Wild-type (WT) C57BL/6 mice were obtained from the Federal University of Minas Gerais (UFMG) animal facility. ZBP1 knockout (KO) mice were provided by Prof Shizuo Akira from Osaka University (Japan). STING KO mice were described earlier (23). All mice were housed in a pathogen-free laboratory facility. Male and female mice aged 8-12 weeks were utilized for the study. All experimental protocols were reviewed and approved by the Animal Studies Committee (protocol CEUA/UFMG 69/2020).

Brucella abortus strain 2308 was acquired from our laboratory collection. The bacterium was cultured in Brucella broth (BB) medium (BD Pharmingen, San Diego, CA) for 3 days at 37°C under constant agitation before use. The optical density (OD) of the culture was measured at 600 nm using a spectrophotometer to determine the bacterial number in the solution.

BMDMs were generated and cultured as described previously (24). Briefly, bone marrow cells from ZBP1 KO and C57BL/6 mice were harvested from the tibias and femurs were differentiated into macrophages using DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA), 20% L929-cell conditioned medium (LCCM), 1% HEPES (Life Technologies) and 100 U/ml penicillin-streptomycin (Life Technologies), at 37°C in 5% CO₂. At day 4 of culture, 10 mL of fresh medium was added. At day 7, cells completely differentiated into macrophages were detached and seeded in 24-well plates at a density of 5 x 10⁵ cells/well for use in experiments.

Cultured macrophages from ZBP1 KO and C57BL/6 mice were infected in vitro with B. abortus at the multiplicity of infection (MOI) of 100:1 in DMEM with 1% FBS for the indicated times at 37°C in 5% CO₂. B. abortus DNA was purified using the Illustra bacteria genomic Prep Mini Spin Kit (GE Healthcare, Buckinghamshire, United Kingdom) according to the manufacturer’s instructions. Then, the purified bacterial DNA was transfected (1 μg/mL) using FuGENE HD (Promega, Madison, WI) accordingly to manufacturer instructions. Culture supernatants and cell lysates were harvested and stored at -80°C until use.

Macrophages from ZBP1 KO and C57BL/6 mice were transfected with siRNA from siGENOME SMARTpools (Dharmacon, Lafayette, CO) using the GenMute siRNA transfection reagent according to the manufacturer’s instructions (SignaGen, Rockville, MD). siGENOME SMARTpool siRNA specific for mouse STING (M-055528-01) and a control siRNA pool were used (D-001206-14-05). Forty-eight hours after transfection, culture medium was replaced and macrophages were infected as described above.

Macrophages from ZBP1 KO and C57BL/6 mice treated as described above were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to obtain total RNA accordingly to manufacturer guidelines. Then, RNA was treated with DNase I (Invitrogen) to remove genomic DNA followed by reverse transcription of 1 μg of total RNA using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare, Chicago, IL) according to the manufacturer’s instructions. Real-time RT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) on a QuantStudio3 real-time PCR instrument (Applied Biosystems), using the following cycling parameters: 60°C for 10 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 60°C for 1 min, and a dissociation stage of 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec, and 60°C for 15 sec. The appropriate primers were used to amplify a specific fragment corresponding to specific gene targets as follows: BiP F: 5’-AGGATGCGGACATTGAAGAC-3’, R: 5’-AGGTGAAGATTCCAATTACATTCG-3’; XBP1(s) F: 5’-GAGTCCGCAGCAGGTG-3’, R: 5’-GTGTCAGAGTCCATGGGA-3’; IFN-β F: 5’-GCCTTTGCCATCCAAGAGATGC-3’, R: 5’-ACACTGTCTGCTGGTGGAGTTC-3’; IFN-α4 F: 5’-CCTGTGTGATGCAGGAACC-3’, R: 5’-TCACCTCCCAGGCACAGA-3’; β-actin F: 5’-GGCTGTATTCCCCTCCATCG-3’, R: 5’-CCAGTTGGTAACAATGCCATGT-3’. All data are presented as relative expression after normalization to the β-actin gene.

Macrophage supernatants from ZBP1 KO and C57BL/6 mice were harvested from treated cells for cytokine, NO and lactate dehydrogenase (LDH) measurements. The murine cytokines (IL-1β, IL-6, IL-12 and TNF-α) were detected using ELISA kits (R&D systems, Minneapolis, MN), according to the manufacturer’s instructions. To evaluate NO production, the concentration of nitrite (NO₂ ⁻) was assessed using the Griess reagent method as previously described (25). The LDH activity was measured using the CytoTox96 LDH release kit (Promega, Madison, WI), according to the manufacturer’s instructions.

Supernatants from treated macrophages were harvested and cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with 1:100 protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Then, equal volume of supernatants or equivalent amounts of protein of cell lysates were loaded onto 12% SDS-polyacrylamide gels. Following electrophoresis, bands were transferred to nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden) according to standard techniques. Membranes were blocked in Tris-buffered saline (TBS) with 0.1% Tween-20 containing 5% nonfat dry milk for 1 hr and then incubated at 4°C overnight with primary antibodies (IL-1β, clone 3A6; IRF-1, clone D5E4; BiP, clone C50B12; β-actin, clone 13E5; Cell Signaling Technology, MA, Danvers) (ZBP1, clone Zippy-1; p20 subunit of caspase-1, clone Casper-1; Adipogen, San Diego, CA). The membranes were washed three times for 5 min in TBS with 0.1% Tween 20 and incubated for 1 hr at room temperature with the appropriate HRP-conjugated secondary antibody (Cell Signaling Technology). Proteins were visualized using Luminol chemiluminescent HRP substrate (Millipore, Burlington, MA) in an Amersham Imager 600 (GE Healthcare). Densitometry analysis was performed using ImageQuant TL Software (GE Healthcare) and band intensities were normalized to β-actin. Data were relativized to the level of WT macrophages infected with B. abortus for 8 h assigned arbitrarily with the value of 1.0.

ZBP1 KO and C57BL/6 mice were infected i.p. with 1 x 10⁶ colony formation units (CFU) of B. abortus in 0.1 ml of saline (NaCl 0.9%). After 2 and 4 weeks post-infection, mice were sacrificed and spleens were used to determine the number of bacteria by CFU counting. For the measurement of viable intracellular bacteria in vitro, infected macrophages were washed twice with PBS and then lysed for 10 min at room temperature in 1 mL of PBS containing 0.1% Triton X-100 under manual agitation. To assess B. abortus CFU, spleens and cells lysates were serially diluted in saline and plated in duplicate on BB agar. Plates were incubated for 3 days at 37°C and CFU number was determined.

Spleen cells were harvested from infected mice and treated with an ammonium-chloride-potassium buffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM ethylenediaminetetraacetic acid [EDTA]; pH 7.2) to lyse red blood cells. After washing, the cells were resuspended in RPMI medium (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 25 mM HEPES, 10% heat-inactivated FBS, 100 U/mL penicillin G sodium, and 100 mg/mL streptomycin sulfate, and adjusted to 1 × 10⁶ cells per well in a 96-well plate. Splenocytes were stimulated with B. abortus (MOI of 100:1), 1 µg/ml Escherichia coli LPS (Sigma-Aldrich), or 5 µg/ml Concanavalin A (ConA) (Sigma-Aldrich). Spleen cells were incubated at 37°C in 5% CO₂, and culture supernatants were collected 48 or 72 hours after stimulation to measure TNF-α or IFN-γ, respectively, by ELISA (R&D Systems).

Data analysis and graphing were performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA). All quantitative data are expressed as mean ± standard deviation. The data presented are representative of three independent experiments. A p value less than 0.05 (p<0.05) was considered statistically significant using two-way ANOVA test.

In the context of intracellular Brucella infection, the activation of innate immune sensors can occur through distinct mechanisms leading to type I IFN response (21). To evaluate the impact of ZBP1 in this pathway, BMDMs were obtained from both ZBP1 KO and C57BL/6 mice. Subsequently, these cells were exposed to the virulent B. abortus S2308 strain, and the expression of the IFN-β gene ( Figure 1A ) and IFN-α gene ( Figure 1B ) was assessed. The results revealed a significant reduction in the expression of both type I IFN genes in ZBP1 KO macrophages compared to WT cells. In prior studies, the involvement of ZBP1 in DNA-mediated innate immune responses was recognized, and ZBP1 was designated as a cytosolic DNA sensor (3). Therefore, we examined the influence of transfected Brucella abortus-derived DNA on IFN-β expression through ZBP1 activation, using macrophages from ZBP1 KO and WT mice. As observed in Figure 1C , IFN-β expression induced by transfected DNA was dependent on the presence of ZBP1. Taken together, our data demonstrated that both Brucella infection and bacterial DNA transfection induces type I IFN responses in a ZBP1-dependent manner.

Moreover, we addressed the cooperation between the STING and ZBP1 in driving type I IFN expression during B. abortus infection. Hence, we performed siRNA silencing of STING in ZBP1 KO and WT macrophages. The knockdown of siRNA led to decreased expression of IFN-β in both ZBP1 KO and WT cells compared to cells treated with the control (scramble siRNA) ( Figure 1D ). The data suggest that both STING and ZBP1 contribute to type I IFN responses induced by Brucella-infected macrophages.

Previous data unveiled that B. abortus infection led to UPR induction and enhanced expression of the UPR downstream targets, BiP and XBP1(s). Moreover, it was shown the crucial role of IFN-β in triggering UPR during B. abortus infection (22). Given that ZBP1 plays a pivotal role in type I IFN response, we examined the involvement of this receptor in the UPR activation during B. abortus infection. In this regard, macrophages from ZBP1 KO and WT mice were infected to assess the expression of BiP ( Figure 2A ) and XBP1(s) ( Figure 2B ). The results indicated increased expression of both BiP and XBP1(s) in a ZBP1-dependent manner. Therefore, our data suggest that ZBP1 plays a role in controlling the UPR activation upon B. abortus infection.

IRF-1 operates as a transcriptional regulator, activating the expression of genes associated with protection against invading pathogens (26). IRF-1 functions downstream of IFN expression, participating in the signal transduction pathway initiated by IFN during infection (27). Regarding B. abortus infection, it was previously demonstrated that the expression of IRF-1 depends on the activation of IFNAR by IFN-β (21). Given this context, we evaluated the level of IRF-1 protein expression in macrophages derived from WT and ZBP1 KO mice. We noted a significant increase in IRF-1 protein level in WT cells following bacterial infection compared to the non-infected cells ( Figures 3A, B ). Moreover, this upregulation was partially dependent on ZBP1, as KO macrophages exhibited reduced IRF-1 protein level 16 hours post-infection. Furthermore, we assessed the protein level of BiP upon B. abortus infection ( Figures 3A, C ). It was observed that the increase in BiP protein level occurred at 16 hours post-infection, and this enhancement relies in the presence of ZBP1. This result strengthens the conception that ZBP1 contributes to activate the UPR. Finally, we examined the level of ZBP1 protein stimulated by the infection ( Figures 3A, D ). The results revealed a significant increase in ZBP1 protein level only at 16 hours post-infection. Collectively, our data indicates that ZBP1 stimulated by B. abortus infection drives IFN-β expression potentially contributing to IRF-1 expression.

In addition to the type-I IFN response during ZBP-1 activation, the NF-κB signaling pathway leading to proinflammatory cytokine production also constitutes a line of defense against pathogenic infections (7). In that context, ZBP1 also emerged as a regulator of proinflammatory cytokine production, such as IL-6 and TNF-α (11). Thus, we aimed to evaluate the participation of ZBP1 in the secretion of proinflammatory cytokines by macrophages during B. abortus infection. Macrophages derived from ZBP1 KO mice exhibited similar levels of IL-12, IL-6, and TNF-α in comparison to WT infected macrophages at all time points tested ( Figures 4A–C ). Moreover, considering nitric oxide (NO) as another classical marker of the inflammatory macrophage profile, we assessed NO production in infected cells. The findings revealed that ZBP1 KO infected macrophages displayed no difference in NO production compared to infected cells from WT mice ( Figure 4D ). Taken together, the data suggest that the production of proinflammatory cytokines and NO by macrophages infected with B. abortus occurs independently of ZBP1 activation.

A growing body of evidence showed that ZBP1 induces inflammasome activation and subsequent IL-1β secretion (28–30). Thus, to gain insight into the role of ZBP1 in promoting inflammasome activation in response against B. abortus infection, we assessed the production of IL-1β and caspase-1 processing in macrophages. The data showed no difference in the secretion of IL-1β when comparing macrophages derived from ZBP1 KO mice to those from WT mice ( Figure 5A ). Furthermore, both WT and ZBP1 KO infected macrophages exhibited equivalent levels of pro-IL-1β ( Figure 5B ), and no difference was observed regarding the presence of caspase-1 p20 subunit in cell supernatants ( Figure 5B ), suggesting no influence of ZBP1 in inflammasome assembly and activation.

Cell death is closely interconnected to the host immune response during infection, and ZBP1 was previously linked to cell death measured by lactate dehydrogenase (LDH) release (28). In addition, we demonstrated previously that B. abortus infection induces pyroptosis and LDH release in a process dependent on caspase-11 activation and gasdermin-D cleavage (31). Therefore, we assessed the involvement of ZBP1 in macrophage cell death mediated by bacterial infection through LDH release in a time-lapse experiment. It was observed that LDH release is prominent after 16 hours of infection compared to non-infected cells. Furthermore, there is no difference concerning cell death between WT and ZBP1 KO macrophages at any assessed time point ( Figure 5C ). Thus, the data indicates that ZBP1 is not essential for the cell death induced by B. abortus in infected macrophages.

Previously, our group demonstrated that mice deficient in the IFN-αβ receptor controlled Brucella infection more efficiently than wild-type animals (32). This data indicated the detrimental role of type I IFN signaling to the host during B. abortus infection. Since IFN-β expression is partially diminished in ZBP1 KO mice, we explored the role of ZBP1 in host defense against B. abortus. Thus, we infected both WT and ZBP1 KO mice and then assessed the bacterial load in the spleen at 2 and 4 weeks post-infection (wpi) ( Figure 6A ). As observed, there is no difference between WT and ZBP1 KO mice concerning CFU counts in spleen at both analyzed time points post-infection. In addition, to further explore the role of ZBP-1 in vivo, we analyzed the production of proinflammatory cytokines during B. abortus infection. Splenocytes from wild-type and ZBP1 KO infected mice were stimulated with live bacteria, ConA, or LPS as controls, and cytokine secretion was determined. Analysis of all stimuli demonstrated that ZBP1 KO-infected mice produced similar levels of the proinflammatory cytokines TNF-α ( Figure 6B ) and IFN-γ ( Figure 6C ) compared to WT infected animals.

Furthermore, we evaluated the CFU counts following 8 hours and 16 hours of infection in macrophages derived from ZBP1 and WT mice ( Figure 6D ). As observed, there is no difference in bacterial replication within macrophages at any of the analyzed time points. Therefore, these data suggest that ZBP1 is not essential for controlling either Brucella infection in vivo or bacterial replication inside macrophages.