Coinfection of Porcine Circovirus 2 and Pseudorabies Virus Enhances Immunosuppression and Inflammation through NF-κB, JAK/STAT, MAPK, and NLRP3 Pathways

Figures

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  Figure 1. Coinfection of PCV2 and PRV affects in PK-15 cells. Viral genomic copies were evaluated using real-time PCR at indicated hour post-infection. The data are presented as the means ± SD. **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (A) PCV2. (B) PRV.

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  Figure 2. Coinfection of PCV2 and PRV modulates IFNs. Western blotting was performed using Interferon alpha 1 Antibody, Interferon beta/IFNB, IFN-γ, IL28/29 (H-1), and Anti-β-Actin Antibody as primary antibodies, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. Unprocessed original images can be found in Supplementary Figure S1.

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  Figure 3. Coinfection of PCV2 and PRV regulates IRFs. (A,B) Expression levels of IRFs. The mRNA levels of IRF3 (A) and IRF7 (B) were evaluated via real-time PCR. (C) Protein levels of IRF7. Western blotting was performed using Rabbit Anti-IRF7 antibody and Anti-β-Actin Antibody as primary antibodies, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. (D,E) Expression levels of ISGs. The mRNA levels of ISG15 (D) and ISG56 (E) were evaluated via real-time PCR. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. The data are presented as the means ± SD. Unprocessed original images can be found in Supplementary Figure S2.

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  Figure 4. Coinfection of PCV2 and PRV suppresses STAT1-related JAK/STAT pathways. (A–D) Expression levels of JAK/STATs. The relative mRNA levels of JAK1 (A), SOCS1 (B), SOCS3 (C), and STAT1 (D) were detected using real-time PCR. (E,F) Protein levels of STAT1 and IRF9. Western blotting was performed using p-STAT1, Anti-STAT1 Antibody, IRF9 Antibody, and Anti-β-Actin Antibody as primary antibodies, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. (G) Expression levels of IRF9 were detected using real-time PCR. The data are presented as the means ± SD. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. Unprocessed original images can be found in Supplementary Figure S3.

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  Figure 5. Coinfection of PCV2 and PRV modulates the NF-κB signal pathway. (A,B) Expression levels of iκB and p65. The relative mRNA levels of iκB (A) and p65 (B) were examined using real-time PCR. The data are presented as the means ± SD. **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (C–E) Protein levels of p65 and iκB. Western blotting was conducted using P-IΚBα (Ser32 Ser36), IΚB-α, NFκB p65 Polyclonal Antibody, Phospho-NFκB p65 (Thr276) Polyclonal Antibody, and Anti-β-Actin Antibody as primary antibody, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. Unprocessed original images can be found in Supplementary Figure S4.

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  Figure 6. Coinfection of PCV2 and PRV modulates expressions of host pro-inflammatory factors. (A–D) Expression levels of pro-inflammatory factors. The relative mRNA levels of IL1α (A), TNFα (B), IL6 (C), and IL1β (D) were evaluated using real-time PCR. The data are presented as the means ± SD. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (E) Protein levels of pro-inflammatory factors. Western blotting was performed using IL1α, IL1B Antibody, IL6, TNFα, and Anti-β-Actin Antibody as primary antibodies, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. Unprocessed original images can be found in Supplementary Figure S5.

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  Figure 7. Coinfection of PCV2 and PRV activates the NLRP3 pathway. (A,B) Expression levels of NLRP3 and COX2. The relative mRNA levels of NLRP3 (A) and COX2 (B) were examined via Real-time PCR. The data are presented as the means ± SD. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (C) Protein levels of NLRP3 and COX2. Western blotting was performed using NLRP3, Cox-2, and Anti-β-Actin Antibody as primary antibodies, respectively. HRP-labeled Goat Anti-mouse IgG (H+L) and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. Unprocessed original images can be found in Supplementary Figure S6.

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  Figure 8. Coinfection of PCV2 and PRV activates inflammatory and immune responses via p38 and JNK1/2. (A–C) Expression levels of MAPKs. The relative mRNA levels of p38 (A), ERK1/2 (B), and JNK1/2 (C) were examined via real-time PCR. The data are presented as the means ± SD. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (D–G) Protein levels of MAPKs. Western blotting was conducted using p-p38 (Thr180/Tyr182), P38, JNK (FL), p-JNK (Thr 183/Tyr 185), ERK p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb, and Anti-β-Actin Antibody as primary antibody, respectively. HRP-labeled Goat Anti-mouse IgG (H+L), HRP-labeled Donkey Anti-Goat IgG (H+L), and HRP-labeled Goat Anti-rabbit IgG (H+L) were used as the secondary antibody. β-actin was used as a control. The average expression level of the target protein in each group is shown below each lane. The protein amount of the PK-15 group is set to 1, and the values of other groups are the ratio with the PK-15 group. Unprocessed original images can be found in Supplementary Figure S7.

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  Figure 9. Proposed immune and inflammatory reactions caused by coinfection of PCV2 and PRV. Arrow (→), enhance; T line (⊥) means inhibition.