| Macrophages are the main participants in innate and acquired immunity,playing an important role in tissue homeostasis,angiogenesis,and innate metabolism.Macrophages are key models for understanding the regulatory mechanisms of immune response and diagnosing or treating various diseases.Pigs are the most important agricultural animals and valuable preclinical research animal models.However,there is currently no unified method for isolating and differentiating pig macrophages,and there is no systematic study comparing pig macrophages obtained by different methods.In order to provide a theoretical reference value for the diagnosis and molecular mechanisms of porcine PRRSV,ASFV,Toxoplasma gondii,and other diseases,this study induced M1 macrophages(named M1＿IFN)through two methods γ+ LPS and M1＿GM-CSF)and M2 macrophages(M2＿IL4+IL10 and M2＿MCSF,respectively),and compared the transcriptome characteristics between and within macrophage subtypes.Through GSEA analysis,this study explored the correlation between macrophage characteristics and transcriptome in pig macrophages infected with various pathogens.The pig macrophage model provided in this study can help diagnose pig diseases caused by different pathogen infections and analyze the role of macrophages in molecular mechanisms.The macrophage model described in this article is helpful for the diagnosis of different pig diseases,including porcine reproductive and respiratory syndrome virus(PRRSV),African swine fever virus(ASFV),Toxoplasma gondii(T.gondii),porcine circovirus type 2(PCV2),Haemophilus parasuis serovar 4(HPS4),Mycoplasma hyopneumoniae(Mhp),Streptococcus suis serotype 2(SS2),and LPS from Salmonella enterica serotype minnesota Re 595.The main results are as follows:(1)Using RNA-seq technology,the study identified 620 genes that were highly expressed in M1 macrophages and 110 genes that were highly expressed in M2.Among them,207 genes with high expression of M1＿IFNγ+LPS and 309 genes with high expression of M1＿GM-CSF;391 genes with high expression of M2＿IL4+IL10,and 211 genes with high expression of M2＿M-CSF.Then,we performed functional enrichment analysis on differential genes and enriched 20 pathways respectively in the comparison between two groups,such as white cell migration,inflammatory response,positive regulation of immune response,etc.(2)The DEGs identified by the study conduct protein interaction networks through String to identify the Hub gene.Among them,compared with M1＿GM-CSF the phagocytic gene expression of M1＿IFNγ+LPS was down-regulated(i.e.,CD68,MRC1,PPARG,A2M)or up-regulation of phagocytosis inhibitory factors(CAMP),up-regulation of antibacterial genes(S100A9,CAMP,LTF,RETN),and up-regulation of migration adhesion genes(S100A9,SELP).However,M1＿GM-CSF(CCL2,CD28,ACE)and M1＿IFNγ+LPS(S100A9,SELP,RETN)have different pro-inflammatory genes,M1＿GM-CSF also found anti-inflammatory genes(A2M,MRC1,FCER1A).M2＿IL4+IL10(PPARG,ADIPOQ,FLT1,HMOX1)and M2＿M-CSF(IL6,CD4,IL10,PTGS2,FOS)have different anti-inflammatory genes.(3)We used GSEA to analyze the correlation between the DEGs identified from macrophages infected with different pathogens and the DEGs of macrophages cultured in vitro.The results indicated that genes from the SS2-infected macrophages were most significantly enriched in the M1＿IFNγ+LPS set,genes from the PPRSV,ASFV,Mhp,and LPS infected were most significantly enriched in the M1＿GM-CSF,genes from the PCV2 and T.gondii Me49 infections were most significantly enriched in the M2＿M-CSF set,and genes from the different species of T.gondii infections were most significantly enriched in the M2＿IL4+IL10 set.In conclusion,this study explored the correlation and difference between and within different pig macrophage subtypes obtained by different methods through transcriptome technology and identified the correlation between pig macrophage subtypes obtained by us and pig diseases through GSEA technology. |
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