Fig 1: The anti-CSFV activity of TNF involves JAK/STAT signaling. (a,b) The PEDSV.15 cells were treated with pTNF (a) or IFN-β (b) in the presence of ruxolitinib (2 µM) or DMSO for six hours prior to CSFV-luc infection (MOI, 0.1 TCID50/cell) and measurement of firefly luciferase activity 22 h later. The values are shown as the percentage of the DMSO control in the absence of pTNF or IFN-β, respectively. (c,d) JAK/STAT inhibitor Compound Library screens were performed with CSFV-luc-infected cells pre-stimulated with the medium, LPS or pTNF in the presence of the individual JAK/STAT inhibitors at the concentration of 0.5 µM (c) or 5 µM (d). The data due to cytotoxic and antiviral activity of the compounds were eliminated from the analysis. (e) The PEDSV.15 cells were treated with pTNF or mock in the presence or absence of ruxolitinib for four hours, and the IFN-β mRNA to 18S ribosomal RNA ratio was quantified by means of RT-qPCR and plotted as log10 fold induction. (f) The PEDSV.15 cells were transfected with an NF-κB-dependent firefly luciferase gene reporter plasmid and treated with IFN-β, pTNF, LPS in the presence of the NF-κB signaling inhibitor TPCA-1 or the JAK/STAT inhibitor ruxolitinib. The NF-κB activity is represented as fold luciferase induction related to the medium treatment. The data represent the means and the standard deviations of three independent experimental replicas. The differences were considered to be statistically significant at p < 0.05 using the Student’s t-test (ns, nonsignificant).
Fig 2: The anti-CSFV activity of TNF requires IFNAR1, while IRF3 is dispensable. PEDSV.15 cells lacking functional IFNAR1 or IRF3 were generated using CRISPR/Cas9-based genome editing by targeting early exons with two separate gRNAs each. (a,c) PCR amplifications of the IFNAR1 (a) and IRF3 (c) loci are shown for the parent cells and for two knockout cell clones each. A Cas9-exposed clone with a functional receptor (IFNAR1-WT#4) served as the negative control. (b,d) The IFNAR1- (b) or IRF3-KO cells (d) were stimulated with pTNF, LPS, IFN-β or the medium for seven hours followed by infection with CSFV-luc at a MOI of 0.1 TCID50/cell for 22 h before cell lysates were processed for firefly luciferase measurement. The data in (b,d) represent the means and the standard deviations of six independent experimental replicas. Significant differences compared with the medium (p < 0.05) were calculated with one-way ANOVA and post hoc tests (the p-values are indicated; ns, nonsignificant).
Fig 3: CSFV infection does not interfere with TNF- or LPS-mediated induction of IFN-β mRNA in PEDSV.15 cells. The PEDSV.15 (a,b), IRF1-KO#2 (a) and IRF3-KO#4 cells (b) were mock-infected (–) or infected with CSFV vEy-37 (+) for 3 days and stimulated subsequently for 2.5 h with pTNF, LPS or p(I:C) at the indicated concentrations or left untreated (medium). After stimulation, IFN-β mRNA induction normalized with 18S RNA was quantified by RT-qPCR. (c) Parallel cultures of the mock- (–) or CSFV-infected (+) PEDSV.15, IRF1-KO#2 and IRF3-KO#4 cells were lysed and the proteins were separated by SDS–PAGE and analyzed by means of Western blotting for IRF3, CSFV Npro and β-actin expression. The data in (a,b) show the means and the standard deviations of three independent experimental replicas. Statistically significant differences were determined with the unpaired, two-tailed Student’s t-test (the p-values are indicated; ns, nonsignificant).
Fig 4: TNF antagonizes CSFV infection in porcine aortic endothelial cells and in primary porcine MDM, but not in PK-15 and SK-6 cells. (a) The PEDSV.15, PK-15 and SK-6 cells were treated with mTNF (5 ng/mL) or the medium for five hours. After the washing step with the medium, the cells were infected with CSFV-luc at a MOI of 0.1 TCID50/cell. The firefly luciferase activity in cell culture lysates was determined 20 h after infection. (b,c) Similarly, the PEDSV.15 cells were treated with increasing concentrations of mTNF prior to CSFV-luc infection and quantification of luciferase activity from cell lysates (b) or viral titers from cell culture supernatants (c) 20 h later. (d) The parallel mock- (0) and mTNF-treated CSFV-luc-infected PEDSV.15 cell cultures were analyzed for cell viability using the alamarBlue cell viability assay at the indicated times and the values are shown as percentage of untreated cells. (e) The antiviral effect of mTNF in the PEDSV.15 cells was assessed in time-of-addition experiments. (f) The pretreated cells were infected and cultured in the presence of mTNF until termination of the experiments 20 h after infection. (g) Porcine MDM were stimulated for six hours with increasing concentrations of mTNF prior to CSFV-luc infection (MOI 0.2 TCID50/cell) and measurement of luciferase activity in cell culture lysates 20 h later. (h) Viability of the TNF-treated and infected MDM was assessed in parallel as in (d). (i) The PEDSV.15 cells were preincubated for six hours with mTNF, pTNF and LPS in the presence of increasing concentrations of adalimumab, a human TNF-neutralizing antibody, prior to CSFV-luc replication analysis as described above. In panels (a,b,e–g), the firefly luciferase activity is shown as the percentage of the medium control in the absence of TNF. The data represent the mean of three (i), four (a–c,e,f), five (g,h) or 12 (d) experimental replicas with error bars indicating the standard deviations. Statistically significant differences (p < 0.05) were determined using the unpaired, two-tailed Student’s t-test (ns, nonsignificant).
Fig 5: The anti-CSFV activities of LPS and TNF are IRF1-dependent. (a) PEDSV.15 cells expressing nonfunctional IRF1 were generated with CRISPR/Cas9-based genome editing. (b) Two independent knockout clones (#2 and #12) and the Cas9-exposed negative control (IRF1-WT#1) with intact IRF1 were stimulated with pTNF, LPS, IFN-β or the medium for seven hours followed by infection with CSFV-luc at a MOI of 0.1 TCID50/cell for 22 h before the cell lysates were processed for firefly luciferase measurement. (c,d) The effect of two hours of stimulations with LPS (c) or pTNF (d) on the expression of IFN-β mRNA normalized to 18S ribosomal RNA was assessed in the PEDSV.15 and IRF1-KO#2 cells. The data in (b) represent the means and the standard deviations of six independent experimental replicas and significant differences compared with the medium (p < 0.05) were calculated with one-way ANOVA and post hoc tests (p-value indicated; ns, nonsignificant). The data in (c,d) represent the means and the standard deviations of three independent experimental replicas. Significant differences compared with the medium (p < 0.05) were calculated with the unpaired, two-tailed Student’s t-test (the p-values are indicated; ns, nonsignificant).
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