Caspase-1 inhibits IFN-β production via cleavage of cGAS during M. bovis infection

Yi Liao a, b, Chunfa Liu c, Jie Wang d, Yinjuan Song a, Naveed Sabir a, Tariq Hussain a, Jiao Yao a, Lijia Luo a, Haoran Wang a, Yongyong Cui e, Lifeng Yang a, Deming Zhao a, Xiangmei Zhou a,*
a Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100093, China
b College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu, 610041, China
c National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, China
d CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China
e Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA

A R T I C L E I N F O

Chen, 2018; Liao et al., 2019). M. bovis can also activate caspase-1 via the AIM2 and NLRP7 inflammasomes(Yang et al., 2013; Zhou et al.,
* Corresponding author at: National Veterinary Drug Safety Evaluation Center, China Agricultural University, Room 308, 2 Yuanmingyuan West Road, Haidian District, Beijing, China.
E-mail address: [email protected] (X. Zhou).
https://doi.org/10.1016/j.vetmic.2021.109126 Received 25 April 2020; Accepted 13 May 2021
Available online 15 May 2021
0378-1135/© 2021 Published by Elsevier B.V.2016).

Keywords:
M. bovis Caspase-1 IL-1β IFN-β cGAS

A B S T R A C T

Mycobacterium bovis (M. bovis) infection triggers cytokine production via pattern recognition receptors. These cytokines include type I interferons (IFNs) and interleukin-1β (IL-1β). EXcessive type I IFN levels impair host resistance to M. bovis infection. Therefore, strict control of type I IFN production is helpful to reduce pathological damage and bacterial burden. Here, we found that a deficiency in caspase-1, which is the critical component of the inflammasome responsible for IL-1β production, resulted in increased IFN-β production upon M. bovis infection. Subsequent experiments demonstrated that caspase-1 activation reduced cyclic GMP-AMP synthase (cGAS) expression, thereby inhibiting downstream TANK-binding kinase 1 (TBK1)- interferon regulatory factor 3 (IRF3) signaling and ultimately reducing IFN production. A deficiency in caspase-1 activation enhanced the bacterial burden during M. bovis infection in vitro and in vivo and aggravated pathological lesion formation.
Thus, caspase-1 activation reduced IFN-β production upon M. bovis infection by dampening cGAS-TBK1-IRF3 signaling, suggesting that the inflammasome protects hosts by negatively regulating harmful cytokines.

1. Introduction

Mycobacterium infection induces innate immune responses in host macrophages. Macrophages utilize pattern recognition receptors (PRRs) to probe damage-associated molecular patterns (DAMPs) and pathogen- associated molecular patterns (PAMPs), which include the nucleic acids of Mycobacterium. The retinoic acid-inducible gene I (RIG-I) receptor recognizes mycobacterial RNA released into the macrophage cytosol and induces IFN-β secretion through the MAVS-IRF7 pathway(Cheng and Schorey, 2018). For intracytoplasmic DNA recognition, interferon (IFN)-γ inducible protein 204 (IFI204) has been found to recognize Mycobacterium bovis (M. bovis) DNA in the cytoplasm and trigger stimulator of IFN genes (STING)-interferon regulatory factor 3 (IRF3) pathway-dependent IFN-β production(Chunfa et al., 2017). Further- more, cyclic GMP-AMP synthase (cGAS), another cytoplasmic receptor, recognizes mycobacterial or mitochondrial DNA (mtDNA) and catalyzes the production of cGAMP, which acts as a second messenger to activate the STING-IRF3 pathway, leading to IFN-β release(Collins et al., 2015). Although type I IFNs are key cytokines against viruses, excessive IFN levels can be detrimental to the host during M. bovis infection(Dorhoi et al., 2014). Therefore, hosts have developed subtle mechanisms to negatively regulate type I IFNs.
Mycobacterial infection also causes NLRP3 inflammasome activation (Zhou et al., 2015). The NLRP3 inflammasome is a complex composed of the receptor NLRP3, the adaptor ASC and the catalytic subunit caspase-1. Upon stimulation with multiple DAMPs or PAMPs, including a dispersed trans-Golgi network, reactive oXygen species (ROS), and mtDNA, oligomerization of the NLRP3 inflammasome triggers the pro- teolytic activity of caspase-1, which is indispensable for the catalytic cleavage and secretion of prointerleukin (IL)-1β and IL-18(Chen and In addition, caspase-1 can induce a lytic programmed cell death progress known as pyroptosis. Interestingly, caspase-1 is also involved in the proteolytic processing of innate immune response-associated pro- teins. Jabir et al. reported that caspase-1 activated by Pseudomonas aeruginosa infection could cleave TRIF, resulting in reduced a autophagy and type I IFN production(Jabir et al., 2014). Wang et al. reported that caspase-1-dependent cleavage of cGAS attenuated type I IFN production during DNA virus infection (Wang et al., 2017).
Although type I IFNs help control the spread of viral infection, the excessive production of these cytokines is harmful during M. bovis infection (Wang et al., 2019). In contrast, inflammasome activation and subsequent production of caspase-1 and IL-1β help hosts resist Myco- bacterium infection while suppressing antiviral cytokines (Yang et al., 2013).
Because viruses and mycobacteria can simultaneously activate type I IFN and inflammasomes, mutual regulation between these two path- ways is necessary for hosts to resist pathogenic microorganisms.
Indeed, previous studies have shown that there is a mutual inhibitory effect between these two pathways. IFN-I has been reported to inhibit inflammasome activation (Reboldi et al., 2014), and many PRRs, such as AIM2 (Liu et al., 2016), NLRX1 (Lei et al., 2012; Guo et al., 2016; Allen et al., 2011), NLRP6 (Anand et al., 2012), NLRC3 (Schneider et al., 2012), and NLRC5 (Tong et al., 2012), negatively regulate IFN-I production.
It has been reported that caspase-1 can cleave cGAS and thus decrease IFN-β production. In addition, AIM2 inflammasome activation by M. bovis infection can inhibit IFN-β expression(Liu et al., 2016), but it is not clear whether caspase-1 cleaves cGAS during this process.
Here, we report the inhibitory effect of caspase-1 on IFN-β produc- tion via cGAS cleavage during M. bovis infection. In M. bovis-infected bone marrow-derived macrophages (BMDMs), cGAS was cleaved by caspase-1, resulting in decreased TBK1 phosphorylation and reduced IRF3 nuclear transfer, eventually leading to decreased IFN-β expression. Consequently, after feeding with a caspase-1 inhibitor, mice exhibited increased IFN-β production, relatively severe lesions, and an increased bacterial burden. Our study therefore revealed the mechanism by which caspase-1 regulates the cGAS-STING pathway during M. bovis infection. Because M. tuberculosis complex infection is often associated with viral coinfection, our findings suggest that the regulation of inflamma- somes and IFNs by medicine should be explored during coinfection and that efforts should be made to avoid medicines causing insufficient or excessive innate immune responses.

2. Materials and methods

2.1. Animal infection model
SiX-week-old female C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Forty mice were randomly divided into four groups: (1) DMSO group (n 10); (2) VX-765 group (n 10); (3) DMSO M. bovis infection group (n 10) and (4) VX-765 M. bovis infection group (n 10). Mice were infected by nasal drip with 200 CFU of bacterial solution. Animals in the VX-765 group and the VX-765 M. bovis group were orally administered VX- 765 (25 mg/kg/day), and mice in the DMSO group and the DMSO
M. bovis infection group were administered DMSO. Mice were eutha- nized 3–6 weeks post infection. Serum, lung, liver and spleen samples were collected aseptically for the subsequent experiments. Serum, lung homogenate and spleen homogenate were used for analysis by enzyme- linked immunosorbent assays (ELISAs), and the remaining lung and liver tissue was used for histopathological examination and CFU assay.

2.2. Cell culture
Mice were sacrificed in a humane manner and immersed in 75 % ethanol for disinfection of the surface. The tibia, femur and humerus were aseptically separated, and the tissue around the bone was washed away. The end of the bone was cut off, and the bone marrow was washed into a centrifuge tube with RPMI 1640 medium (Solarbio, Beijing, China) and centrifuged at 1,000 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in RPMI 1640 medium con- taining 10 ng/mL M-CSF (PeproTech, Rocky Hill, NJ, USA) and 10 % fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cell sus- pension was transferred to a petri dish (Corning, New York, NY, USA) and cultured for 7 days. After 7 days, the cells were transferred to a cell culture plate (Corning) and cultured overnight for subsequent experiments.

2.3. Bacterial culture and infection
M. bovis was donated by the China Institute of Veterinary Drug Control (CVCC, Beijing, China). Bacteria were cultured in a shaking incubator at 37 ◦C until the midlogarithmic growth phase.
For establishment of a cellular infection model, M. bovis was infected with BMDMs at a multiplicity of infection (MOI) of 10. After 2 h, the bacterial solution was removed, and the cells were washed with PBS and cultured in fresh medium. This time was defined as 0 h post infection. For inhibition of caspase-1, VX-765 was added to the medium 1 h before infection. For activation of caspase-1, LPS (200 ng/mL) was added to the medium for 6 h of pretreatment, and then, 5 mM ATP was added for 1 h. The supernatants and cell samples were collected at the indicated times for the subsequent experiments.

2.4. CFU assay
For determination of the CFUs, the cell lysate or tissue homogenate was serially diluted and inoculated onto 7H11 medium (BD Biosciences, Franklin Lakes, NJ, USA), which contains OADC (Solarbio) and 0.05 % Tween-80 (Difco, Leeuwarden, Netherlands), and after three weeks, the number of colonies was counted.

2.5. Reagents
A rabbit monoclonal anti-mouse cGAS antibody (D3O8O) and rabbit monoclonal anti-mouse phospho-TBK1/NAK antibody (D52C2) were obtained from Cell Signaling Technology (Danvers, MA, USA). A rabbit polyclonal anti-mouse IRF3 antibody (11312-1-AP), rabbit polyclonal anti-mouse Histone-H3 antibody (17168-1-AP), and mouse monoclonal anti-mouse GAPDH antibody (60004-1-Ig) were obtained from Pro- teintech (Wuhan, Hubei, China). A goat anti-rabbit secondary antibody (ZB-5301) and goat anti-mouse secondary antibody (ZB-5305) were obtained from Beijing ZSGB Biotechnology (Beijing, China). LPS (L8880) and ATP (IA0590) were purchased from Solarbio. Belnacasan (VX-765) (S2228) was purchased from Selleckchem (Houston, TX, USA). cGAMP (SML1229) was purchased from Sigma-Aldrich (Milwaukee, WI, USA). A FITC-labeled goat anti-rabbit IgG (H L) antibody (A0562) and DAPI dihydrochloride (C1002) were purchased from Beyotime (Shanghai, China).

2.6. Isolation of nuclear and cytoplasmic fractions
For isolation of nuclear fractions, DMDM cell lysates were processed by nuclear and cytoplasmic protein extraction kits (Beyotime) according to the manufacturer’s instructions.

2.7. Western blotting
Following the manufacturer’s instructions, the Fast Protein Precipi- tation and Concentration Kit (Boster Biotech, Wuhan, China) was used for total cellular protein extraction. First, 5 SDS sample buffer was added to the protein extract and boiled for 10 min. The proteins were separated by SDS-PAGE on 8–15 % gels, and then electrotransferred to Immobilon-P Transfer Membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary and secondary antibodies on a 4 ◦C shaker overnight and then washed with TBS-Tween solution at 37◦C for 30 min. The blots were visualized with an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

2.8. Immunofluorescence assay
Slides with DMEM attached to the surface were fiXed with immu- nostaining fiXative solution (Beyotime) for 10 min, and then washed 3 times with PBS for 5 min each. Cells were immersed in 37 ◦C blocking solution (Beyotime) for 1 h. Primary antibodies were added according to the manufacturer’s instructions and incubated overnight at 4 ◦C. Wash 3 times for 5 min with PBS. Secondary antibodies were added according to the manufacturer’s instructions, incubated at 37 ◦C for 1 h, and washed 3 times with PBS for 5 min each. DAPI (1:10) was added, incubated at 37◦C for 5 min, and washed 3 times with PBS for 5 min each. The slides were sealed with gum and examined with an OLYMPUS microscope.

2.9. ELISA
For determination of the concentrations of IL-1β, IFN-β, IL-6 and TNF-α in the samples, the cell supernatants or the animal tissues were tested by using ELISA kits (Cusabio, Wuhan, Hubei, China) according to the manufacturer’s protocol.

2.10. Cell viability assay
BMDMs were seeded in 96 wells plate at a density of 4 105 cells per well, pretreated with VX-765 and infected with M. bovis in accordance with the protocol mentioned above. For cell viability analysis, the assay was performed by using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, USA) in accordance with the manufacturer’s guidelines.

2.11. Statistical analysis
Statistical analyses were performed by using GraphPad Prism 6 software. P-values < 0.05 were considered significant. Student’s t-test was used to compare two groups, and one-way ANOVA followed by the post hoc Tukey’s test was used for comparisons of multiple groups. The data shown in the manuscript represent three independent experiments. The error bars indicate the standard deviation (SD).

3. Result

3.1. Caspase-1 reduces IFN-β production
Caspase-1 has been reported to inhibit IFN-β during viral infections (Wang et al., 2017).The AIM2 inflammasome has been reported to inhibit IFN-β during M. bovis infection(Liu et al., 2016).
To determine whether caspase-1 is involved in inhibiting IFN-β production, we pretreated BMDMs with VX-765, a caspase-1 inhibitor, and then infected the BMDMs with M. bovis. cGAMP, an agonist of IFN-β production, served as the positive control. The inhibition of caspase-1 resulted in an increase in IFN-β production in M. bovis-infected macro- phages but not in cGAMP-treated cells (Fig. 1A). In addition, VX-765 treatment reduced cell mortality (Fig. 1B), suppressed the production of IL-1β and promoted the production of tumor necrosis factor (TNF)-α and IL-6 during M. bovis infection (Fig. 1C).
Our results suggest that caspase-1 inhibits IFN-β production in vitro during M. bovis infection. To determine whether caspase-1 contributes to inhibiting IFN-β production in vivo, C57BL/6 mice administered VX-765 or DMSO were infected with M. bovis. At 3 weeks or 6 weeks post infection, animals were sacrificed, and the serum, lungs and spleen were collected aseptically. M. bovis infection triggered significant

Fig. 1. Caspase-1 reduces IFN-β production. (A) ELISA analysis of IFN-β in the supernatants of M. bovis-infected BMDMs with or without VX-765 treatment. (B) Cell viability analysis of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (C) ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatants of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (D) ELISA analysis of IL-1β in lung or spleen homogenate and serum from M. bovis-infected C57BL/6 mice administered VX-765 or DMSO. (E) ELISA analysis of IFN-β, TNF-α and IL-6 in lung or spleen homogenate and serum from M. bovis-infected C57BL/6 mice administered VX-765 or DMSO. Data are representative of at least three independent in vitro experiments. The asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.=not significant). UNT = untreated; cGAMP = cyclic guanosine monophosphate-adenosine monophosphate, positive control for IFN-β production, 1 μg/mL, 3 h; VX-765, inhibitor of caspase-1 activation, 20 μM, 1 h prior to infection in vitro, 25 mg/kg/day in vivo. enhancements in the production of IL-1β, IFN-β, TNF-α and IL-6 in these organs. In addition, VX-765 treatment reduced the production of IL-1β (Fig. 1D) and increased the production of IFN-β, TNF-α and IL-6 (Fig. 1E).
Collectively, these results show that during M. bovis infection, caspase-1 negatively regulates IFN-β production in vitro and in vivo.

3.2. Caspase-1 inhibits the TBK1-IRF3 pathway
M. bovis infection can trigger the activation of the TBK1-IRF3 pathway (Cui et al., 2016; Li et al., 2019). The activation of TBK1 is marked by phosphorylation. Phosphorylated TBK1 recruits and catalyzes IRF3 phosphorylation. Then, phosphorylated IRF3 enters the nu- cleus and initiates the expression of IFN-β.
Previous studies have demonstrated that the inflammasome sensor AIM2 can reduce IFN-β expression by inhibiting the TBK1-IRF3 pathway (Liu et al., 2016). To examine whether caspase-1 is involved in the inhibition of TBK1 during M. bovis infection, we detected the expression of phosphorylated TBK1 via western blotting. We observed higher phos- phorylated TBK1 levels in BMDMs treated with the caspase-1 inhibitor VX-765 than in cells not treated with VX-765 during M. bovis infection (Fig. 2A and B). Then, we used an immunofluorescence assay to confirm these results (Fig. 2C).
We performed western blotting and an immunofluorescence assay to detect the nuclear translocation of IRF3, a process that indicates the activation of IRF3. It is conceivable that if caspase-1 can inhibit IRF3 activation during M. bovis infection, an increased amount of IRF3 will enter the nucleus under treatment with the caspase-1 inhibitor VX-765. As expected, more IRF3 was observed in the nucleus of VX-765-treated BMDMs than in that of cells not treated with VX-765 during M. bovis infection (Fig. 2D, E and F).
To further explore the role of caspase-1 in inhibiting the TBK1-IRF3 pathway, we used agonistic stimulation of caspase-1 with lipopolysac- charide (LPS) adenosine triphosphate (ATP). We observed that pre- treatment with LPS ATP significantly reduced the phosphorylation of TBK1 during M. bovis infection (Fig. 2G). Similarly, LPS ATP treatment before M. bovis infection also significantly reduced the translocation of IRF3 into the nucleus (Fig. 2H). Collectively, our results suggest that caspase-1 may reduce IFN-β production by inhibiting the TBK1-IRF3 pathway.

3.3. Caspase-1 is involved in cGAS cleavage
Previous studies have shown that caspase-1 can inhibit IFN-β

Fig. 2. Caspase-1 inhibits the TBK1-IRF3 activation. (A) Immunoblot analysis of p-TBK1 in lysates of BMDM at the indicated timepoints post M. bovis infection. (B) Immunoblot analysis of p-TBK1 in lysates of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (C) Immunofluorescence analysis of p-TBK1 in BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (D) Immunoblot analysis of IRF3 in nuclear fractions of BMDMs at the indicated timepoints post M. bovis infection. (E) Immunoblot analysis of IRF3 in nuclear fractions of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (F) Immunofluorescence analysis of IRF3 in nuclear fractions of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (G) Immunoblot analysis of p-
TBK1 in lysates of BMDMs with or without LPS + ATP treatment at 72 h post M. bovis infection in the absence or presence of VX-765. (H) Immunoblot analysis of IRF3 in nuclear fractions of BMDMs with or without LPS + ATP treatment at 72 h post M. bovis infection in the absence or presence of VX-765.Data are representative of at least three independent experiments. The asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.=not significant). UNT = untreated; cGAMP = cyclic guanosine monophosphate-adenosine monophosphate, positive control for IFN-β production, 1 μg/mL, 3 h; VX-765, inhibitor of caspase-1 activation, 20 μM, 1 h prior to infection in vitro; LPS + ATP, positive control for caspase-1 activation (LPS: 200 ng/mL, 6 h; ATP: 5 mM, 1 h). production by cleaving cGAS. To verify whether the caspase-1 activated in M. bovis infection is cleaved by cGAS, we first measured the expres- sion of cGAS after M. bovis infection. We observed that the expression of cGAS was decreased significantly at 72 h post M. bovis infection, indi- cating that M. bovis infection may cause cGAS cleavage (Fig. 3A). Next, we tested the effect of caspase-1 on the decrease in cGAS expression by using VX-765. Compared with no VX-765 treatment, the use of VX-765 inhibited the decrease in cGAS expression, indicating that cGAS may be cleaved by caspase-1 during M. bovis infection (Fig. 3B). To further verify the cleavage effect of caspase-1 on cGAS, we activated caspase-1 with LPS ATP treatment and then performed infection with M. bovis. We found that the expression of cGAS was further reduced, and this phenotype could be reversed by the use of VX-765 (Fig. 3C).
In summary, our results suggest that M. bovis infection may inhibit the TBK1-IRF3 pathway by including cGAS cleavage, resulting in reduced IFN-β expression.

3.4. Caspase-1 inhibits bacterial survival and reduces pathological severity
Caspase-1 can trigger the production of IL-1β, which is a key cytokine in the response against tuberculosis(Yamada et al., 2000; Juffermans et al., 2000). To explore the effect of caspase-1 on the pathological damage caused by tuberculosis, the mice were infected with M. bovis nasally and then fed VX-765 at a dose of 25 mg/kg/d. The mice were weighed weekly. At 3 or 6 weeks post infection, the mice were sacrificed, the lungs and spleens were weighed to calculate the organ index. The lungs were prepared for pathological observation. Our results showed that compared with mice not fed VX-765, VX-765-fed mice had signifi- cantly reduced weight from 3 weeks post M. bovis infection (Fig. 4A), significantly increased lung and spleen organ index at 6 weeks post infection (Fig. 4B), massive lung and liver lesions (Fig. 4C).
Previous studies have shown that silencing the AIM2 inflammasome can promote M. bovis survival(Liu et al., 2016). To explore the effect of caspase-1 on M. bovis proliferation and progression, we used a colony-forming unit (CFU) assay to detect the quantity of bacteria in the lungs and livers of infected mice fed VX-765. The results showed that compared with mice not fed VX-765, those fed VX-765 showed a significantly increased quantity of bacteria in the lungs and livers during
M. bovis infection (Fig. 4D). To confirm this result, we performed ex- periments with BMDMs, and the results showed that more bacteria proliferated in BMDMs pretreated with VX-765 than in untreated BMDMs (Fig. 4E).
In summary, our results indicate that caspase-1 can inhibit the pro- liferation and progression of M. bovis in hosts and reduce pathological damage.

4. Discussion

Although M. bovis can activate caspase-1 through a variety of inflammasomes and caspase-1 can inhibit IFN-β production, the mech- anism involving caspase-1 in regulating IFN-β during M. bovis infection has not been thoroughly explored. In experiments, we observed that under the condition of caspase-1 inhibitor feeding, mice infected with
M. bovis exhibited increased IFN-β production, relatively large patho- logical lesions, and an enhanced bacterial burden. IFN-β has shown harmful effects on hosts in tuberculosis, characterized by the recruit- ment of neutrophils to the lungs and the lack of development of a Th1 immune response. IL-1β is the critical cytokine for hosts to resist
Mycobacterium. Previous studies have suggested that mice deficient in IL-1β signaling have an increased susceptibility to mycobacteria with enhanced mortality and an increased bacterial burden (Yamada et al.,2000; Juffermans et al., 2000; Fremond et al., 2007). Because treatment with caspase-1 inhibitors reduces IL-1β levels, a deteriorated phenotype may be caused by enhanced IFN-β production and reduced IL-1β levels. However, some reports indicate that IL-1β expression may increase tuberculosis susceptibility (Zhang et al., 2014), In addition, study on humanized mouse shows that the production of IL-1β in mice with tuberculosis relapse was suppressed in the setting of HIV co-infection (Huante et al., 2020). To reconcile these contradictory results, we speculated that the role and production of IL-1β during mycobacterium infection may be affected by many factors, such as host genetic diversity, abundance of cytokines and concomitant diseases et al. It might be novel strategy for the prevention and treatment of tuberculosis to clarify and manipulation these factors in future. In vitro, we further demonstrated that caspase-1 negatively regulated the cGAS-TBK1-IRF3 pathway and inhibited IFN-β production through cGAS cleavage. Interestingly, it has been reported that AIM2 restricts STING-TBK1-dependent IFN-β pro- duction by competitively conjugating with M. bovis DNA (Liu et al.,2016). In addition, previous studies have shown that caspase-1 limits the IFN-β response to cytosolic DNA by activating gasdermin D (Banerjee et al., 2018). Therefore, the mechanisms by which inflammasomes

Fig. 3. Caspase-1 is involved in cGAS cleavage. (A) Immunoblot analysis of cGAS in lysates of BMDMs at the indicated timepoints post M. bovis infection. (B) Immunoblot analysis of cGAS in lysates of BMDMs with or without VX-765 treatment at 72 h post M. bovis infection. (C) Immunoblot analysis of cGAS in lysates of BMDMs with or without LPS + ATP treatment at 72 h post M. bovis infection in the absence or presence of VX-765. Data are representative of at least three independent experiments. The asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.=not significant). UNT = untreated; cGAMP = cyclic guanosine monophosphate-adenosine monophosphate, positive control for IFN-β production, 1 μg/mL, 3 h; VX-765, inhibitor of caspase-1 activation, 20 μM, 1 h prior to infection in vitro; LPS + ATP, positive control for caspase-1 activation (LPS: 200 ng/mL, 6 h; ATP: 5 mM, 1 h).

Fig. 4. Caspase-1 inhibits bacterial survival and reduces pathological severity. (A) Body weight curve of mice post M. bovis infection. (B) Organ index of lungs and spleens from M. bovis-infected mice administered VX-765 or DMSO at 3 weeks or 6 weeks post infection. (C) Pathological lesions (H&E staining) in the lung and liver of mice infected with M. bovis for 3 weeks or 6 weeks in the presence or absence of VX-765. (D) Bacterial load of lungs and livers from M. bovis-infected mice administered VX-765 or DMSO at 3 weeks or 6 weeks post infection. (E) Bacterial load of M. bovis-infected BMDMs in presence or absence of VX-765. Data are representative of at least three independent experiments. The asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.=not significant). VX-765, inhibitor of caspase-1 activation, 25 mg/kg/day in vivo. negatively regulate the innate immune response may be diverse. Indeed, inflammasomes exploit numerous parallel or redundant pathways to limit the type I IFN response. NLRX1 acts as a negative regulator of IFN-β secretion by either inhibiting the interaction between MAVS and RIG-I (Lei et al., 2012; Allen et al., 2011) or disrupting the STING-TBK1 interaction (Guo et al., 2016). These findings revealed the existence of multiple IFN-β-modulating mechanisms, highlighting the importance of avoiding excessive IFN-β production. The type of IFN-β-modulating mechanism active in a host may depend on the pathogen, cell status, and immune pathway activation.
Mitochondrial damage can induce inflammasome activation and IFN-β production. After mitochondrial damage, mtDNA is released into the cytoplasm. As a common ligand, mtDNA can be recognized by
NLRP3 and cGAS, which activate the inflammasome and STING path- ways, respectively, resulting in IL-1β or IFN-β secretion. It would be interesting to explore the following questions: will cGAS and NLRP3 compete with mtDNA to trigger their respective downstream pathways as critical hallmarks of a new regulatory mechanism? What factors determine the priority of cGAS versus NLRP3 binding to mtDNA? Recently, it has been reported that caspase-1 is essential for inhibiting the activation of the cGAS-STING pathway triggered by DNA virus infection. Here, we identify a similar regulatory mechanism: caspase-1 is critical in limiting cGAS-STING activation during M. bovis infection. Previous studies have also shown that during Francisella novicida infection, caspase-1 triggers negative regulation of the cGAS-STING pathway via K+ effluX mediated by gasdermin D. It will be of interest to determine the role of caspase-1-dependent gasdermin D activation in the process of modulating IFN-β production during M. bovis infection.
The caspase-1 inhibitor VX-765 is already being studied in a phase II clinical trial. Importantly, type I IFNs have significant antiviral and antitumor effects and is exploited for treatment of viral infections and cancer (Snell et al., 2017; Lazear et al., 2019). In addition, type I IFNs are also involved in the suppression of inflammasomes (Guarda et al., 2011;
Mayer-Barber et al., 2011) and used to treat multiple sclerosis, Behcet’s syndrome and familial Mediterranean fever caused by excessive IL-1β production (Inoue et al., 2012; Ureten et al., 2004). Our findings suggest that the use of type I IFNs or VX-765 in tuberculosis patients with or without these diseases may cause deleterious effects and, in some cases, worsen the condition. Therefore, this side effect should be carefully evaluated in clinical treatment.
Our results showed that the treatment of VX-765 increased TNF-α production post M. bovis infection. However, previous studies suggested that treatment of VX-765 can reduce the production of TNF-α (Xu et al., 2019). We speculated that these contradictory results might be due to the different mechanisms inducing TNF-α during different condition. During mycobacterial infection, cGAS-TBK1-NF-κB axis may be trig- gered, resulting in TNF-α production. Thus the treatment of caspase-1 inhibitor, VX-765, may enhance TNF-α production by aggravating cGAS-TBK1-NF-κB axis. However, on the context of other pathogen infection, IL-1R-NF-κB axis may be responsible for TNF-α production. Thus the use of VX-765 may reduce IL-1β secretion and IL-1R-NF axis activity, resulting decreased TNF-α production.

5. Conclusion

In this study, we found that caspase-1 activation can reduce IFN-β production upon M. bovis infection by dampening cGAS-TBK1-IRF3 signaling in vitro. In addition, our results showed that a deficiency in caspase-1 activation enhanced the bacterial burden during M. bovis infection in vitro and in vivo and aggravated pathological lesion for- mation. These findings contribute to the understanding of the mutual balancing mechanism between inflammasomes activation and cGAS- TBK1-IRF3 signaling during mycobacterial infection and present the basis for investigating caspase-1 regulation as a therapeutic strategy for infectious diseases.

Ethics statement
All protocols and procedures were performed according to the Chi- nese Regulations of Laboratory Animals—The Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of People’s Republic of China) and Laboratory Animal Requirements of Environ- ment and Housing Facilities (GB 14925–2010, National Laboratory Animal Standardization Technical Committee). The license number associated with their research protocol was 20110611–01 and the animal study proposal was approved by The Laboratory Animal Ethical Committee of China Agricultural University.

Author contributions
YL performed the experiments and wrote the manuscript. CL, JW and YC inspired experimental design and taught key experimental tech- niques. JY, YS, LL and HW helped in cell culture and animal infection. TH and NS assisted in the English grammar check. LY, XZ and DZ guided the performance of experiments and reviewed the manuscript critically before submission.

Funding
This work was supported by the National Natural Science Foundation of China (grant No. 31873005); China Agriculture Research System (grant CARS36); the National Key Research and Development Program (grant 2017YFD0500901); the MOSTRCUK international cooperation

Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments
We thank CVCC for donating the M. bovis strain. We acknowledge National Animal TSE Lab, for provision of BSL-3 Laboratories facilities and experimental instruments. We are also grateful to professors of China Agricultural University for their suggestions.

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