Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing

Abstract

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

Assessment of DNA methylation in porcine immune cells reveals novel regulatory elements associated with cell-specific gene expression and immune capacity traits

“…Pigs were housed in BSL2 rooms at the National Animal Disease Center (Ames, IA) and all animal procedures were performed in compliance with and with approval by the Institutional Animal Care and Use Committee. Blood was drawn and peripheral blood mononuclear cells (PBMCs) were isolated as described in Herrera-Uribe et al 2021 [ 18 ]. For granulocyte isolation, blood was collected in BD Vacutainer ACD solution A tubes and subjected to dextran sedimentation using 6% Dextran/0.9% NaCl solution at room temperature for 45–60 min.…”

Hepatic galectin-3 is associated with lipid droplet area in non-alcoholic steatohepatitis in a new swine model

“…In phase 2b, this drug was safe but not associated with significant reduction in fibrosis compared with placebo except for a subgroup of patients without oesophageal varices 67 . Since LGALS3, galectin-3, has emerged as a canonical 62 and noncanonical inflammasome activator in macrophages 68 and has been found in myeloid-derived cells 23 , it is plausible that activation LGALS3 in Kupffer cells is an early event in the cascade of events taking place in the progression from hepatic simple steatosis into NASH. However, in rats, hepatic stellate cells production of galectin-3 contributes to the expansion of hepatic progenitor cells 69 .…”

“…RNAseq provides accurate transcript levels and their isoforms 18 . Selected examples of its use in swine include characterization of microRNA in adipose tissue, inflammation-related genes in NASH-induced in Bama minipigs 19 , 20 , meat quality 21 or immune response in peripheral or intestinal cells 22 , 23 .…”

Integrated single-cell RNA-seq and DNA methylation reveal the effects of air pollution in patients with recurrent spontaneous abortion

“…The whole decidua represents an extremely complex mixture of cell types and estimates of gene expression in mixed samples are inherently inaccurate because it is difficult to adjust for differences in cell composition [ 45 ]. At the maternal–fetal interface, decidual NK cells and macrophages account for a large number of decidual leukocytes during early pregnancy.…”