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

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.


INTRODUCTION
The innate immune system detects pathogen-associated molecular patterns (PAMPs) or dangerassociated molecular patterns (DAMPs) via germline-encoded pattern recognition receptors (PRRs) (1). Subsequently, innate immune responses are activated, and inflammatory cytokines, such as interferons (IFNs), proinflammatory cytokines, and chemokines, are generated. DAMPs and PAMPs comprise self-and foreign-derived double-stranded DNA in the cytosol (2). Stimulator of interferon genes (STING) is an ER-resident adaptor protein that is critical in mediating the signaling triggered by cytosolic nucleic acids (3,4). After activation by an agonist, STING undergoes a conformational change resulting in the recruitment of TANK binding kinase (TBK1) to STING (5,6). TBK1 subsequently phosphorylates IFN-regulated factor 3 (IRF3) and nuclear factor-κB (NF-κB), which translocate into the nucleus and stimulate expression of type I IFN and proinflammatory cytokines (7). Given the importance of the STING-mediated pathway in the activation of innate immunity and host protection from pathogens, harnessing the innate immunity activated by STING agonists is a promising strategy for antiviral and antitumor therapeutics (8,9). G10 is a synthetic small molecule that indirectly activates human STING and triggers IRF3-dependent IFNs expression but not NF-κB activation, thereby protecting against infection with emerging alphaviruses (10).
Pigs are a validated model for use in biomedical research fields, such as xenotransplantation and immune disorders (32,33). However, the interplay between type I IFN and inflammasomes has not been well documented in pigs to date. Here, we report that G10 triggers the activation of a STING-dependent type I IFN response and the NLRP3 inflammasome in porcine cells. G10 provides priming and activating signals, both of which are required for NLRP3 inflammasome activation. Furthermore, the NLRP3 inflammasome negatively regulates the type I IFN response in porcine cells after G10 treatment. Our results reveal a new mechanism through which G10 activates the NLRP3 inflammasome in porcine cells.

Nuclear and Cytoplasmic Extraction
Nuclear and cytoplasmic extraction was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific) according to the manufacturer's instructions. The extracted fractions were subjected to immunoblotting analysis.

ASC Speck Oligomerization Assay
Cells were lysed in homogenization buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, and 320 mM sucrose) supplemented with protease inhibitor cocktail via passage through a 21-gage needle 30 times. The lysates were subjected to centrifugation at 1500 rpm for 10 min. The supernatants were diluted with 1 volume of CHAPS buffer (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl 2 , 0.5 mM EGTA, and 0.1% CHAPS) supplemented with protease inhibitor cocktail and were centrifuged at 5000 rpm for 10 min. The pellets were washed three times with ice-cold PBS and then re-suspended in 30 µl of CHAPS buffer supplemented with 2 mM DSS crosslinker (21655, Thermo Fisher Scientific). After incubation at 37 • C for 20 min, the reaction was quenched by the addition of Laemmli buffer, and the samples were subjected to SDS-PAGE.

Immunofluorescence Analysis
Cells were grown on coverslips (12-545-80, Thermo Fisher Scientific) in 12-well plates and fixed with PBS/4% paraformaldehyde at room temperature for 30 min. The cells were then permeabilized with PBS/0.1% Triton X-100 at room temperature for 3 min. After being washed twice with PBS, the cells were incubated with PBS/10% FBS supplemented with the primary antibody at room temperature for 1 h. After being washed three times with PBS, the cells were labeled with 10% FBS/PBS supplemented with fluorescent secondary antibody (#A-11034, Thermo Fisher Scientific) at room temperature for 1 h. Images were captured under a Zeiss LSM 800 confocal microscope and processed in ImageJ software for quantitative image analysis.

Dual Luciferase Reporter Assays
Cells cultured in 24-well plates were co-transfected with pCMV-Renilla (normalization plasmid) and p65-Luc (luciferase reporter plasmid) with Lipofectamine 3000 (L3000015, Invitrogen). At 24 h post transfection, luciferase reporter assays were performed with the Dual-Luciferase Reporter Assay System (E1910, Promega) according to the manufacturer's instructions. The luminescence signal was detected with a Fluoroskan Ascent FL Microplate Fluorometer (Thermo Fisher Scientific).

Chromatin Immunoprecipitation Assays
Cells grown in 10-cm dishes were cross-linked with DMEM containing 1% formaldehyde for 15 min, and then the crosslinking was stopped by the addition of 125 mM glycine for 5 min. After being washed twice with PBS, the cells were incubated in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 5 mM MgCl 2 , and 0.5% NP40) supplemented with protease inhibitor cocktail on ice for 10 min and centrifuged at 2000 rpm for 5 min. The cell pellets containing chromatin were suspended in SDS lysis buffer (50 mM Tris-HCl, pH 7.9, 10 mM EDTA, and 0.5% SDS) supplemented with protease inhibitor cocktail and sonicated into fragments with an average length of 1 kb. Chromatin immunoprecipitation (ChIP) assays were performed with IgG or antibody against Sp1. Primer specific for p65 promoter was as follows: Fw: 5 -CCCCTCGGTGCCTTCT-3 and Rv: 5 -CGATGGGTGCACGCTA-3 .

Caspase-1 Activity Assays
Caspase-1 activity was assessed with a Caspase-Glo 1 Inflammasome Assay kit (G9951, Promega) with cell lysates treated as indicated, according to the manufacturer's instructions.

The Tissue Culture Infective Dose Assays
Vero cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well. On the next day, the cells were inoculated with serially diluted viruses (10 −1 -10 −12 -fold) for 1 h at 37 • C. The excess viral inoculum was removed by washing with PBS. Then, 200 µl of DMEM/2% FBS was added to each well, and the cells were further cultured for 3-5 days. The cells demonstrating the expected cytopathic effects were observed daily, and the tissue culture infective dose (TCID 50 ) value was calculated with the Reed-Muench method.

Plaque Assays
Vero cells were cultured just to confluency in six-well plates and inoculated with serially diluted viruses (10 −1 -10 −7 -fold) for 1 h at 37 • C. The excess viral inoculum was removed by washing with PBS. Then, 4 ml of DMED/1% methylcellulose (M8070, Solarbio) was added to each well, and the cells were further cultured for 4-5 days. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min before the plaques were counted.

Statistical Analysis
GraphPad Prism 7 software was used for data analysis. Data are shown as mean ± standard deviations from three independent experiments. Statistical significance between two groups was analyzed with two-tailed unpaired Student's t-test or oneway ANOVA.

G10 Activates the Type I IFN Response in Porcine Cells
G10 is a human-specific STING agonist that activates the IFN response ( Figure 1A) (10). We aimed to determine whether G10 might activate porcine STING-mediated type I IFN activation. We found that G10 up-regulated the transcription of IFN-β mRNA in both porcine PK15 kidney epithelial cells and 3D4/21 alveolar macrophages, in a manner dependent on STING and its downstream effectors TBK1 and IRF3, but not on IFNAR1 (Figures 1B,C). Ablation of Sting abolished G10-stimulated IFN-β secretion in PK15 and 3D4/21 cells ( Figure 1D). We further detected the expression of IFN-stimulated gene 15 (ISG15) under G10 treatment by using RT-qPCR analysis. As shown in Figure 1E, challenge of Sting −/− , Tbk1 −/− , Irf3 −/− , and Ifnar1 −/− PK15 cells with G10 had no effect on ISG15 transcription. This finding was also confirmed in Sting −/− and cells were seeded in 12-well plates at a density of 1 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 TCID 50 assays. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA.
We next assessed the antiviral activity of G10 against pseudorabies virus (PRV), a member of the subfamily Alphaherpesvirinae in the family Herpesviridae, and the causative pathogen of Aujeszky's disease in pigs (35). G10 treatment inhibited PRV infection in porcine PK15 and 3D4/21 cells (Figure 1G). STING deficiency in PK15 and 3D4/21 cells abrogated the antiviral activity of G10 and enhanced PRV infection ( Figure 1G). These findings demonstrate that G10 acts on STING, resulting in activation of type I IFN and antiviral activity in porcine cells.

G10 Activates P65 Gene Transcription in Porcine Cells
G10 activates human STING and triggers IRF3-dependent IFNs expression but not NF-κB activation. Unexpectedly, porcine 3D4/21 and PK15 cells showed significant P65 mRNA up-regulation in response to G10 in a dose-dependent manner (Figure 2A). Knockout of STING inhibited G10-, but not LPS-induced p65 transcription in 3D4/21 and PK15 cells (Figure 2A). Replenishment of STING expression in Figure S1A).
We next sought to address how G10 transcriptionally activates porcine p65 expression. We analyzed the promoter of the porcine p65 gene and found three consensus binding sites for the transcriptional factor Sp1 ( Figure 2B). Promoter mutation analysis with dual luciferase reporter assays indicated that the second Sp1 binding site (−1300) in the p65 promoter was essential for G10-mediated induction of p65 expression in a STING-dependent manner in 3D4/21 and PK15 cells (Figures 2C,D). However, LPS-stimulated p65 transcription was not dependent on these three Sp1 binding sites or on STING (Figures 2C,D). In addition, knockdown of Sp1 with RNA interference affected the expression of P65 mRNA in 3D4/21 and PK15 cells under G10 treatment (Figures 2E,F). Knockdown of Sp1 did not prevent LPS-induced p65 transcription, thus suggesting that G10 and LPS regulate p65 transcription through different mechanisms in porcine cells ( Figure 2F). Furthermore, ChIP assays showed that Sp1 was recruited to the promoter of p65 after G10 treatment, in a manner dependent on either STING or Sp1 ( Figure 2G). Collectively, these data suggest that G10 induces p65 transcription through STING and Sp1 in porcine cells.

G10 Activates the Porcine NF-κB Signaling Pathway
On the basis of the above findings, we attempted to determine whether G10 might activate the porcine NF-κB signaling pathway. We first addressed whether G10 induced the translocation of P65 into the nucleus. G10 did not stimulate P65 translocation into the nucleus, as indicated by immunofluorescence in human THP-1 cells (Supplementary Figure S1B). However, treatment of wild-type (WT) 3D4/21 and PK15 cells with G10 significantly promoted STING-dependent nuclear localization of P65 ( Figure 3A). However, the percentage of cells with nuclear localized P65 in WT and Sting −/− 3D4/21 and PK15 cells was unchanged by LPS treatment (Figure 3A). LPS, but not G10, induced P65 translocation into the nucleus in Sting −/− 3D4/21 cells, as indicated by immunoblotting of nuclear and cytoplasmic extracts (Figure 3B).
We next investigated the transcription of the NF-κB target genes Il-1β and Il-18 (36). G10 treatment significantly induced the transcription of IL-1β and IL-18 mRNA in 3D4/21 and PK15 cells, in a manner dependent on STING (Figures 3C,D). Knockdown of Sp1 abrogated G10-but not LPS-induced transcription of IL-1β and IL-18 mRNA (Figures 3E,F). Furthermore, we ablated p65 with CRISPR/Cas9 editing in 3D4/21 and PK15 cells (Figure 3G and Supplementary Figure S1C). The mRNA levels of IL-1β and IL-18 were not upregulated by the treatment of G10 and LPS in p65 −/− 3D4/21 and PK15 cells, thus suggesting that G10-and LPS-induced expression of IL-1β and IL-18 mRNA was dependent on P65 (Figures 3H,I). These data indicate that G10 activates the NF-κB signaling pathway in porcine cells.
To further establish the role of G10 in IL-1β and IL-18 secretion in porcine cells, we assessed the secretion of these cytokines in cells with Sp1 or P65 expression interference. Knockdown of Sp1 by RNA interference decreased G10induced IL-1β and IL-18 secretion in 3D4/21 and PK15 cells (Figures 4C,D). IL-1β and IL-18 secretion was constitutively up-regulated in response to LPS + Nig treatment, regardless of Sp1 deficiency (Figures 4C,D). In addition, P65-deficient 3D4/21 and PK15 cells, compared with WT cells, did not respond to G10 or to LPS + Nig in terms of induction of IL-1β and IL-18 secretion (Figures 4E,F). G10 promoted mature IL-1β secretion in 3D4/21 cells in a STING-dependent manner, as indicated by immunoblotting analysis of mature IL-1β ( Figure 4G). Transfection of STING-Flag plasmid into Stingdeficient 3D4/21 cells resulted in G10-induced IL-1β and IL-18 secretion, to levels comparable to those in WT cells ( Figure 4H). Together, these data demonstrate that STING-mediated NF-κB activation is essential for G10-induced IL-1β and IL-18 secretion in porcine cells.

G10-Induced ASC Oligomerization and
Caspase-1 Activation Promote IL-1β and IL-18 Maturation in Porcine Cells IL-1β and IL-18 maturation requires nucleation of the adaptor protein ASC, which controls Caspase-1 activation  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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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.
Frontiers in Immunology | www.frontiersin.org 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 × 10 5 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 × 10 5 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. cells were seeded in 12-well plates at a density of 1 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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 × 10 5 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. and subsequent cleavage of pro-IL-1β and pro-IL-18 (37). We observed that G10 treatment induced bright fluorescent ASC specks in WT but not Sting −/− 3D4/21 and PK15 cells, whereas induction of ASC specks by LPS + Nig treatment was independent of STING ( Figure 5A). Moreover, STING was responsible for G10-mediated induction of ASC oligomerization in PK15 cells, as indicated by immunoblotting analysis (Figure 5B).

G10 Activates the NLRP3 Inflammasome in Porcine Cells
Because our data suggested that G10 is a bona fide inflammasome activator, we sought to investigate which PRRs might be involved in G10-mediated activation of inflammasomes. We first analyzed the mRNA expression of inflammasome effectors under G10 treatment. As indicated by RT-qPCR analysis, G10 did not induce NLRP1 transcription in 3D4/21 and PK15 cells (Supplementary Figures S2A,B). However, NLRP3 mRNA was significantly up-regulated by G10 treatment in WT but not in Sting −/− 3D4/21 and PK15 cells (Figure 6A). Because NLRP3 inflammasome activation leads to NLRP3 aggregation (38), we detected NLRP3 aggregation by immunofluorescence. We observed that multiple puncta of NLRP3 appeared after G10 treatment in WT but not STING-deficient 3D4/21 and PK15 cells (Figure 6B). We stimulated 3D4/21 cells with G10 to observe whether STING co-localized to NLRP3. No obvious co-localization was observed when cells were treated with DMSO (Supplementary Figure S2C). G10 treatment induced multiple puncta of STING formation, some of which colocalized with NLRP3 (Supplementary Figure S2C). These results demonstrated that G10 activates porcine NLRP3.

Inhibition of the NLRP3 Inflammasome Enhances G10-Induced Type I IFN in Porcine Cells
Type I IFN and inflammasomes are reciprocally regulated, in a process essential for immune homeostasis (42). Therefore, we defined the role of the NLRP3 inflammasome in the regulation of type I IFN in response to G10 in porcine cells. Ablation of p65 further increased G10-, but not LPS-induced IFN-β and ISG15 mRNA transcription, as well as IFN-β secretion in 3D4/21 and PK15 cells (Supplementary Figures S3A,B and Figure 8A). Inhibition of the NLRP3 inflammasome by MCC950, or of Caspase-1 by VX765, prevented G10-mediated induction of IL-1β and IL18 secretion and enhanced IFN-β secretion in 3D4/21 and PK15 cells (Supplementary Figures S3C,D and  Figures 8B,C). In addition, G10-induced Nlrp3 −/− , Asc −/− , Caspase-1 −/− , and p65 −/− 3D4/21 and PK15 cells generated more IFN-β than WT and Ifnar1 −/− cells (Figures 8D,E). These data demonstrate that inhibition of the NLRP3 inflammasome enhances G10-induced type I IFN expression in porcine cells.

DISCUSSION
In this study, we demonstrated that G10 activates the porcine type I IFN response, in line with previous observations in human cells. Intriguingly, we further demonstrate that G10 primes the NLRP3 inflammasome through up-regulation of NLRP3, pro-IL-1β, and pro-IL-18 through STING, in contrast to the process in human cells. Furthermore, G10 induces potassium efflux and the oligomerization of NLRP3 and ASC, thus resulting in the formation of active Caspase-1, which in turn induces the secretion of mature IL-1β and IL-18. Treatment of NLRP3 inflammasome-deficient cells with G10 enhanced type I IFN expression and effectively inhibited viral infection. Our data indicate that type I IFN is negatively regulated by the NLRP3 inflammasome in porcine cells (Figure 9).
Nuclear factor-κB, a central mediator of immune and stress responses, is activated by multiple intra-and extracellular stimuli (43,44). Our work shows that G10 can activate the NF-κB pathway in porcine cells, but this activation does not occur in human cells. We found that G10 induces porcine p65 transcription through STING and Sp1. A previous study has indicated that the human p65 promoter lacks both TATA and CCAAT consensus sequences and contains three consensus binding sites for Sp1 (45). Human cytomegalovirus (HCMV) infection increases Sp1 expression and activates transcription of p65 through its promoter Sp1-binding sites (46,47). Because cGAS and STING are key sensors of HCMV in primary human monocyte-derived DCs and macrophages (48,49), our data may explain the mechanism through which HCMV infection activates p65 transcription. We also show that LPS-induced 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 × 10 5 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 TCID 50 assays. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA.  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.
p65 transcription is independent of STING and Sp1 in porcine cells. LPS activates NF-κB through Toll-like receptor 4-mediated signaling and causes Sp1 protein degradation by an LPS-inducible Sp1-degrading enzyme (50). Therefore, these findings combined with our current findings suggest that NF-κB is regulated by diverse stimuli through distinct mechanisms in different species.
Several studies have suggested that STING is involved in inflammasome activation. The cyclic dinucleotides 3 5diadenylate and 3 5 -diguanylate are bacterial second messengers that activate STING-mediated innate immune responses (51). They stimulate robust secretion of IL-1β through NLRP3 inflammasome signaling that is independent of STING (52). The intrinsic STING agonist cGAMP is catalyzed by cGAS from GTP and ATP (53), and it induces inflammasome activation through a STING, AIM2, NLRP3, ASC, and Caspase-1 dependent process in human and mouse cells (54). Cyclic dinucleotides from prokaryotes and eukaryotes are involved in different pathways of STING-mediated activation of inflammasomes. Nevertheless, our data demonstrate that STING is essential for G10-mediated activation of the canonical NLRP3 inflammasome in porcine cells. We did not determine whether AIM2 is involved in G10induced NLRP3 inflammasome activation, because AIM2 is not present in pigs (55). Importantly, given that STING is a promising target for cancer immunotherapy, our results suggest that the roles of STING agonist-induced type I IFN in inflammasome activation should be assessed in clinical trials.
The sustained robust inflammation may lead to collateral damage due to the overproduction of inflammatory cytokines (56). Aside from the antimicrobial functions of IFNs, aberrant IFN production is associated with a variety of autoimmune disorders (57). We demonstrated that the NLRP3 inflammasome negatively regulates type I IFN, which may be essential for immune homeostasis in pigs. Multiple reports have demonstrated that the NLRP3 inflammasome can induce and regulate the development of adaptive immunity (58). Both IL-1β and IL-1β are involved in T cell activation and memory cell formation (59)(60)(61)(62). Additionally, IL-1β and IL-18 have adjuvant capacity. Moreover, IL-1β enhances humoral immunity (63), and IL-18 augments IgE antibody production (64). Therefore, these beneficial aspects on the NLRP3 inflammasome may support the use of G10 as an effective vaccine adjuvant in the pig industry.

CONCLUSION
Stimulator of interferon genes is an ER-resident transmembrane protein that integrates cytosolic dsDNA-triggered activation of innate immune responses. G10 is a synthetic agonist of human STING that stimulates only STING-dependent IFN expression. However, we found that G10 acts on STING and activates both type I IFN and the canonic NLRP3 inflammasome. G10 activates the NLRP3 inflammasome in porcine cells through simultaneous priming and activation. Our data indicate that the G10-activated NLRP3 inflammasome negatively regulates type I IFN, a process that may be essential for immune homeostasis in pigs.

SUPPLEMENTARY MATERIALS
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.575818/full#supplementary-material FIGURE S1 | G10 activates porcine NF-κB signaling pathway. (A) WT and Sting −/− 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, Sting −/− 3D4/21 cells were transfected with plasmid for expression of STING-Flag (4 µg) for 24 h. Then 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 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) THP-1 cells were seeded and differentiated in 12-well plates with coverslips at a density of 2 × 10 5 per well. On the next day, cells were treated with 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. ns, no significance. (C) Schematic representation of the porcine p65 genomic structure and DNA sequencing results for the indicated knockout cells. Protospacer sequence is shown in red. The PAM sequence is framed by black boxes. (D) THP-1 cells were seeded and differentiated in 12-well plates with coverslips at a density of 2 × 10 5 per well. On the next day, cells were treated as in A. The medium was then harvested and IL-1β secretion was quantified by ELISA. ***P < 0.001 determined by two-tailed Student's t-test. (E,F) Schematic representations of the porcine Asc (E) and Caspase-1 (F) genomic structure and DNA sequencing results for the indicated knockout cells. Protospacer sequences are shown in red. The PAM sequences are framed by black boxes.  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. (C) 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were untreated or treated with G10 (20 µM), MCC950 (10 µM), and VX765 (10 µM) as indicated for 24 h. The medium was then harvested and IL-1β and IL-18 secretion was quantified by ELISA. ***P < 0.001 determined by two-tailed Student's t-test. ns, no significance. (D) PK15 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in C. The medium was then harvested and IL-1β and IL-18 secretion was quantified by ELISA. **P < 0.01 determined by two-tailed Student's t-test. ns, no significance. (E) WT and Ifnar1 −/− 3D4/21 cells were seeded in 12-well plates at a density of 1 × 10 5 per well. On the next day, cells were treated as in C. Virus was harvested by three freeze-thaw cycles and PRV titer was assessed with TCID 50 assays. **P < 0.01, ***P < 0.001 determined by one-way ANOVA. (F) PRV titer was assessed with plaque assays from E. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA.