Genome-Wide Analysis of Antiviral Signature Genes in Porcine Macrophages at Different Activation Statuses

Abstract

Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153–5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20–50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis.

Antiviral Regulation in Porcine Monocytic Cells at Different Activation States

“…Animal procedures and the isolation of alveolar macrophages and peripheral blood mononuclear cells (PBMCs) were conducted as described previously (29,(31)(32)(33)(34)(35)(36)(37). PBMCs were isolated using a 60% Ficoll-Paque Plus gradient (GE Healthcare, Piscataway, NJ).…”

“…Mediators and conditions for polarization of porcine monocytic cells were applied as previously described (1)(2)(3)(4)(5)29). In brief, Ms and DCs were stimulated with the mediators LPS, IFN-␥, IL-4, IL-10, IFN-␣, and IFN-␤ at 20 ng/ml, and blood monocytes (BMs) were stimulated with either GM-CSF at 50 ng/ml or M-CSF at 100 ng/ml for 30 h. All mediators (purchased from R&D Systems, Minneapolis, MN, or Sigma-Aldrich, St. Louis, MO) were dissolved in 1ϫ Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen) containing 1% bovine serum albumin (BSA) (fraction V, cold ethanol precipitated; Sigma) and applied (1:100) to the cultured cells.…”

“…The expression of marker genes relative to each activation status and antiviral state was revealed using next-generation transcriptome sequencing (RNA-Seq) for genome-wide screening and confirmed family-wide using real-time reverse transcription-PCR (RT-PCR) assays. For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29).…”

“…For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29). In brief, 3 ϫ 10 7 cells at each activation status (including the control DPBS mock stimulation) were pooled from three technical replicates representing cells obtained from four outbred pigs.…”

“…Few studies have focused on the activation status of porcine monocytic cells or how cell activation status modulates antiviral immunity (27,28). Notably, in this study we focused on examining Gene Ontology (GO) analysis based on comparative transcriptomes revealed in macrophages at different activation statuses upon viral infection, rather than a transcriptomic comparison between infected and noninfected tissues/cells, which has been well documented in previous studies (29,30). In this context, we used a multiplex cytokine assay and transcriptomic sequencing (RNA-Seq) analysis to profile gene response pathways in porcine monocytic cells polarized to typical activation statuses and antiviral states.…”