| Porcine circovirus type 2(PCV2) is the major pathogen of porcine circovirus associated diseases(PCVAD), such as postweaning multisystemic wasting syndrome(PMWS). PCV2 infected pigs show immunosuppressive characteristics, and PCV2 infection lead the pigs sensitive to other pathogens, as well as lead the vaccines lost the affections, to further make the mortality rate rise and enlarge the economic losses of pig industry. Monocyte-macrophage cells are the main cell types for body’s defense and pathogen presentation of the immune system. PCV2 infects and persists in the cytoplasm of these cells to affect the anti-microorganism activities. This long-term coexistent with the cells can be used to be an excellent mechanism for PCV2 to evade immune recognition, replicate and further pathogenic to the host. However, the mechanism of how PCV2 regulates the functional activity of macrophages is still largely unknown. In this study, PCV2 strains were isolated from the nature PMWS pigs. Then PCV2 were infected to porcine alveolar macrophages(PAMs) to investigate the kinetics and regulatory mechanism of interleukin(IL)-10 and IL-12p40 expression. The impact and regulatory mechanism of the interactional components of PCV2 and the host cells for IL-10 production were preliminary determined. The results were as follow.1. The whole genomes of two PCV2 strains(SXXY and SXBJ) were amplified from tissues of nature PMWS pigs. The PCV2 strains were identified to be PCV2 a and PCV2 b genotypes. The PCV2 genome DNAs were recycled in vitro then transfected PK-15 cells. After 5 passages, two infectious PCV2 viruses were obtained.2. The PCV2 ORF1, ORF2 and ORF3 genes were cloned into pET-32 a vectors. The recombined plasmids were then transfected into E. coli BL21. IPTG were used to induce the expression of PCV2 Rep, Cap and ORF3 proteins. SDS-PAGE assays showed the PCV2 Rep, Cap and ORF3 proteins at the sizes of about 52 KDa, 39 KDa and 29 KDa were correctly expressed. The proteins were collected to immunize rabbits. The serum of the rabbits were harvested and purified. The purified polyclonal antibodies were specific and the titers were higher than 1 : 10 000.3. The PCV2 ORF1, ORF2 and ORF3 genes were cloned into pShuttle-CMV. The recombined shuttle plasmids were linearized by Pme I, then transfected into E. coli BJ5183 containing pAdeasy-1 by electro-transformation. The recombined adenovirus plasmids were linearized by Pac I and transfected into 293 A cells to generate recombined adenoviruses expressing PCV2 Rep, Cap and ORF3 proteins. RT-PCR and western blotting confirmed the expression of PCV2 Rep, Cap and ORF3 proteins by the recombined adenoviruses at the sizes of about 52 KDa, 39 KDa and 29 KDa.4. PCV2 and PCV1 were infected to PAMs, respectively, ELISA and dual luciferase activity assays results found that PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1(p < 0.01). Quantitative-PCR results showed that PCV2 upregulated IL-10 mRNA expression twice upon infection(p < 0.05,p < 0.01), and the IL-10 mRNA expression in the latter phase was significantly higher than the earlier phase(p < 0.05). Western blotting and ChIP assays investigated that upon initial PCV2 infection, the p50/p50 mediated NF-κB pathway and Akt1 mediated PI3K/Akt pathway were activated, the p38 MAPK and ERK pathways were further activated in the latter phase, while PCV1 could only activate NF-κB pathway in the latter phase of infection. Recombined adenoviruses expressing PCV2 Rep, Cap and ORF3 proteins were infected to PAMs, the results showed that Cap and Rep upregulated IL-10 expression(p < 0.01), while ORF3 did not. Western blotting results showed Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways. Downregulation or knockout of the interacting protein of Cap, gC1 qR, significantly suppressed the PCV2-induced PI3K/Akt and p38 MAPK activation, resulting in significantly decrease or block of IL-10 production in PCV2-infected cells(p < 0.05, p < 0.01).5. PAMs were harvested from the piglets infected by PCV2, flow cytometry results showed the CD68 positive PAMs of the whole cells was 55.3% in PCV2 infected piglets, and 87.6% in control piglets. Flow cytometry assays investigated that the marker of M2 macrophages, CD206, was significantly upregulated in PAMs from PCV2-infected piglets for 2 weeks(p < 0.05), and further upregulated in PAMs from PCV2-infected piglets for 4 weeks(p < 0.01), while CD163 was not. And Q-PCR results found that the mRNAs of M1 macrophages were significantly downregulated(p < 0.01), while the mRNAs of M2 macrophages were significantly upregulated(p < 0.01). Flow cytometry and Q-PCR assays showed the LPS/IFN-γ induced IL-12p40 expression in PCV2-infected PAMs was repressed both in transcriptional and post-transcriptional levels. Screen of related miRNAs by Q-PCR, the results showed miR-23a/b were significantly upregulated in PCV2-infected PAMs(p < 0.05, p < 0.01). The miR-23a/b mimics could inhibit the LPS/IFN-γ induced IL-12p40 expression(p < 0.05) and the miR-23a/b inhibitors abated the inhibition of PCV2 to LPS/IFN-γ induced IL-12p40 expression(p < 0.05).This study isolated 2 PCV2 strains, obtained anti PCV2 Rep, Cap and ORF3 polyclonal antibodies and recombined adenoviruses expressing PCV2 Rep, Cap and ORF3 proteins. Furthermore, the study demonstrated PCV2 induced IL-10 high level expression through activation of p50/p50 mediated NF-κB, Akt1 mediated PI3K/Akt, p38 MAPK and ERK pathways. The PCV2 Cap interacting protein gC1 qR is the key cellular regulator molecule mediated PCV2 induced activation of PI3K/Akt and p38 MAPK pathways and IL-10 high level expression. PCV2 infection polarized the PAMs to M2 macrophages, and inhibited LPS/IFN-γ induced IL-12p40 expression at post-transcriptional level by upregulating of miR-23a/b. The results illuminated the characteristic and regulatory mechanisam of PCV2 induced IL-10/IL-12p40 expression in PAMs, and established the foundation for the research of pathogenesis of PCV2. |
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