The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells

Abstract

Pigs have anatomical and physiological characteristics comparable to those in humans and, therefore, are a favorable model for immune function research. Interferons (IFNs) and inflammasomes have essential roles in the innate immune system. Here, we report that G10, a human-specific agonist of stimulator of interferon genes (STING), activates both type I IFN and the canonical NLRP3 inflammasome in a STING-dependent manner in porcine cells. Without a priming signal, G10 alone transcriptionally stimulated Sp1-dependent p65 expression, thus triggering activation of the nuclear factor-κB (NF-κB) signaling pathway and thereby priming inflammasome activation. G10 was also found to induce potassium efflux- and NLRP3/ASC/Caspase-1-dependent secretion of IL-1β and IL-18. Pharmacological and genetic inhibition of NLRP3 inflammasomes increased G10-induced type I IFN expression, thereby preventing virus infection, suggesting negative regulation of the NLRP3 inflammasome in the IFN response in the context of STING-mediated innate immune activation. Overall, our findings reveal a new mechanism through which G10 activates the NLRP3 inflammasome in porcine cells and provide new insights into STING-mediated innate immunity in pigs compared with humans.

Keywords: NLRP3 inflammasome; STING agonist; innate immunity; interferon; negative regulation.

FIGURE 1

G10 elicits a type I IFN response in porcine cells. (A) Schematic representation of the STING-mediated type I IFN response. (B) WT, Sting^–/–, Tbk1^–/–, Irf3^–/–, and Ifnar1^–/– PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with G10 at the indicated concentrations for 24 h. Total mRNA was then reverse-transcribed to cDNA and IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (C) WT, Sting^–/–, and Ifnar1^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in B. IFN-β mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (D) WT and Sting^–/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in B. The medium was then harvested and IFN-β secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E) WT, Sting^–/–, Tbk1^–/–, Irf3^–/–, and Ifnar1^–/– PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in B. ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (F) WT, Sting^–/–, and Ifnar1^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in B. ISG15 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G) WT and Sting^–/– PK15 and 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were infected with PRV-QXX (MOI = 1) and simultaneously treated with G10 as in B. Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID₅₀ assays. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA.

FIGURE 2

G10 stimulates Sp1-dependent p65 transcription in porcine cells. (A) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10 (0.6–20 μM), and LPS (1 μg/ml) for 24 h. Total mRNA was then reverse-transcribed to cDNA and P65 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (B) Diagrams of the P65 promoter and the various mutants, with the Sp1 binding sites indicated. (C) WT and Sting^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were transfected with 0.1 μg/well p65–Luc variants and 0.02 μg/well pCMV-Renilla. At 24 h post transfection, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 6 h. p65 promoter activity was assessed with dual luciferase reporter assays. ***P < 0.001 determined by two-tailed Student’s t-test. (D) p65 promoter activity was assessed with dual luciferase reporter assays in WT and Sting^–/– PK15 cells the same as in C. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E) 3D4/21 and PK15 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were transfected with indicated siRNA for 48 h. Sp1 protein was assessed with immunoblotting analysis. β-actin served as loading control. (F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were transfected with indicated siRNA for 48 h. Then cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. Total mRNA was then reverse-transcribed to cDNA and P65 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. ***P < 0.001 determined by two-tailed Student’s t-test. (G) WT and Sting^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, 3D4/21 cells were transfected with siSp1-3 for 48 h. Then WT, Sting^–/–, and siSp1-3 transfected 3D4/21 cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml). Sp1 ChIP assays were performed at 24 h post treatment. ***P < 0.001 determined by two-tailed Student’s t-test.

FIGURE 3

G10 activates the NF-κB signaling pathway in porcine cells. (A) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. Translocation of P65 into the nucleus (DAPI) was assessed by immunofluorescence analysis with antibody against P65. Quantification of cells with nuclear localized P65 is shown on the right (n = 30 cells). Scale bar, 10 μm. ***P < 0.001 determined by two-tailed Student’s t-test. (B) WT and Sting^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in A. Phospho-P65 and P65 were assessed with immunoblotting analysis in the cytosol and nuclei fraction. β-actin (indicating cytosol) and Lamin B1 (indicating nuclei) served as loading controls. (C,D) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with G10 at the indicated concentration for 24 h. Total mRNA was then reverse-transcribed to cDNA and IL-1β (C) and IL-18 (D) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E,F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were transfected with indicated siRNAs for 48 h. Then, cells were treated as in A. Total mRNA was then reverse-transcribed to cDNA and IL-1β (E) and IL-18 (F) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G) P65 protein was assessed with immunoblotting analysis in WT, p65^–/– 1#, and p65^–/– 2# 3D4/21 and PK15 cells. β-actin served as loading control. (H,I) WT, p65^–/– 1#, and p65^–/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in A. Total mRNA was then reverse-transcribed to cDNA and IL-1β (H) and IL-18 (I) mRNAs were assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test.

FIGURE 4

G10 promotes IL-1β and IL-18 secretion in porcine cells. (A,B) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at indicated concentrations for 24 h. The medium was then harvested and IL-1β (A) and IL-18 (B) secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (C,D) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were transfected with indicated siRNA for 48 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at indicated concentrations for 24 h. The medium was then harvested and IL-1β (C) and IL-18 (D) secretion was quantified by ELISA. ***P < 0.001 determined by two-tailed Student’s t-test. (E,F) WT, p65^–/– 1#, and p65^–/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in C. The medium was then harvested and IL-1β (E) and IL-18 (F) secretion was quantified by ELISA. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G) WT and Sting^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in C. The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β by immunoblotting analysis. (H) WT and Sting^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, Sting^–/– 3D4/21 cells were transfected with plasmid for expression of STING-Flag plasmid (4 μg) for 24 h. Then, cells were treated as in A. The medium was then harvested and IL-1β and IL-18 secretion was quantified by ELISA. **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test.

FIGURE 5

G10 induces ASC oligomerization and Caspase-1 activation in porcine cells. (A) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations for 24 h. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown on the right (n = 30 cells). Scale bar, 10 μm. ***P < 0.001 determined by two-tailed Student’s t-test. (B) WT and Sting^–/– PK15 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in A. ASC oligomerization was assessed by immunoblotting analysis. (C,D) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations in the absence (PBS) or presence of Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (E,F) WT and Asc^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10 and LPS + Nig at the indicated concentrations for 24 h. The medium was then harvested and IL-1β (E) and IL-18 (F) secretion were quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (G,H) WT and Caspase-1^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in E. The medium was then harvested and IL-1β (G) and IL-18 (H) secretion were quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test.

FIGURE 6

G10 activates the NLRP3 inflammasome in porcine cells. (A) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations for 24 h. Total mRNA was then reverse-transcribed to cDNA and NLRP3 mRNA was assessed by RT-qPCR analysis. The results were normalized to the level of β-actin expression. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (B) WT and Sting^–/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were transfected with plasmid for expression of NLRP3-Flag (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS + Nig (1 μg/ml + 2.5 μM) for 24 h. NLRP3 activation was assessed by immunofluorescence analysis with antibody against Flag. Scale bar, 10 μm. (C,D) WT, Sting^–/–, Nlrp3^–/–, Asc^–/–, and Caspase-1^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, LPS + Nig, LPS + ATP, and VX765 at the indicated concentrations for 24 h. The medium was then harvested and IL-1β (C) and IL-18 (D) secretion were quantified by ELISA. ***P < 0.001 determined by two-tailed Student’s t-test. (E) WT and Nlrp3^–/– 3D4/21 and PK15 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated as in B. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown on the right (n = 30 cells). Scale bar, 10 μm. ***P < 0.001 determined by two-tailed Student’s t-test. (F) WT and Nlrp3^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in B. ASC oligomerization was assessed by immunoblotting analysis. (G) WT and Nlrp3^–/– 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10, and LPS + Nig at the indicated concentrations in the absence (PBS) or presence of Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-tailed Student’s t-test. (H) WT and Nlrp3^–/– 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated as in B. The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β, pro-Caspase-1, and cleaved Caspase-1 (P20) by immunoblotting analysis.

FIGURE 7

G10-mediated activation of the NLRP3 inflammasome requires potassium flux in porcine cells. (A–D) 3D4/21 (A,B) and PK15 (C,D) cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with G10 (20 μm), LPS + Nig (1 μg/ml + 2.5 μM), and CL097 (70 μM) in the absence or presence of KCl (10–80 mM) for 24 h. The medium was then harvested and IL-1β (A,C) and IL-18 (B,D) secretion were quantified by ELISA. *P < 0.05, **P < 0.01 determined by two-tailed Student’s t-test. ns, no significance. (E) 3D4/21 cells were seeded in 12-well plates with coverslips at a density of 1 × 10⁵ per well. On the next day, cells were transfected with plasmid for expression of ASC-GFP (2 μg) for 24 h. Then, cells were treated with vehicle (DMSO), G10 (20 μm), and LPS + Nig (1 μg/ml + 2.5 μM) in the absence (PBS) or presence of KCl (80 mM) and NaCl (10 mM) for 24 h. ASC oligomerization was assessed by fluorescence microscopy. Quantification of cells with ASC specks is shown at the bottom (n = 30 cells). Scale bar, 10 μm. ***P < 0.001 determined by two-tailed Student’s t-test. ns, no significance. (F) 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with G10 (20 μm) and LPS + Nig (1 μg/ml + 2.5 μM) as indicated in the absence (PBS) or presence of KCl (45 mM) and Caspase-1 inhibitor YVAD-CHO (5 μM) for 24 h. Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit. ***P < 0.001 determined by two-tailed Student’s t-test. (G) 3D4/21 cells were seeded in 60-mm dishes at a density of 4 × 10⁵ per dish. On the next day, cells were treated with vehicle (DMSO), G10 (20 μm), and LPS + Nig (1 μg/ml + 2.5 μM) in the absence (PBS) or presence of KCl (80 mM) for 24 h. The medium was harvested to analyze mature IL-1β (P17) secretion, and the cells were harvested to analyze pro-IL-1β, pro-Caspase-1 and cleaved Caspase-1 (P20) by immunoblotting analysis.

FIGURE 8

Inhibition of the NLRP3 inflammasome augments G10-induced type I IFN and antiviral activity in porcine cells. (A) WT, p65^–/– 1#, and p65^–/– 2# 3D4/21 and PK15 cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO), G10 (20 μM), and LPS (1 μg/ml) for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. **P < 0.01, ***P < 0.001 determined by one-way ANOVA. (B,C) 3D4/21 (B) and PK15 (C) cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with G10 (20 μM), MCC950 (10 μM), and VX765 (10 μM) as indicated for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA. (D,E) WT, Sting^–/–, Nlrp3^–/–, Asc^–/–, Caspase-1^–/–, p65^–/– 1#, and Ifnar1^–/– 3D4/21 (D) and WT, Sting^–/–, Nlrp3^–/–, Asc^–/–, Caspase-1^–/–, p65^–/– 1#, and Ifnar1^–/– PK15 (E) cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated with vehicle (DMSO) or G10 (20 μM) for 24 h. The medium was then harvested and IFN-β secretion was quantified by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA. ns, no significance. (F,G) 3D4/21 (F) and PK15 (G) cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were infected with PRV-QXX (MOI = 1) and simultaneously treated as in B. Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID₅₀ assays. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA. (H,I) WT, Sting^–/–, Nlrp3^–/–, Asc^–/–, Caspase-1^–/–, p65^–/– 1#, and Ifnar1^–/– 3D4/21 (H) and WT, Sting^–/–, Nlrp3^–/–, Asc^–/–, Caspase-1^–/–, p65^–/– 1#, and Ifnar1^–/– PK15 (I) cells were seeded in 12-well plates at a density of 1 × 10⁵ per well. On the next day, cells were treated as in D. Virus was harvested by three freeze–thaw cycles and PRV titer was assessed with TCID₅₀ assays. *P < 0.05, **P < 0.01 determined by one-way ANOVA.

FIGURE 9

Schematic model showing G10 activation of the NLRP3 inflammasome in porcine cells. In human cells, G10 activates only type I IFN. In porcine cells, G10 also activates the NF-κB signaling pathway, which is a priming signal for NLRP3 inflammasome activation. G10 induces potassium efflux and triggers NLRP3 inflammasome activation, which negatively regulates type I IFN.