Single-Cell Transcriptomic And Chromatin Accessibility Atlas Of Peripheral Blood Mononuclear Cells Reveal The Immune Cell Heterogeneity Of Pigs

Pig is one of the most important economic animals in China,and Pigs are similar to humans in anatomical size and structure,immunology,genome,and physiology,and thus it has become an important animal model for the research applications of complex human diseases.However,compared with researches on the human immune system,the current understanding of the marker genes of porcine immune cells is relatively lacking,and there is also a lack of systematic research on the heterogeneity of porcine immune cells.In addition,there are significant differences in stress resistance and disease resistance between domestic pig breeds in my country and imported commercial pig breeds from the Western.Although there have been some studies attempted to explain the mechanism in recent years,the cellular and molecular mechanisms underlying the differences in immune characteristics between Chinese and foreign pig breeds are still not fully parsed in a large extent.The single-cell sequencing technology developed in recent years can reveal the cell heterogeneity at the single-cell level,and has an increasingly wide range of applications in immunology and other fields.Porcine peripheral blood mononuclear cells(PBMC)belong to a group of heterogeneous immune cells,mainly including lymphocytes(T cells,B cells and NK cells),monocytes and dendritic cells,which are crucial to the body’s immunity and widely for immunological research and clinical applications.In this study,PBMC was used as the main model,and single-cell transcriptomic and epigenetic techniques were used to study the heterogeneity of porcine immune cells and the differences in immune capacity between Chinese and foreign pig breeds.It has been reported that there are differences in resistance to various pathogens between the Chinese domestic pig breed Meishan pig and the introduced commercial breed Duroc pig,which may be related to the differences in innate immune characteristics between breeds.Therefore,this study used scRNA-seq technology to study the PBMCs of Meishan pigs and Duroc pigs to explore the mechanism of porcine immune cell heterogeneity and differences in immune characteristics between breeds firstly.Chromosome accessibility detection at the single-cell level to explore the regulatory network of heterogeneous immune cells in porcine PBMC;and gene interference,RNA-seq and ChIP-seq technology to explore its transcriptional regulatory function in monocyte-macrophages.The main results of this study are summarized as follows:1.Analysis of the mechanism of immune differences between breeds based on routine transcriptome sequencing of PBMC from Meishan and Duroc pigsIn order to explore the underlying molecular mechanism of the differences in immune characteristics between Chinese local pig breeds and European commercial pig breeds,this study firstly performed routine transcriptome sequencing of PBMCs of Meishan and Duroc pigs.Differential gene expression analysis identified thousands of differentially expressed genes.GO and KEGG enrichment analysis showed that the up-regulated genes in Meishan pigs were significantly related to immune response,immune defense and other biological processes and signaling pathways.GSEA analysis also obtained similar results.Afterwards,61 hub genes were screened out by WGCNA combined with differentially expressed gene analysis.Through immune infiltration analysis of differentially expressed genes in PBMCs of the two groups,it was inferred that the proportion of monocytes and neutrophils in Meishan pigs was higher than that of Duroc pigs,while the proportion of T regulatory cells was lower.Moreover,the correlation analysis of Hub genes obtained by WGCNA analysis found that the highly expressed genes were significantly positively correlated with monocytes and neutrophils,and significantly negatively correlated with T regulatory cells,while the lowly expressed genes were the opposite.These results suggest that differences in immune characteristics between Meishan and Duroc pigs are closely related to genes related to innate immune defense response and immune cell differentiation,and may also be closely related to the differences in immune cell composition in PBMC.2.Analysis of immune cell heterogeneity in PBMCs of Meishan and Duroc pigs based on single-cell transcriptome sequencingIn order to explore the heterogeneity of porcine immune cells and its association with the differences in immune characteristics between breeds,this study used scRNA-seq technology to perform single-cell transcriptome sequencing of PBMCs of Meishan pigs and Duroc pigs.A total of 63,710 single-cell transcriptome profiles were successfully obtained,and 20 cell subsets were identified by cluster analysis,covering αβ T cells,γδ T cells,B cells,myeloid cells,NK cells,and DC cells.Hundreds of marker genes were identified for each cell population.Interestingly,the proportion of γδ T lymphocytes in pig PBMCs was much higher than that in humans and mice,and CD4+CD8+ double positive T cells called memory effector T cells were found.Furthermore,an integrated comparison with human PBMC data showed that humans possess a distinct monocyte subpopulation with a gene expression profile consistent with the recently discovered "Mono4" subpopulation.The above shows that although pig and human immune cell populations are generally similar,they each have some unique immune cell subsets.Further comparison of the PBMC data of Meishan pigs and Duroc pigs showed that the proportion of monocytes and a B cell subset(B1)was higher in the former,while the proportion of γδ T and NK cells was higher in the latter,indicating that the two breeds There were differences in the composition of immune cells in PBMCs.In addition,by comparing the enriched genes of each immune cell subset between the two breeds,it was found that the genes highly expressed in monocytes in Meishan pigs were significantly associated with immune responses such as granulocyte chemotaxis,interferon-γ and monocyte migration.In the γδ T cell cluster,the highly expressed genes in Meishan pigs were found to be significantly related to immune function,and the negative function of negative regulation of immune system processes also appeared.It is closely related to immune functions such as T cell differentiation,activation of myeloid lymphocytes and adaptive immune system;and highly expressed genes in Duroc pigs are significantly enriched in cell adhesion regulation,osteoblast differentiation regulation and positive regulation of protein transport,etc.,the adverse function of negative regulation of immune system processes also appears.Consistent with the results of conventional RNA-seq,these results suggest that immune cell composition and gene expression patterns underlie the differences in immune properties between Meishan pigs and Duroc.3.Analysis of immune cell heterogeneity in porcine PBMC based on single-cell ATAC sequencingTo further understand the heterogeneity of porcine immune cells,this study performed scATAC-seq on Duroc pig PBMCs and successfully mapped 21,855 single-cell chromatin accessibility for the first time.Pig PBMCs were clustered into 19 subpopulations based on scATAC-Seq data with similar structures to cell clusters from scRNA-Seq data,scRNA-Seqbased cell markers were used as a reference,of which 16 subpopulations were annotated successfully,including αβ T cells,γδ T cells,B cells,myeloid cells,NK cells and DC cells,etc,but the other 4 cell subsets(γδ T2,CD8+T3,plasma and platelets)were not annotated as their low proportion of numbers in PBMC.Further motif enrichment analysis of each cell subset peak revealed significantly enriched motifs for each cell subset in porcine PBMC,ranging in number from 83 to 633.Some of which were known to target specific immune cells transcription factors important for the development of T cells,such as EBF1 in B lymphocytes.SP11,CEBPA and CEBPD in the CEBP family in monocytes,and the family of KLFs in theγδ T cell cluster,including KLF2,KLF3,KLF4,KLF5,KLF9.The scATAC-seq data in this study help to further dissect the heterogeneity of immune cells in porcine PBMCs and predict important transcription factors associated with each immune cell subset.4.To explore the regulatory mechanism of the transcription factor CEBPD in monocytes and macrophages based on ChIP-SeqIn this study,combining the differentially expressed genes of PBMC between Meishan pig and Duroc pig breeds,the differentially expressed genes of each immune cell subset in PBMC,and the transcription factors regulated by each immune cell,we speculate that CEBPD plays an important role in monocyte development and immune regulation.It plays an important role and may be involved in the regulation of differences in immune characteristics among varieties.Therefore,the gene CEBPD and transcription factor CEBPD enriched in monocytes were initially screened to explore their regulatory mechanism in monocytes and macrophages.In this study,by interfering with the CEBPD gene at the mRNA and protein levels on human monocytes(THP-1)and porcine alveolar macrophages(3D4/12),RNA-seq was performed to obtain CEBPD knockdown.The gene expression pattern of macrophages,and it was found that the changes in gene expression of monocytes and macrophages after interference were involved in various immune-related signaling pathways,such as Neurodegeneration pathways,Alzheimer’s disease,Human papillomavirus infection,and MAPK signaling pathways,Herpes simplex virus type 1 infection and other diseases immunerelated pathways.Combined with the ChIP-seq analysis results of CEBPD on THP-1 and 3D4/12,the changes in gene expression patterns after knockdown were revealed,and the target genes directly regulated by CEBPD in monocytes were identified.The transcriptional regulatory network of nuclear cells and macrophages.In summary,this study improves the understanding of the heterogeneity of porcine immune cells,and analyzes the close relationship between the differences in immune characteristics between Chinese and foreign pig breeds and the heterogeneity of immune cells.The cell clusters,immune markers and core regulatory factors identified in this study not only provide scientific guidance and basis for the evaluation of the excellent disease resistance and stress resistance characteristics of local pig breeds in China,but also provide theories and data for related research on pig immunology,and help advance future biomedical research in human immunity and xenotransplantation.