Porcine Circovirus Type 2 Induces Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) Downregulation to Promote Interleukin-1β Upregulation in Porcine Alveolar Macrophage

Figure 1. Porcine circovirus type 2 (PCV2) infection in porcine alveolar macrophages (PAMs). (A) PAMs were infected with PCV2b and processed for immunofluorescence assay at 1, 24, and 48 hpi. PCV2b was visualized by the ODs-psdAb probe against the Cap protein (red), the nucleus was stained with DAPI (blue). (B) Cap protein in PCV2b-infected PAMs was also detected by Western blot assay based on Cap protein (VLPs) immunized porcine serum. (C) PCV2 copy numbers were analyzed by qPCR at the time-points indicated. Results are mean ± SE of three independent experiments.

Figure 2. Changes in mRNA expression levels of inflammatory cytokines and receptors. PCR array assay system was used to assess the mRNA expression levels of interleukins (A), chemokines (B), tumor necrosis factor (C), colony-stimulating factors (D), and receptors of chemokine (E) in PAMs incubated with PCV2 and controls after 1, 24, and 48 h. The Ct value of actin-β in PCV2-infected and control groups (F). Fold regulation was obtained by comparing the relative expression intensity of PCV2-infected and uninfected control PAMs. Results are mean ± SE of three independent experiments. * represents p < 0.05.

Figure 3. Validation of PCR array data by porcine cytokine array system. PCV2-induced changes in the secreted levels of IL-1β, IL-10, IL-8, GM-CSF, and TNF-α in the culture supernatants of control and PCV2-infected PAMs were measured using a RayBiotech fluorescent Quantibody® porcine cytokine array system. Data are mean ± SE of three independent experiments. * represents p < 0.05, ** represents p < 0.01.

Figure 4. Downregulation of single-immunoglobulin interleukin-1 related receptor (SIGIRR) in PAMs after PCV2 infection. The expression of SIGIRR in PAMs after PCV2 infection were examined using real-time qPCR. The expression of SIGIRR mRNA was normalized to actin-β and presented as fold change relative to the control (A). The Ct value of actin-β in PCV2-infected and control groups (B). The expression of SIGIRR in protein level was detected by Western blot (C). Densitometric analysis was performed to quantify the ratio of SIGGIR to actin-β. Results are mean ± SE of three independent experiments. * represents p < 0.05, ** represents p < 0.01.

Figure 5. NF-κB activation is critical for PCV2-induced IL-1β production in PAMs. PAMs were infected with PCV2 at an MOI of 1 for 1, 12, 24, and 48 h, and the p-p65 and p65 expression were measured by Western blotting (A). PAMs were treated with DMSO, BAY11-7082 (1 µM) for 1 h then infected with PCV2 at an MOI of 1. The supernatants were collected at 12 hpi and analyzed for IL-1β by ELISA (B). Total cells were harvested and analyzed for IL-1β mRNA expression by real-time RT-PCR (C). DMSO, the solubilization buffer of BAY11-7082, was used as a control. Results are mean ± SE of three independent experiments. ** represent p < 0.01.

Figure 6. The downregulation of SIGIRR promotes IL-1β overexpression. SIGIRR was knocked off in porcine alveolar macrophage cell line 3D4/2. (A) Graphical representation of targeted gRNA at porcine SIGIRR loci. Targeted genomic loci are indicated in green, protospacer adjacent motifs (PAM) sequences are marked in red. (B) Sanger sequencing of genomic PCR products encompassing the genomic-editing site from cells g1, g2, and g3. The wild type reference sequence is shown on the top. The bottom is a targeted sequence with deletions. the size of deletions (▼) on the right of each allele. (C) Western blotting analysis of SIGIRR protein in cells containing CRISPR-Cas9 system plasmids that were selected by puromycin. (D) Western blotting analysis of SIGIRR protein in cells containing expression plasmid pCDH-SIGIRR that was selected by puromycin. (E) IFA analysis of SIGIRR in g1 (SIGIRR-/-), overexpression cells (SIGIRR+/+), and wild type cells. (F) The IL-1β secretion was measured in wild type, SIGIRR-/-, and SIGIRR+/+ cells at 12 h after PCV2 inoculation. (G) The IL-1β mRNA levels were detected in wild type, SIGIRR-/-, and SIGIRR+/+ cells at 12 h after PCV2 inoculation. Results are mean ± SE of…

Figure 7. SIGIRR-/- cells have an increased NF-κB activation compare with SIGIRR+/+ cells. SIGIRR-/- and SIGIRR+/+ 3D4/2 cells were infected with PCV2 at MOI of 1 for 12 h, and the SIGIRR, p-p65, and p65 expression were measured by Western blotting (A). Densitometric analysis was performed by Image J software and results were normalized to β-actin (B). Results are mean ± SE of three independent experiments. * p < 0.05.