The Molecular Mechanism Of Pcv2 Inhibiting Expression Of IL-12p40 Induced By Pathogen Associated Molecular Patterns In Porcine Alveolar Macrophages

Porcine circovirus type 2?PCV2?is the main pathogen of porcine circovirus associated disease?PCVAD?.It has been found that piglets infected with PCV2 are often accompanied by co-infection of other pathogens.However,the immune mechanisms of PCV2-infected animals that are susceptible to multiple pathogens are unknown.As an important pro-inflammatory cytokine,interleukin-12?IL-12?plays a crucial role in the body's resistance to infection by pathogenic microorganisms.The expression of IL-12p40 in the spleen and lymph nodes of PCV2-infected piglets was significantly down-regulated,and the expression of IL-12p40 in alveolar macrophages of piglets inoculated with PCV2 was also decreased.However,it is not clear what mechanism by which PCV2 infects piglets results in inhibition of IL-12p40 expression in immune organs.In this study,the Rep and Cap proteins of PCV1and PCV2 were exchanged to construct four mutant strains of PCV1-Rep2,PCV1-Cap2,PCV2-Rep1 and PCV2-Cap1.The PK-15 cells were transfected respectively and serially passaged 5 times to obtain 4 strains of PCV mutant strains,which provided materials for subsequent studies.Levels of IL-12p40 expression induced by LPS/IFN-?or LPS/R848 were detected in porcine alveolar macrophages infected with Mock,PCV1 and PCV2.Further,pig alveolar macrophages were infected with PCV2 Rep and Cap-expressing recombinant adenoviruses and PCV-mutant strains,and the key components,key signaling pathways and mi RNAs of the inhibition of IL-12p40 expression by PCV2 were identified.This experiment clarifies the molecular mechanism by which PCV2 infection inhibits IL-12p40 expression in PAMs by other pathogen associatedmolecular patterns and provides theoretical data for further elucidating the mechanism of PCV2 infection leading to immune suppression in piglets.The following experimental results were obtained.1.By the chimeric vector construction method,Rep1 and Cap1 of PCV1 were replaced with Rep2 and Cap2 of PCV2,respectively;Rep2 and Cap2 of PCV2 were replaced with Rep1 and Cap1 of PCV1,respectively.This study successfully constructed PCV1-Rep2,PCV2-Cap1,PCV1-Cap2,and PCV2-Rep1 four PCV mutant strains.2.After ex vivo or in vitro infection of porcine alveolar macrophages with Mock,PCV1,and PCV2,cells were stimulated by LPS/IFN-?or LPS/R848.IL-12p40 expression was detected by ELISA and Q-PCR.The results showed that PCV2 infection of porcine alveolar macrophages ex vivo or in vitro significantly inhibited IL-12p40 expression induced by LPS/IFN-?or LPS/R848 compared to Mock or PCV1 infected groups.3.The porcine alveolar macrophages were infected with the recombinant adenoviruses rAd-Rep and rAd-Cap expressing PCV2 Rep and Cap,respectively,and the porcine alveolar macrophages were stimulated by LPS/IFN-?or LPS/R848.The results showed that both Rep and Cap significantly inhibited the expression of IL-12p40 induced by LPS/IFN-?or LPS/R848,but Cap protein inhibited more significantly than Rep.At the same time,PCV or PCV mutant strains were used to infect pig alveolar macrophages.The results showed that the mutants PCV1-Cap2 and PCV2-Rep1 significantly inhibited IL-12p40 expression induced by LPS/IFN-?and LPS/R848.This result shows that Cap protein of PCV2 inhibits IL-12p40expression more strongly than Rep protein.4.PCV2 was infected with wild-type(gC1qR^+/+)and gC1qR gene knockout(gC1qR^-/-)porcine alveolar macrophages,and Mock showed that deletion of gC1qR gene did not affect LPS/IFN-?or LPS/R848-induced IL-12p40 expression.It was shown that gC1qR is involved in the process of PCV2 inhibition of IL-12p40 production.5.The cells transfected with Akt1,p38 MAPK and ERK1 siRNAs were infected with PCV2 and stimulated by LPS/IFN-?or LPS/R848.The results showed that in cells transfected with Akt1 and p38 MAPK-specific siRNA,the inhibitory effect of PCV2 on IL-12p40 was significantly reduced,whereas the expression of IL-12p40 was not significantly changed in cells transfected with ERK1-specific siRNA.6.In PCV2-infected PAMs,miR-23a,miR-23b,miR-29a,and miR-29b were significantly up-regulated.Recombinant adenoviral rAd-Cap can upregulate these mi RNA expressions,while rAd-Rep cannot,gC1qR knockout(gC1qR^-/-)porcine alveolar macrophages are inhibited by upregulation of these miRNAs.PCV2-induced miR-23a,mi R-23b and miR-29b expression was inhibited in Akt1-inhibited cells,and inhibition of PCV2-induced miR-29a and miR-29b expression was inhibited in p38 MAPK-infected cells.Inhibition of ERK1 had no significant effect on PCV2-induced up-regulation of miR-23a,mi R-23b,miR-29a,and mi R-29b.7.Cells were treated with mimics or specific inhibitors of miR-23a,miR-23b,miR-29a,and miR-29b,respectively.After infection with PCV2,they were stimulated by LPS/IFN-?,and IL-12p40 secretion was detected by ELISA.The results showed that after mimics pretreated cells,IL-12p40 induced by LPS/IFN-?or LPS/R848 was significantly reduced.However,only miR-29a and miR-29b mimics significantly decreased IL-12p40 mRNA levels compared with the control group.The specific inhibitors of miR-23a and miR-29b could significantly reverse the PCV2-induced IL-12p40 suppression in protein levels and miR-29b could promote IL-12p40 transcription,whereas miR-23b and miR-29a specific inhibitors did not significantly improve IL-12p40 expression no matter in either protein or mRNA level.Notably,treatment with both miR-23a and miR-29b specific inhibitors could more significantly increase the LPS/IFN-?induced IL-12p40 secretion in PCV2 infected PAMs than miR-23a or miR-29b inhibitors treatment alone and show a same effect as the inhibitors mix of 4 miRNAs?miR-23a,miR-23b,miR-29a,and miR-29b?.In summary,four PCV mutant strains PCV1-Rep2,PCV1-Cap2,PCV2-Rep1 and PCV2-Cap1 were obtained.This study provides certain evidences that PCV2 infection suppress other pathogens-induced IL-12p40 expression at both transcriptional and post-transcriptional levels through the Cap and gC1qR interaction-mediated PI3K/Akt1 and p38 MAPK pathways activation and miR-23a and miR-29b upregulation.These findings might help us to further understand the relative immune mechanisms determining the susceptibility of PCV2-infected animals.