Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing

Authors: HERRERA-URIBE, JUBER - Iowa State University; WIARDA, JAYNE - Orise Fellow; SIVASANKARAN, SATHESH - Iowa State University; DAHARSH, LANCE - Iowa State University; LIU, HAIBO - Iowa State University; Byrne, Kristen; Smith, Timothy - Tim; Lunney, Joan; Loving, Crystal; TUGGLE, CHRISTOPHER - Iowa State University

Submitted to: Frontiers in Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2021
Publication Date: 6/23/2021
Citation: Herrera-Uribe, J., Wiarda, J., Sivasankaran, S.K., Daharsh, L., Liu, H., Byrne, K.A., Smith, T.P., Lunney, J.K., Loving, C.L., Tuggle, C.K. 2021. Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing. Frontiers in Genetics. 12. Article 689406. https://doi.org/10.3389/fgene.2021.689406.
DOI: https://doi.org/10.3389/fgene.2021.689406

Interpretive Summary: Pigs are an important protein source supporting global food security. A major goal of biological research is using genetic information (ie, genotype) to predict the complex physical composition of an individual or individual cells (ie, phenotype). Identifying and cataloging the genes expressed in specific cell types is critical to understanding genotype to phenotype, and informing methods to screen for disease resilience or growth parameters. Cells in the blood are expected to represent the physiological state of an animal, and potentially predict outcomes when animals face stress. Thus, blood immune cell gene expression is valuable in development of tools for predictive modeling of response to stress or expression. While whole blood gene expression is often used, it’s nearly impossible to ascribe expression of genes to a specific immune cell. Thus, two different platforms were used to sort or partition different porcine blood immune cells for subsequent sequencing of RNA and assessing gene expression to specific cells or even individual cells. Gene expression in pig immune cells was comparable to mouse and human, though several cell types in pigs were not represented in a human dataset. A deeper understanding of porcine immune cells was gathered and the necessary framework improving analysis of whole blood data. The data will be publicly available to the international research community as a resource for further exploration.

Technical Abstract: Pigs are an important protein source supporting global food security and a valuable human biomedical model. To extensively catalog transcriptomic profile of peripheral blood immune cells in this important species, two technically distinct approaches were taken and data integrated. First, we used cell sorting to isolate eight cell types from peripheral blood mononuclear cells (PBMCs), 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 and expression patterns of 230 genes were highly correlated with results from¬ NanoString assays. Pairwise comparisons between cell types revealed specific expression of many genes for Myeloid and NK cells, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes (SEG, q less than 0.05 and two-fold above average expression) across all 8 cell types. Gene Ontology analysis for the top 25 percent of SEG showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations (Spearman’s rank P less than 2.2e-16) between pig cells and corresponding human cell populations (Haemopedia). Second, single-cell RNA-sequencing (scRNA-seq) of PBMC was performed to more highly resolve distinct population transcriptomes. Across seven PBMC samples partitioned and sequenced, 28,810 cells distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, greater than 10 B cell clusters including plasmablasts, conventional CD4 and CD8 '' T cells, NK cells, and 4 clusters of gamma, delta T cells. Signature gene sets from the Haemopedia human PBMC bulk RNA-seq data were assessed for relative enrichment in genes expressed in pig cells, and substantial overlap in gene expression between specific pig and human PBMC populations was detected. Integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed 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 of porcine PBMCs, including highly resolved comparisons to human datasets. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine FAANG project available to the research community, as well as further study of, and development of new reagents for, porcine immunology.