Study On The Expression Profile Of MRNA And LncRNA In Porcine Alveolar Macrophages During M1/M2 Polarization

Porcine alveolar macrophages(PAMs)are the first line of defense in the body’s innate immunity.Their strong plasticity provides an important cell model for the analysis of the immune regulation mechanism of pigs(Sus scrofa).lncRNA is involved in immune-related processes.It’s a potential regulator of macrophage polarization.In order to establish different polarized subtypes of porcine alveolar macrophages,we used three primary PAMs isolated from 30-day-old healthy white piglets as experimental materials,using lipopolysaccharide(LPS)plus interferon gamma(IFN-γ)and interleukin 4(IL-4)for polarization induction,and selected the stimulation time as 0 h,6 h,12 h and 24 h to construct M1 and M2 cell models(each treatment group contains three biological replicates).21 cell samples were obtained for strand-specific transcriptome sequencing,and the mRNA expression profile changes of two different polarization subtypes of PAMs at each time point were analyzed and compared.Differentially expressed genes were screened for functional enrichment analysis.At the same time,the PAMs genome-wide long non-coding RNA(lncRNA)was screened and identified,the basic characteristics were compared with mRNA,the target gene was predicted and analyzed,the co-expression trend analysis and the target gene function were analyzed.The main results of this study are as follows:(1)The differential expression analysis of PAMs samples from the M1 and M2 polarization subtype groups at 6 h,12 h and 24 h time points showed that compared with the M0 control group,the M1 polarization subtype group was screened to 1038,3118,and4826 differentially expressed genes,respectively.In the M2 polarization subtype group,509,3399 and 5183 differentially expressed genes were screened respectively.In addition,the number of up-regulated and down-regulated genes of the two polarized subtypes compared with the M0 control group increased with the increase of stimulation time.(2)The GO and KEGG annotation and functional enrichment analysis of the above differential genes showed that the early differentially expressed genes of M1 type polarization of PAMs are mainly involved in the immune response,such as the production of interleukin 12 and various interferons and the inhibition of the anti-inflammatory factor interleukin 10.Generate and activate NFκB signaling pathways and chemokine-mediated signaling pathways,regulate STAT protein tyrosine phosphorylation and positively regulate natural killer cell proliferation,promote inflammatory processes,and initiate defense against pathogens and other related pathways.The differentially expressed genes at the 12 h time point are mainly involved in activating chemokine-mediated signaling pathways,inhibiting the production of anti-inflammatory factor interleukin-10,and positively regulating the proliferation of natural killer cells,positively regulating the JNK cascade,promoting T cell proliferation,strengthening the redox process,and inhibiting related pathways such as cell proliferation and cell cycle arrest.At the 24 h time point,differentially expressed genes are mainly involved in the pro-inflammatory response,and are significantly enriched in NF-κB transcription factor activity,promotion of T cell proliferation and differentiation in the thymus,cell cycle,autophagy and apoptosis.(3)In the M2-type polarization of PAMs,the different genes stimulated by IL-4 for 6hours are mainly involved in the choline metabolism,VEGF signaling pathway,MAPK signaling pathway,FcγR-mediated phagocytosis,PI3K-Akt signaling pathway,etc.The12 h time point differential genes are mainly involved in HTLV-I infection,chemokine signaling pathway,AGE-RAGE signaling pathway and Fanconi anemia pathway in diabetic complications.At the 24 h time point,the differential genes of M2 type PAMs samples were significantly enriched in B cell receptor signaling pathway,sphingolipid signaling pathway,Toll-like receptor signaling pathway,small cell lung cancer,alcoholism,and NF-κB signaling pathway.(4)During the M1 polarization process,the 390 differential genes up-regulated at three time points are most significantly enriched in the interaction pathways between cytokine and cytokine receptors.Among them,the genes involved are CD40,CXCL9,and CXCL9.IL2 RA,IL12B,CXCL10,IL23 A,TNFSF10,IL15,CSF3,IL1 B,IL12RB1,CCL8,IL23 R,CCL2,IL10,CXCL8,Sus＿scrofa＿new Gene＿12336,TNFSF13 B,CCL20,LOC110258579,IL15 RA and IL10CL11;The 150 differentially genes down-regulated at three time points are most significantly enriched in the Notch signaling pathway,among which the participating genes are DTX1,DLL1,DTX4 and NUMBL.During the M2 polarization process,the 121 differential genes up-regulated and expressed by PAMs at three time points were most significantly enriched in the EGFR tyrosine kinase inhibitor resistance pathway,and the participating genes were NRG1,MAPK3,Sus＿scrofa＿new Gene＿1215 and PLCG1;The 147 differential genes that were down-regulated at the three time points were most significantly enriched in the PI3K-Akt signaling pathway,and the participating genes were Sus＿scrofa＿new Gene＿1306,LPAR5,DDIT4,JAK2,SPP1,LPAR1,CSF1,PIK3R5,HGF,LAMC2 and GNG8.(5)Transcripts assembled from 21 samples were identified and predicted by lncRNA,and a total of 23995 lncRNAs with high reliability were obtained.Co-expression trend analysis showed that lncRNAs in the two polarized subtypes were divided into two data sets with a co-up-regulation trend and a co-down-regulation trend.During M1 type polarization,the lncRNA target genes whose co-expression trend is up-regulated are mainly enriched in the Jak-STAT signaling pathway,phosphatidylinositol signaling system and natural killer cell-mediated cytotoxicity;the lncRNA target genes whose co-expression trend is down-regulated are mainly enriched in mismatch repair,butyrate metabolism and fatty acid biosynthesis.In the process of M2 type polarization,the lncRNA target genes whose co-expression trend is up-regulated are mainly enriched in the formation of the dorsoventral axis,the biosynthesis of pantothenate and coenzyme,and a carbon pool composed of folic acid;the lncRNA target genes whose co-expression trend is down-regulated are mainly enriched in the metabolism of selenium compounds,the biosynthesis of terpenoid skeletons,the biosynthesis of glycosaminoglycans-heparan sulfate/heparin and other pathways.The above transcriptome research results on the polarization process of porcine alveolar macrophages provide a reference direction for the later identification of key polarization factors,and provide basic data for the screening of molecular markers in pig disease resistance breeding.