| Porcine reproductive and respiratory syndrome(PRRS)is one of the swine diseases that have caused significant economic losses in the pig industry in the past two decades.The causative agent of PRRS is PRRS virus(PRRSV)and the virus mainly infects macrophages,which leads to changes in immune response and immunosuppression.The purpose of this study is to analyze the different polarization states of bone marrow-derived macrophages induced by highly pathogenic PRRSV(HP-PRRSV)HuN4 strain and its attenuated vaccine strain HuN4-F112 and to further explore the difference of immune response mechanism between them.In this study,HuN4 and its attenuated vaccine strain HuN4-F112 were inoculated in vivo.It appears that the body temperature of pigs increased significantly after HuN4 infection and they all died within 10 days.Contrary to it,HuN4-F112 infected piglets did not show any clinical signs and could be further protected after challenged with HuN4.Then,the peripheral blood cells were detected by flow cytometry,it was found that HuN4 infection caused a decrease in the number of CD3~+T lymphocytes,but a significant increase in the number of Myelomonocytes.On the contrary,the number of CD3~+T lymphocytes increased significantly after HuN4-F112 infection.Combined with the quantitative detection of mRNA of related factors,it shows that HuN4 infection can increase the expression of CD163,as well as M-CSF,which is an important cytokine that promotes macrophage activation.Also,it seems that the expression of antigen presentation molecules has decreased significantly after HuN4infection,indicating that the changes in the number of monocytes caused by HuN4 may be related to macrophages.Due to the lack of phenotypic genes to identify different functional subsets of porcine macrophages,bone marrow-derived macrophages were isolated and polarized in vitro.Bone marrow cells collected from femur and tibia were activated by M-CSF to form M0 macrophages.And then M0 macrophages were further stimulated by LPS/IFN-?to form M1 macrophages,while Th2 related factors,like IL-4,IL-10,IL-13 can induce M0 macrophages to M2 phenotype with upregulation of anti-inflammatory cytokines.It was observed by microscopes that the size of polarized macrophages was larger and more granular than those of M0 macrophages,and all of them had stronger phagocytosis ability,which has proved the purity of the isolated macrophages.And compared to M1 macrophages,M2 is characterized by its small antennae and pseudopodia.Subsequently,M1 and M2 specific phenotypes were analyzed by RNA-seq,the gene expression was further identified by real-time PCR and Western Blot.The results showed that IDO1 and STX11 were up-regulated in M1 macrophages,while the expression of TP53INP1 and GK genes in M2 macrophages was relatively increased.Therefore,we identified IDO1and STX11,TP53INP1 and GK as biomarkers of M1 and M2 macrophages,respectively.In order to analyze phenotypes of BMDM induced by HuN4 and HuN4-F112,the expression of biomarkers of M1 and M2 macrophages were identified at both mRNA and protein levels.It was found that the expression of IDO1 and STX11 increased significantly after HuN4 infection,but the expression decreased when it comes to TP53INP1 and GK.On the other hand,the attenuated HuN4-F112 strain could not show increased expression of IDO1 and STX11 at protein levels.And this indicates that HuN4has the tendency of promoting the expression of M1 biomarkers.In addition,virus titer,IFA and Western Blot results showed that the replication ability of HuN4 was stronger than that of F112.And It was found that HuN4 infection could inhibit the phagocytosis and antigen presentation ability of macrophages,and then affect the immune response of T lymphocytes.This study provides a theoretical basis for explaining the pathogenic mechanism of HuN4 and the protective mechanism of vaccine strain F112,and puts forward the idea of regulating immune response by regulating the phenotypes of macrophages in vivo. |
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