Essential Role Of Porcine Circovirus Type 2 Infection In Regulation Of Macrophage Polarization And Bacterial Coinfection

Porcine circovirus type 2(PCV2)is the primary causative agent of porcine circovirus–associated disease(PCVAD)that is one of the most important viral infectious diseases affecting swine industry worldwide.PCV2 modulates the host immune function to lead to immunosuppression and diseases.PCV2 infected pig show mild clinical symptoms,and coinfections with other swine pathogens including pathogenic bacteria such as Actinobacillus pleuropneumoniae(APP)can exacerbate PCVAD.PCVAD is widespread throughout the world and leads to immense economic losses in the global swine industry.Understanding of collaborative pathogenicity of PCV2 and bacteria is significant for treatment and control of PCVAD in clinical.Although some factors have been reported,the exactly molecular mechanisms of PCV2-induced secondary bacterial infections are still unclear.Macrophages are the major target cells of PCV2,which are typically polarized by the dichotomy of classical(M1)and alternative(M2)activation statuses.It has been shown that macrophage activation plays an important role in coinfection of virus and bacteria.However,it remains unknown how PCV2 regulates macrophage activation and whether PCV2-induced macrophage activation plays role in coinfection of PCV2 and bacteria.By using murine bone marrow-derived macrophages(BMDMs)and mice as the research model,we found that PCV2b infection strikingly induced expression of a subset of M1-associated genes in BMDM and peritoneal cells.Moreover,PCV2b infection also decreased expression of M2-associated genes.In addition,flow cytometric analysis showed that PCV2b infection induced more M1macrophage population in peritoneal cells.Further study found that PCV2 infection more markedly increased M1-associated genes and inhibited M2-associated genes in primary porcine alveolar macrophages.Together these results showed that PCV2b infection induced M1 macrophage polarization state.To deeply study the regulatory mechanisms of PCV2b infection induced M1 macrophage polarization,PCV2b infected BMDMs obtained from Myd88-deficient mice,we found that Myd88^–/–clearly suppressed M1 gene expression induced by PCV2b,suggesting that PCV2b infection induced M1 gene expression via Myd88-mediated signaling pathways.Further assay proved that induction of M1 genes by PCV2b infection is dependent on activation of NF-?B and JNK signaling pathways.In addition,pretreatment of TSA,an inhibitor of HDACs,restored expression of M2 genes inhibited by PCV2b,indicating that histone modification may plays a role in expression of M2 genes inhibited by PCV2b.Further assay proved that decrease of M2 genes by PCV2b via targeting histone deacetylation and methylation.Moreover,Rep and Cap of PCV2b decreased expression of Jmjd3,is known to act as H3K27demethylases.To study the role of PCV2b infection skewed M1 status to promote cocurrent infection of other pathogens.By using BMDMs as research models,we found that PCV2b priming significantly promoted invasion of APP and Salmonella typhimurium(STM)in vitro.However,Myd88-deficient or pretreatment of Bay11,an inhibitor of NF-?B,decreased bacterial invasion induced by PCV2 infection.In mice,PCV2b priming facilitates bacterial infection and synergetic infection of these pathogens exacerbates inflammatory responses,which may contribute to PCV2 pathogenesis.Taken together,PCV2b infection induces M1 macrophage polarization via activating NF-?B and JNK signaling pathways and suppression of M2 genes via histone methylation and deacetylation.PCV2 infection skewed macrophages toward a M1 status to lead to coinfection with other pathogenic bacteria.Hence,our study reveals a novel mechanism of PCV2induced-immunosuppression and provides theoretical guidance for the clinical treatment of PCVAD,and provides potential targets for drug development.