Suppression of lymphocyte apoptosis in spleen by CXCL13 after porcine circovirus type 2 infection and regulatory mechanism of CXCL13 expression in pigs

Figures

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  Figure 1. Pathological changes in the spleen tissue of LW and YL pigs. Tissue slices were stained by HE; the white bar indicates 100 μm. A Mock-infected LW pigs; B PCV2-infected LW pigs; C mock-infected YL pigs; D PCV2-infected YL pigs.

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  Figure 2. Apoptotic cells in the spleen tissue of LW and YL pigs analyzed by TUNEL staining. The white bar indicates 100 μm. A Mock-infected LW pigs; B PCV2-infected LW pigs; C mock-infected YL pigs; D PCV2-infected YL pigs.

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  Figure 3. PCV2 genomic DNA copies in spleen tissues of PCV2-infected LW and YL pigs. The PCV2 genomic DNA copies in spleen were measured through absolute qPCR. Multiplying the value on the Y-axis by 106 indicates the copy number of PCV2 genomic DNA in 1 g of spleen tissue. Data are represented as the mean value ± SE of three experiments. ***Indicates significance at the P value threshold level of 0.001.

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  Figure 4. Twelve DEGs validated by RT-qPCR and expression changes of CXCL13 protein in porcine spleens after PCV2 infection. A Twelve candidate genes validated by RT-qPCR in LW and YL pigs. The HPRT1 gene was used as an internal control. Data are represented as the mean value ± SE of three experiments. Different lowercase letters indicate significance at the P value threshold level of 0.05. B Expression changes of CXCL13 protein in porcine spleens after PCV2 infection. The GAPDH gene was used as an internal control. LW-u, mock-infected LW pigs; LW-i, PCV2-infected LW pigs; YL-u, mock-infected YL pigs; YL-i, PCV2-infected YL pigs. Data are represented as the mean value ± SE of three experiments. *Indicates significance at the P value threshold level of 0.05.

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  Figure 5. CXCL13 can suppress the apoptosis of murine splenocytes during PCV2 infection. Splenocytes were freshly isolated from murine spleen and treated with PCV2 at an MOI of 0.1 TCID50 alone or along with CXCL13 (1 μg/mL). The cells were analyzed by flow cytometry for PI (y axis) and FITC-conjugated annexin V (x axis) at 2, 4, 8, 16, and 24 hpi after treatment. The total percentages of PI− annexin V+ cells (Q4) and PI+ annexin V+ cells (Q2) indicate the apoptosis rate. The nine scatter plots shown on the left are from a single experiment, which was representative of three separately performed experiments. The bar graphs shown on the right indicate mean value ± SE of three experiments. * and *** indicate significance at the P value threshold levels of 0.05 and 0.001, respectively.

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  Figure 6. Transcriptional activity of the CXCL13 promoter cloned from LW and YL pigs. A Luciferase activities of pGL3-CXCL13LW and pGL3-CXCL13YL in PK15 cells at 24 h post-transfection. Luciferase activities of pGL3-CXCL13LW (B) or pGL3-CXCL13YL (C) in PK15 cells with and without PCV2 infection. PCV2-infected groups (pGL3-CXCL13LW-i and pGL3-CXCL13YL-i) were inoculated with PCV2 at an MOI of 0.1 TCID50 and PCV2-uninfected groups (pGL3-CXCL13LW-u and pGL3-CXCL13YL-u) were treated with equivalent PK15 cell lysate at 6 h post-transfection. Luciferase activity was detected at 0, 12, 24, 36, and 48 hpi. D Detailed delineation of the promoter regions of the porcine CXCL13 gene by 5′ deletion analysis. The left panel shows a schematic diagram for firefly luciferase reporter constructs containing the indicated genomic sequences upstream of CXCL13. The right panel shows the luciferase activity of the corresponding deletion construct. At least three separate experiments were performed with each plasmid DNA preparation. Data are represented as the mean value ± SE of three experiments. * and *** indicate significance at the P value threshold levels of 0.05 and 0.001, respectively.

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  Figure 7. Effect of each SNP on the transcriptional activity of the CXCL13 promoter. A Eight SNPs discovered in the critical promoter region of porcine CXCL13. B Luciferase activities of pGL3-CXCL13YL and site-directed mutants of pGL3-CXCL13YL in PK15 cells at 48 h post-transfection. C–G Luciferase activities of each site-directed mutant of pGL3-CXCL13YL in PK15 cells with and without PCV2 infection. PCV2-infected groups (pGL3-(−3065T)-i, pGL3-(−2991G)-i, pGL3-(−1014G)-i, pGL3-(−712T)-i, and pGL3-(−604C)-i) were inoculated with PCV2 at an MOI of 0.1 TCID50, and PCV2-uninfected groups (pGL3-(−3065T)-u, pGL3-(−2991G)-u, pGL3-(−1014G)-u, pGL3-(−712T)-u, and pGL3-(−604C)-u) were treated with equivalent PK15 cell lysate at 6 h post-transfection. Luciferase activity was detected at 0, 12, 24, 36, and 48 hpi. Each bar represents the mean value ± SE of three experiments. *, **, and *** indicate significance at the P value threshold levels of 0.05, 0.01, and 0.001, respectively.

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  Figure 8. CXCL13 is a direct target of ssc-miR-296-5p. A Diagram of the predicted targeting site within the 3′-UTR of CXCL13 (3′UTR-wt). The mutated 3′-UTR (3′UTR-mut) contains mutated sequence that is not complementary to the seed sequence (CCGGGAG) of ssc-miR-296-5p. B Expression of ssc-miR-296-5p in PK15 cells at 24 h post-transfection with 800 nM inhibitor (inhi) or negative control (NC). C Luciferase activity of the constructs harboring the wild-type or mutant 3′-UTR of the CXCL13 gene in PK15 cells with 800 nM ssc-miR-296-5p inhibitor or negative control. Data are represented as the mean value ± SE of three experiments. *, **, and *** indicate significance at the P value threshold levels of 0.05, 0.01, and 0.001, respectively.