Figure 1.
RNA-Seq analysis of DEGs in polarized MФs compared with sham control MФs in PBS post-PRRSV infection.
The heatmaps of 6,624 significant DEGs (left) and numbers of potential signature genes were grouped based on significant up-regulation in only one activation status or co-stimulated in two activation statuses (see table at right, and the supplemented results of DEG statistics in Table S2). FDR (false discovery rate) ≤0.001, fold change ≥2 for DEG significant determination. The color scale under the heatmap illustrates the log2 (fold change) values shown in the heatmap.
Figure 2.
Transcriptomic analysis of selected transcription factor (TF) families.
Members of these TF families have been shown to be critical to the regulation of activation status and antiviral activity in murine monocytic cells. Differential expression of TF families of (A) suppressors of cytokine signaling (SOCS), (B) Kruppel-like factors (KLF), and (C) interferon regulatory factor (IRF) in polarized MФs upon PRRSV infection are shown. (D) The differential expression of IRF family was verified with a real-time RT-PCR assay (the Y-axis scale indicating fold change to M0-PBS). The color scale under each heatmap illustrates the midpoint and range of reads per kilobase per million (RPKM) values of listed transcripts.
Figure 3.
Verification of DEGs at the protein level using a proteomic procedure.
Equal amounts of protein from macrophages at different activation statuses were stained with either red or green fluorescent dyes and co-resolved using a 2D-DIGE procedure (Applied Biomics, Inc., Hayward, CA) to isolate protein spots that significantly increased in macrophages at certain activation statuses and to further identify the isolated proteins by nano LC-MS/MS. Of 16 significantly increased protein spots randomly selected across four activation statuses (black bars) compared with the M0-PBS status (the white bars), 12 (75%) protein spots (WARS, PLEK, RAN, CKB, PLOD3, RNF114, HNRNPU, ATP6V1E1, CANX, MX1, MX2, H2B3A) also showed significant up-regulation at the RNA level, with the other four (SND1, ANXA1, UBE2D3 and MPP5) showing a significant increase only at the protein level. *, FDR ≤0.001 of gene expression and protein ratio ≥2. The number before each gene symbol along the X-axis indicates the protein spot mapped in the gel shown in Figure S2. Gene symbol abbreviations: SND1, staphylococcal nuclease domain-containing protein 1; WARS, tryptophanyl-tRNA synthetase; PLEK, pleckstrin; RAN, Ras-related GTP binding C; CKB, creatine kinase B-type; PLOD3, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3; RNF114, RING finger protein 114; HNRNPU, heterogeneous nuclear ribonucleoprotein U; ANXA1, annexin A1; ATP6V1E1, v-type proton ATPase subunit E 1; UBE2D3, ubiquitin-conjugating enzyme E2 D3; CANX, calnexin; MX, myxovirus resistance gene; H2B3A, histone H2B3A; MPP5, membrane protein palmitoylated 5.
Figure 4.
Pathway analysis of DEGs was annotated against the KEGG database.
A p-value and FDR of <0.05 in the two-sided Fisher’s exact test were considered significant. Selected pathway categories are shown along the vertical axis, and the horizontal axis represents the log10 (p value) of these pathways showing the significant difference among cells at different activation statuses.
Figure 5.
AMPK-mediated pathways for antiviral regulation.
(A) Heatmap of the subset DEGs in the AMP-kinase pathway, which is critical to control of lipid metabolism, are shown. (B) As illustrated in the pathway, most key genes in the M1 statuses (IFNγ- or LPS-induced) or IFNα-antiviral state (MaV) were differentially regulated, leading to a general suppression of lipid metabolism in contrast to a general increase in the M2-IL4 status. In addition, the illustration of AMPK-mediated pathways in other statuses is presented in Figure S3 together with the dose-dependent suppression of PRRSV infection by two AMPK-pathway activators. Legend: green-line box, significant suppression; blue-line box, non-significant suppression; red-line box, significant up-regulation; yellow-line box, non-significant up-regulation; and black-line box, non-significant detection. p (FDR) <0.001, fold change ≥2 for significant determination.
Figure 6.
Epigenetic mechanisms for antiviral regulation.
(A) RNA-Seq analysis of DEGs encoding key enzymes in epigenetic regulation. The heatmaps display family-wide collections of genes encoding DNA/histone methyltransferases, histone deacetylases and histone demethylases. In addition to differential expression analysis, these data also revealed family-wide transcription evidence of most of these porcine epigenetic genes at the mRNA level for the first time. (B) Suppression of PRRSV infection by epigenetic inhibitors at optimized concentrations in MARC-145 cells and porcine MФs. The fluorescent micrographs (inset) show infected cells using a DsRed-labeled PRRSV, whereas the larger bright-field images show cell phenotypes of non-visible cytotoxic effects. The micrographs represent one of three replicates. Summary data are presented below the images.