecretion of cytokines.Flow cytometry was conducted to evaluate the capability of Mo DCs to phagocytose PRRSV antigen. The results suggested that the combination of poly(I: C) and imiquimod showed the best effect of the phagocytosis of Mo DCs catching PRRSV antigen(P<0.05).2. Mice were immunized with different combinations of TLR3/7 ligands and inactivated PRRSV.Mice were immunized with different combinations of TLR3/7 ligands and inactivated PRRSV. Results showed that, in MTT results, the SI was significantly increased in poly(I:C)-inactivated PRRSV antigen group and poly(I: C)-imiquimod-inactivated PRRSV antigen group, and the differences were statistically significant between them(P<0.05). Percentages of both CD4+ and CD8+ T lymphocytes in splenocytes of mice immunized with poly(I:C)-imiquimod-inactivated PRRSV antigen were significantly increased when compared with the other groups(P<0.05). The highest percentages of CD3+ T cells producing IFN-γ and IL-4were detected in poly(I: C)-imiquimod-inactivated PRRSV antigen group(P<0.05). Both Th1-type, Th2-type cytokines and PRRSV-specific neutralization antibody levels were all significantly increased in poly(I: C)-imiquimod-inactivated PRRSV antigen group(P<0.05).3. Proteomics changes of Mo DCs in the process of PRRSV infection.A total of 252 cellular proteins in Mo DCs that were significantly altered at different time periods post-infection were identified. The expressed proteins of significant difference were analyzed by GO database, KEGG database and COG database. The expression of PRRSV receptor proteins such as vimentin and CD163 are significantly changed. Clathrin-depended pathway is one of the important pathway for endocytosis, the expression of clathtin associated proteins such as clathrin heavy chain(CLTC), clathrin light chain(CLTA),phosphatidylinositol-binding clathrin assembly protein(PICALM) and apetela 2(AP2) were significantly increased. The expression of antigen presentation associated protein MHC II molecules SLA-DR was also significantly increased. The expression of regulation of actin cytoskeleton associated proteins IQGAP(IQGAP1 and IQGAP2) were significantly decreased.The expression of JAK-STAT signaling pathway associated proteins STAT1, Mx1, Mx2 and OAS2 were significantly increased. The expression changes of PICALM, STAT1 and Mx1 were confirmed by western blot, which were in accordance with the results of iTRAQ.In order to know the interaction between PRRSV and Mo DCs, the network of differentially expressed proteins(P≤0.05 and ratios ≥1.5 or P≤0.05 and ratios≤0.67) were analyzed by String(Search Tool for the Retrieval of Interacting Genes/Proteins) software.These seed proteins have important functions in ribosome, endoplasmic reticulum for protein processing and metabolic pathway.We show that the synergistic combination of TLR3 and 7 ligands significantly enhances the function of Mo DCs to present inactivated PRRSV antigen through TLR/MyD88-NF-κB signaling pathway. Furthermore, the immune enhancing effect of the combination of TLR3 and 7 ligands is also observed in mice. This study demonstrates that the combination of TLR3 and 7 ligands could be used as novel adjuvant for PRRS inactivated vaccine. The analysis of proteomic analysis by iTRAQ provides a theoretical basis and direction for further understanding PRRSV infection and immune mechanism of Mo DCs. |
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