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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: J Immunol. 2019 Mar 20;202(9):2671–2681. doi: 10.4049/jimmunol.1801233

Brucella abortus cyclic dinucleotides trigger STING-dependent Unfolded Protein Response that favors bacterial replication

Erika S Guimarães *, Marco Túlio R Gomes *, Priscila C Campos *, Daniel S Mansur #, Adara A dos Santos #, Jerome Harms †, Gary Splitter †, Judith A Smith ‡, Glen N Barber §, Sergio C Oliveira *,
PMCID: PMC6478548  NIHMSID: NIHMS1523091  PMID: 30894428

Abstract

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis and meningitis in humans. Signaling pathways triggered by Brucella abortus involves STING that leads to production of type I interferons. Here, we evaluated the pathway linking the Unfolded Protein Response (UPR) and the endoplasmic reticulum resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger c-di-GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I interferon signaling pathway, such as IFN-β, interferon regulatory factor-1 (IRF-1), and guanylate binding proteins. Furthermore, IFN-β is also important for the UPR induction during B. abortus infection. Indeed, IFN-β shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-β favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial c-di-GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-β signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

Introduction

The innate immune system is the first line of defense against invading pathogens (1) as innate immune sensors mediate the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In this scenario, nucleic acids are considered a critical pathogen signature that triggers host pro-inflammatory immune responses (2) including the production of inflammatory cytokines and interferons (IFNs). Stimulator of Interferon Genes (STING) is an ER resident transmembrane protein (3) that regulates type I interferon-dependent innate immunity in response to DNA (4). During intracellular bacterial infection, STING can function as a direct sensor of bacterial cyclic dinucleotides (CDNs) as well as an adaptor molecule in DNA recognition (5). For example, Chlamydia trachomatis and Listeria monocytogenes secrets the second messenger c-di-AMP that binds directly to STING (6, 7) while several other pathogens activate STING in a cGAS-dependent manner (8, 9). Recently we reported that, during B. abortus infection, STING directly detected bacterial CDNs, triggering a type I IFN response that led to the upregulation of several interferon-related genes, including guanylate nucleotide binding proteins (GBPs) (10).

Brucella abortus is a Gram-negative facultative intracellular bacterium that causes brucellosis in a wide range of animals and is considered the most prevalent bacterial zoonosis worldwide (11). Although brucellosis causes abortion and infertility in cattle (12), in humans the disease is characterized by recurrent undulant fever, debilitating arthritis, endocarditis, and meningitis (13, 14), causing considerable economic loss and a major public health burden (11, 12). Better understanding of the host-pathogen interplay that enables Brucella replication is crucial for the development of an effective treatment for brucellosis.

During its intracellular life cycle, B. abortus ensures its survival through formation of Brucella-containing vesicles (BCVs). BCVs traffic from the endocytic compartment and fuse with the endoplasmic reticulum (ER) to generate an ER-derived vacuole, that is permissive to replication (15–17). The ER has critical roles in maintaining cellular homeostasis by processing and folding secretory and membrane proteins (18). Disruption of ER homeostasis, initiated by accumulation of misfolded proteins, induces ER stress and activates a conserved endoplasmic stress response termed the Unfolded Protein Response (UPR). In mammalian cells, three primary ER membrane resident stress sensors, inositol-requiring kinase 1 (IRE1), activating transcription factor (ATF6), and PKR-like endoplasmic reticulum kinase (PERK), govern UPR responses (19, 20). Through UPR targeted gene transcription, including chaperones, that increase the ER protein folding capacity, and through ER-associated degradation (ERAD), the UPR relieve the stress on the ER, preserving cellular homeostasis (20). Failure to restore ER functions results in programmed cell death (19). In addition, the UPR triggers events associated with innate immunity and host defense, linking ER stress to inflammation (21). It is well known that Brucella can activate the UPR (22, 23); however, the mechanisms regulating its activation and the impacts on innate immunity are still poorly understood.

Taking into account Brucella dependence on the ER to establish a replicative niche, we evaluated the pathway linking the UPR and the ER resident transmembrane molecule STING, during B. abortus infection. In this study, we show that B. abortus induces the UPR that is crucial for the production of multiple pro-inflammatory mediators, including several molecules linked to the type I IFN signaling pathway. Moreover, we provide evidence of the mobilization of ER stress responses upon engagement of STING by Brucella CDNs that are known to induce IFN-β (5, 10). Also, we determine that type I IFN signaling is a major component in triggering B. abortus-induced UPR. Finally, we show that the UPR is crucial for B. abortus persistence both in vitro and in vivo.

Material and Methods

Mice

Wild-type C57BL/6 mice were purchased from the Federal University of Minas Gerais, and STING knockout (STING KO) mice was described previously (3). The animals were maintained at the Federal University of Minas Gerais and used at 6–8 weeks of age. All animal experiments were preapproved by the Institutional Animal Care and Use Committee of the Federal University of Minas Gerais (CEUA no. 87/2017).

Bacterial Strains

Bacteria used in this study included the B. abortus virulent strain S2308 (B. abortus) and a variant that constitutively expresses GFP (B. abortus-GFP). The Brucella cyclic dimeric GMP (c-di-GMP) guanylate cyclase mutant strain (Δ1520) that has a deletion on a single Brucella diguanylate cyclase was generated in our laboratory and previously described (10, 24). Before being used for cell infection, bacteria were grown in Brucella broth medium (BD Pharmingen, San Diego, CA, USA) for 3 days at 37 °C under constant agitation.

Cell Culture

Macrophages were derived from bone marrow of C57BL/6 and STING KO mice as previously described (25). Briefly, bone marrow cells were removed from the femurs and tibias of the animals and cultured in 24-well plates (5×105 cells/well for cytokine and western blot analysis and 1×105 cells/well over a sterile coverslip for microscopy analysis) in DMEM (Life Technologies, Carlsbad, CA, USA) containing 10% FBS (HyClone, Logan, UT, USA), 1% HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/mL murine recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech, Rocky Hill, NJ, USA). At day 10 of culture, when the cells had completely differentiated into macrophages, bone marrow-derived macrophages (BMDMs) were infected with B. abortus as described below. WT and STING KO murine embryonic fibroblasts (MEFs) were provided by Dr. G.N. Barber (University of Miami, FL). MEFs, and THP-1, a human monocytic cell line, were maintained in high-glucose DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Carlsbad, CA, USA) at 37 °C in 5% CO2/95% air in a humidified incubator. MEFs were seeded on 24-well plates containing sterile coverslips at 1×104 cells/well a day before the experiment and kept on normal growth medium. THP-1 were seeded on 24-well plates at 5×105 cells/well a day before the experiment and kept on growth medium supplemented with 100nM PMA for THP-1 differentiation for 24 h.

Generation of IFNAR knockout human cell line by CRISPR

A549 human lung epithelial cells were cotransfected with two gRNA/Cas9/GFP plasmids (provided by Horizon, Cambridge, UK) targeting the IFNR1 locus using FuGENE (Promega, Madison-WI, USA). The guide RNAs used were 5’- GCAGCCGCAGGTGAGAGGCG-3’and 5’-CTGCGGCGGCTCCCAGATGA-3’. Then 72 h after transfection, cells were sorted for GFP fluorescence into single cells. Single-cell derived clones were then genotyped and phenotyped to confirm the knockout cells.

Infection with B. abortus

Cultured cells were infected in vitro with virulent B. abortus strain 2308, or B. abortus c-di-GMP guanylate cyclase mutant (Δ1520), at a multiplicity of infection (MOI) of 100:1 in DMEM supplemented with 10% FBS for 24 h. In confocal microscopy experiments, BMDMs were infected with B. abortus-GFP at a MOI of 20:1 or 50:1 in DMEM supplemented with 10% FBS for 24 h, and MEFs cells infected with 1000:1 in DMEM supplemented with 10% FBS for 4 h.

Reagents

For the UPR blockade in vitro, cells were pre-treated with 1 mg/mL tauroursodeoxycholic acid (TUDCA) (Sigma-Aldrich, St. Louis, MO, USA) or with 50 μM 4μ8c, a selective IRE1 inhibitor (Sigma-Aldrich, St. Louis, MO, USA) for 30 min prior to infection with B. abortus (23). For UPR blockade in vivo, mice were treated ad libitum with 200 mg/dL 4-phenylbutyrate (4-PBA) (Sigma-Aldrich, St. Louis, MO, USA) in their drinking water for one week, starting one day prior to infection. As a positive control of UPR activation, cells were treated for 6h with 1 μg/mL of the ER stress inducer Tunicamycin (Tm) (Sigma-Aldrich, St. Louis, MO, USA) a potent N-linked glycosylation inhibitor (26). For IFN-β neutralization, cells were pre-treated for 30 min prior to infection with 10 U/mL of polyclonal antibody against the mouse IFN-β (anti-IFN-β) (PBL Assay Science, Piscataway, NJ, USA). Where indicated cells were treated 24 h prior to infection with 100 U/mL of a mouse recombinant IFN-β (rIFN-β) (Millipore, Burlington, MA, USA).

Knockdown via small interfering RNA

BMDMs were transfected with siRNA from siGENOME SMARTpools (Dharmacon, Lafayette, CO) with the GenMute siRNA transfection reagent according to the manufacturer’s instructions (SignaGen, Rockville, MD). siGENOME SMARTpool siRNAs specific for mouse XBP1 (M-040825-00-0005), mouse Hspa5 (BiP) (M-040337-00-0005), and a control siRNA pool (D-001206-14-05) were used in this study. Forty-six hours after siRNA transfection, cells were infected with B. abortus MOI 100:1 as described above. After 17h, supernatant was collected to measure IL-1β, CXCL10, and IL-6 by ELISA, according to the manufacturer’s instructions.

Transfection experiments

Transient transfections of BMDMs were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in a ratio (in milliliters) of 1:0.50, following the manufacturer’s instructions. Cells were cultured in DMEM and transfected, as indicated with 3 μg/mL of c-di-AMP or c-di-GMP or dsDNA90 (InvivoGen, San Diego, CA, USA) for 24 h.

Real Time RT-PCR

Samples were resuspended in TRIzol (Invitrogen, Carlsbad, CA, USA) to isolate total RNA in accordance with the manufacturer’s instructions. Total RNA was treated with DNase I (Invitrogen, Carlsbad, CA, USA) to remove genomic DNA followed by reverse transcription of 1μg of total RNA using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions. Real-time RT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) on a QuantStudio3 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The appropriate primers were used to amplify a specific fragment corresponding to specific gene targets as described below. The UPR activation engages the induction of targeted gene transcription. Several targets of the UPR are pathway specific, thus activation of the primary axis of the UPR (ATF6, IRE1 and PERK) can be detected by the induction of its downstream targets, such as binding immunoglobulin protein (BiP/GRP78), spliced X-box binding protein 1 (XBP1s), and C/EBP homologous protein (CHOP), respectively. Therefore, the UPR was detected by quantitative real time PCR as previously described (23, 27). For qPCR analysis, 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’; CHOP F: 5’-CATCACCTCCTGTCTGTCTC-3’, R: 5’-AGCCCTCTCCTGGTCTAC-3’; IFN-β F: 5’-GCCTTTGCCATCCAAGAGATGC-3’, R: 5’-ACACTGTCTGCT GGTGGAGTTC-3’; GBP2 F: 5’-CTGCACTATGTGACGGAGCTA-3’, R: 5’-CGGAATCGTCTACCCCACTC-3’; GBP3 F: 5’-CTGACAGTAAATCTGGAAGCCAT-3’, R: 5’-CCGTCCTGCAAGACGATTCA-3’; GBP5 F: 5’-CTGAACTCAGATTTTGTGCAGGA-3’, R: 5’-CATCGACATAAGTCAGCACCAG-3’; β-actin F: 5’-GGCTGTATTCCCCTCCATCG-3’, R: 5’-CCAGTTGGTAACAATGCCATGT-3’. hBiP F: 5’-TGTTCAACCAATTATCAGCAAACTC-3’, R: TTCTGCTGTATCCTCTTCACCAGT-3’; hXBP1(s) F: 5’-CTGAGTCCGAATCAGGTGCAG-3’, R: 5’-ATCCATGGGGAGATGTTCTGG-3’; hIFN-β F: 5’-TCTGGCACAACAGGTAGTAGGC-3’, R: 5’-GAGAAGCACAACAGGAGAGCAA-3’; hβ-actin F: 5’-AGAGCTACGAGCTGCCTGAC-3’, R: 5’-AGCACTGTGTTGGCGTACAG-3’. All data are presented as relative expression after normalization to the β-actin gene, and measurements were conducted in triplicate.

Cytokine measurements

BMDMs were infected with B. abortus for 24 h, and supernatants from cell culture were harvested and assayed for the production of murine IL-6, IL-1β, and CXCL10/IP-10 by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Western blot analysis

BMDMs were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were loaded onto 12% SDS polyacrylamide gel, transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, USA). Membranes were blocked for 1 h at room temperature with TBS containing 0.1% Tween 20 and 5% non-fat dry milk and incubated overnight with 1:1000 primary antibodies anti-IRF-1, -BiP, -phospho-p65, or -β-actin (Cell Signaling Technology, MA, Danvers, USA) at 4 °C. Subsequently, membranes were incubated for 1 h at room temperature with 1:1000 anti-rabbit IgG HRP-conjugated (Cell Signaling Technology, MA, Danvers, USA) secondary antibody. Proteins were visualized using Luminol chemiluminescent HRP substrate (Millipore, Burlington, MA, USA) in an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Immunofluorescence and microscopy analysis

C57BL/6 or STING KO mouse embryonic fibroblasts (MEFs) (1×104) were plated onto 24 wells containing glass coverslips and infected as described above with Brucella-GFP. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Cells were subsequently blocked for 1 h with 3% BSA in PBS at room temperature prior to incubation with anti–GRP78 (BiP) primary antibody (Abcam, Cambridge, UK) at 4 °C overnight. For evaluation of STING activation by B. abortus, MEFs cells were infected with B. abortus (MOI of 1000:1) or transfected with dsDNA90 (3 μg/ml) for 4 h. Cells were processed for immunofluorescence as described above and incubated with a rabbit polyclonal antibody against STING as described previously (10). Anti-rabbit conjugated with Alexa Fluor 546 was used for detection of primary antibodies. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen) and confocal microscopy analysis was performed in a Nikon A1 confocal system. Confocal microscopy analysis was performed 4 h after infection given that at this time-point, BiP-expression and STING activation were enhanced on MEFs cells. Three coverslips were analyzed per sample and representative images were taken using a ×40 objective.

Estimation of intracellular Brucella by confocal microscopy

BMDMs (1×105) were plated onto 24 wells containing glass coverslips and infected as described above with Brucella-GFP. The number of bacteria was assessed in cells infected for 24 h. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Immunolabeling with rhodamine-phalloidin (0.04 mM in 0.5% Triton X-100, 1% BSA in PBS) (Thermo Fisher Scientific, Waltham, MA, USA) was performed to visualize cell shape according to manufacturer’s instructions. Coverslips were mounted on slides using ProLong Gold with DAPI mounting medium (Invitrogen, Carlsbad, CA, USA). Confocal microscopy analysis was performed in a Nikon A1 confocal system. Three coverslips were analyzed per sample and representative images were taken using a ×40 objective. Counts of intracellular bacteria were performed manually by visualization of individual intracellular GFP-expressing Brucella. The number of GFP-expressing bacteria per cell was counted using 50 cells for each condition.

Measurement of Brucella CFU in macrophages

For the measurement of viable intracellular bacteria using CFU, cells 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. Lysates were diluted from 10 to 1000 times in PBS and plated on petri dishes containing Brucella broth agar. Petri dishes were incubated for 3–4 days at 37 °C before CFU counting.

Bacterial enumeration in B. abortus infected mice

Five mice per group were infected intraperitoneally (i.p.) with 1×106 virulent B. abortus in 100μl of PBS. After 1-week post infection, animals were sacrificed, and livers and spleens were used to determine the number of bacterial CFU. Organs harvested from each animal were macerated in 10 mL of saline (NaCl 0.9%). To determine bacterial burden, livers or spleens 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.

Statistical analysis

The results of this study were analyzed using Two-way ANOVA, One-way ANOVA, or Student’s t test, as indicated, with GraphPad Prism 5 computer software (GraphPad Software). A p value <0.05 was considered significant.

Results

B. abortus induces the ATF6 and IRE1 UPR pathways

B. abortus targets the ER to create a safe niche of replication (17), which leads to a remarkable ER reorganization (16, 23). Previous reports suggested that translocation of the T4SS effector VceC induces ER stress during B. abortus infection (22). Moreover, B. melitensis induces the UPR via the Toll/interleukin-1 receptor domain containing protein (TcpB) (23). To investigate the induction of the UPR during B. abortus infection in our model, BMDMs and THP-1 human cells were infected for 24 h and activation of the three main UPR sensors were evaluated. B. abortus infection led to enhanced BiP (Figure 1A), and XBP1(s) expression (Figure 1B) in macrophages but no induction of CHOP was observed (Figure 1C). Moreover, pre-treatment with the ER chaperone TUDCA, a well-known ER stress inhibitor, decreased Brucella-induced BiP (Figure 1D) and XBP1(s) expression (Figure 1E) in macrophages, as well as in THP-1 cells (Figure 1F and 1G). However, TUDCA caused no significant impact on BMDMs viability (Supplementary Figure 2A). Furthermore, pre-treatment with the selective IRE1 inhibitor, 4μ8c, had no impact on BiP expression (Figure 1H). In contrast, 4μ8c treatment decreased Brucella-induced XBP1(s) expression (IRE1 pathway) as expected (Figure 1I). To better understand the kinetics of BiP and XBP1(s) induction, we infected BMDMs derived from C57BL/6 mice and assessed UPR induction at several time points. B. abortus infection triggered the activation of the UPR in a bimodal activation profile: the first peak of BiP (Figure 1J) and XBP1 (Figure 1K) expression was observed at an early time point (3 h post-infection) and a second and more prominent peak at 24 h post-infection.

Figure 1. B. abortus induces UPR signaling.

Figure 1.

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BMDMs derived from C57BL/6 mice or THP-1 human cells were uninfected (NI), infected with B. abortus for 24 h or pre-treated for 30 min with 1 mg/mL TUDCA or with 50 μM 4μ8c and then infected with B. abortus for 24 h and UPR targeted gene expression was determined by qPCR. Tunicamycin (Tm) (1 μg/mL) was used as a positive control for UPR induction where indicated. (A) BiP, (B) XBP1(s), and (C) CHOP gene expression upon B. abortus infection. (D) BiP and (E) XBP1(s) gene expression after TUDCA pre-treatment. (F) BiP and (G) XBP1(s) gene expression after TUDCA pre-treatment in THP-1 cell line. (H) BiP and (I) XBP1(s) expression after 4μ8c pre-treatment in BMDMs. (J) BiP and (K) XBP1(s) gene expression in BMDMs at indicated time points after B. abortus infection. Data are representative of three independent experiments. Significant differences are denoted by an asterisk for p<0.05 compared to NI and by & for p<0.05 compared to B. abortus, one-way ANOVA.

B. abortus-induced UPR regulates several proinflammatory mediators and type I IFN responses

ER stress is strongly associated with several inflammatory diseases, such as diabetes and Crohn’s disease (28, 29). Furthermore, during B. abortus infection, it was previously showed that the UPR is associated with a pro-inflammatory response linked to IL-6 (22, 30) and IL-1β (31) production. To determine whether B. abortus-induced UPR is required to induce other pro-inflammatory mediators, BMDMs were stimulated with B. abortus, with pre-treatment with TUDCA or 4μ8c or subjected to specific gene knockdown by small interference RNA. Our data show that blockage of the UPR by TUDCA treatment interfered with B. abortus-induced secretion of IL-1β (Figure 2A), CXCL10 (Figure 2B), and IL-6 (Figure 2C). Similarly, specific blockage of the IRE1 axis of the UPR by 4μ8c also interfered with B. abortus-induced secretion of IL-1β (Figure 2D), CXCL10 (Figure 2E), and IL-6 (Figure 2F). Furthermore, XBP1 and BiP gene knockdown by siRNA also interfered with IL-1β (Figure 2G), CXCL10 (Figure 2H), and IL-6 (Figure 2I) secretion upon B. abortus infection. Additionally, TUDCA treatment also interfered with phosphorylation of the p65 subunit of NF-κB (Figure 2J), and strongly regulated IRF-1, a transcription factor of the type I IFN pathway. Indeed, besides CXCL10/IP10 secretion, TUDCA treatment reduced IRF-1 (Figure 2K) and IFN-β expression in macrophages (Figure 2L) and in THP-1 cells (Figure 2M), as well as diminished transcripts of GBP2 (Figure 2N), GBP3 (Figure 2O), and GBP5 (Figure 2P) in macrophages. These results indicate that B. abortus-induced UPR has an important role in inducing IFN-β responses and is linked to the production of several molecules associated with type I IFN pathway.

Figure 2. B. abortus-induced UPR regulates several proinflammatory mediators and type I IFN responses.

Figure 2.

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BMDMs derived from C57BL/6 mice were uninfected (NI), infected with B. abortus or pre-treated for 30 min with the UPR inhibitors and then infected with B. abortus. Culture supernatants were harvested 24 h post-infection to measure (A) IL-1β, (B) CXCL10 and (C) IL-6 by ELISA upon 1mg/mL TUDCA treatment; (D) IL-1β, (E) CXCL10 and (F) IL-6 by ELISA upon 50 μM 4μ8c treatment. BMDMs derived from C57BL/6 mice were subjected to specific BiP and XBP1(s) gene knockdown and then infected with B. abortus, and culture supernatants were harvested 24 h post-infection to measure (G) IL-1β, (H) CXCL10 and (I) IL-6 by ELISA. Cell lysates were harvested 24 h post-infection, and (J) pp65 and (K) IRF-1 expression determined by western blot with β-actin being used as loading control. Total RNA was extracted, and qPCR was performed to measure (L) IFN-β in murine macrophages and (M) in THP-1 human cells, and (N) GBP2, (O) GBP3, and (P) GBP5 expression in BMDMs. Data are representative of three independent experiments. Significant differences compared to B. abortus are denoted by an asterisk for p<0.05, one-way ANOVA.

Brucella-induced UPR is STING-dependent and is triggered by bacterial c-di-GMP

Inflammation is generally triggered when pattern recognition receptors (PRRs) detect PAMPs or DAMPs during microbial infections. STING is an ER resident transmembrane protein that participates during sterile ER stress by regulating IFN-β expression (21). UPR regulation of the type I IFN pathway and the surprisingly early induction of the UPR prompted us to investigate the potential link between the UPR and STING during B. abortus infection. To evaluate the role of STING in UPR responses, BMDMs derived from C57BL/6 and STING KO mice were infected with B. abortus. BiP (Figure 3A) and XBP1(s) expression (Figure 3B) were reduced, and CHOP unaffected (Figure 3C) in STING KO cells, revealing that UPR induction was severely impaired in STING KO compared to C57BL/6 infected BMDMs. Furthermore, western blot (Figure 3D) and immunofluorescence analysis (Figure 3E with quantification in 3F) confirmed that BiP induction was compromised in STING KO cells. Based on previous experience (10), the decreased UPR was not related to insufficient infection of the STING KO macrophages, since these animals were more susceptible to Brucella. Given the importance of STING in regulating the UPR, we infected C57BL/6 MEFS with B. abortus-GFP and transfected with dsDNA90 and observed STING translocation through confocal microscopy. After 4h of infection or transfection, STING rapidly underwent trafficking from the endoplasmic reticulum to the perinuclear-associated endosomal regions of the cell. However, pre-treatment with TUDCA blocked this event in infected cells (Figure 3G with quantification in 3H). Taken together, these results show that STING plays a crucial role in inducing the UPR during B. abortus infection. Moreover, STING can function as a direct sensor of bacterial-derived CDNs, as well as an adaptor molecule in genomic DNA recognition (5). Our group recently demonstrated that B. abortus produces c-di-GMP that activates STING-dependent type I IFN responses (10). To determine if B. abortus CDNs are able to trigger the STING-dependent UPR, we infected BMDMs derived from C57BL/6 and STING KO mice with B. abortus wild-type or with a Brucella guanylate cyclase mutant strain (Δ1520). Interestingly, B. abortus Δ1520 led to a reduced UPR induction in comparison with B. abortus wild-type strain (Figure 4A, 4B and Supplementary Figure 1A), which was not due to differences in bacteria uptake (Supplementary Figure 2B), indicating that bacterial CDNs influence UPR induction. Furthermore, B. abortus Δ1520 led to a reduced CXCL10 secretion in comparison with B. abortus wild-type strain (Figure 4C). To confirm CDNs involvement in UPR induction, we transfected BMDMs from C57BL/6 and STING KO mice with well-known STING agonists. C-di-GMP, c-di-AMP and dsDNA90 were all capable of inducing the UPR in C57BL/6 derived macrophages; however, they prompted lower levels of UPR induction in STING KO macrophages (Figure 4D and Supplementary Figure 1B), indicating that bacterial CDNs are required for triggering the UPR response during B. abortus infection and confirming STING’s involvement in sensing bacterial CDNs and triggering the UPR. Taken together these results indicate that STING is required to induce the UPR during B. abortus infection and involves the bacterial metabolite c-di-GMP.

Figure 3. Brucella-induced UPR is STING-dependent and is triggered by bacterial c-di-GMP.

Figure 3.

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BMDMs derived from C57BL/6 and STING KO mice were uninfected (NI) or infected with B. abortus wild-type for 24 h. (A) BiP, (B) XBP1(s), and (C) CHOP gene expression determined by qPCR after infection. (D) Cell lysates were harvested 24 h post-infection and BiP expression was determined by western blot with β-actin being used as loading control. (E) C57BL/6 and STING KO MEFs were infected with B. abortus-GFP (MOI 100:1) for 24 h and processed for fluorescence microscopy analysis. Images are representative of all experiments. GFP-expressing bacteria are shown in green, BiP staining is in red, and DAPI (DNA) is in blue. Scale bar, 20 μm. (F) % of BiP-expressing cells in relation to total cells. (G) C57BL/6 MEFs were uninfected (NI), infected with B. abortus-GFP (MOI 1000:1), pre-treated with TUDCA and then infected with B. or transfected with 3 μg/mL dsDNA90 for 4 h and processed for fluorescence microscopy analysis. Images are representative of all experiments. GFP-expressing bacteria are shown in green, STING staining is in red, and DAPI (DNA) is in blue. Scale bar, 20 μm. (H) % of STING-activated cells in relation to total cells. Data are representative of three independent experiments. Significant differences are denoted by an asterisk for p<0.05, compared to NI and by & for p<0.05 compared to B. abortus, two-way ANOVA for panels A-C and F and one-way ANOVA for panel H.

Figure 4. IFN-β production enhances UPR activation in response to B. abortus infection.

Figure 4.

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BMDMs derived from C57BL/6 and STING KO mice were uninfected (NI) or infected with B. abortus wild-type or with Δ1520 mutant for 24 h. (A) BiP and (B) XBP1(s) gene expression determined by qPCR and (C) CXCL10 secretion determined by ELISA. (D) BMDMs derived from C57BL/6 and STING KO mice were transfected with 3 μg/mL c-di-AMP, c-di-GMP or dsDNA90 for 24 h. Total RNA was extracted, and qPCR performed to measure BiP gene expression. (E) BMDMs derived from C57BL/6 and STING KO mice were uninfected (NI) or infected with B. abortus wild-type for 24 h and IFN-β gene expression determined by qPCR. (F) BMDMs derived from C57BL/6 and STING KO mice were uninfected, infected with B. abortus or, where indicated, pre-treated for 17h with 100 U/mL mouse recombinant IFN-β (rIFN-β) and XBP1(s) gene expression determined by qPCR. BMDMs derived from C57BL/6 mice were uninfected (NI), infected with B. abortus or pre-treated with 10 U/mL IFN-β neutralizing antibody (anti-IFN-β) for 30 min prior to infection. (G) BiP and (H) XBP1(s) gene expression determined by qPCR after the infection. Culture supernatants were harvested 24 h post-infection to measure (I) IL-6 and (J) IL-1β by ELISA. (K) Residual CFU in BMDMs derived from C57BL/6 mice untreated or pre-treated for 17h with 100 U/mL rIFN-β. (L) BMDMs derived from C57BL/6 were infected with B. abortus-GFP (MOI 20:1) for 24 h and processed for fluorescence microscopy analysis. Images are representative of all experiments analyzed. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape evaluation is in red, and DAPI (DNA) is in blue. Scale bar, 30 μm. (M) Number of GFP-expressing bacteria counted in 50 cells for each condition. Data are representative of three independent experiments. Significant differences are denoted by an asterisk for p<0.05 and by an # for p<0.05 compared to C57BL/6 B. abortus, two-way ANOVA for panels A-F, one-way ANOVA for G-J, and Student’s t test for K and M.

IFN-β enhances UPR activation in response to bacterial infection and favor B. abortus replication

Since STING has a crucial role for UPR induction and STING KO mice showed reduced IFN-β expression levels compared to C57BL/6 mice (Figure 4E), we evaluated the role of type I IFN in UPR responses during B. abortus infection. Interestingly, BMDMs treated with a mouse recombinant IFN-β (rIFN-β) showed enhanced XBP1(s) expression, during infection in both C57BL/6 and STING KO macrophages (Figure 4F). Additionally, BMDMs were infected with B. abortus for 24 h, with or without pre-treatment with an IFN-β neutralizing antibody (anti-IFN-β). Blockage of the IFN-β signaling reduced BiP (Figure 4G), and XBP1(s) expression (Figure 4H), as well as IL-6 (Figure 4I) and IL-1β secretion (Figure 4J). Furthermore, we demonstrated that priming BMDMs with rIFN-β enhanced the number of B. abortus CFU in vitro (Figure 4K). In accordance with these data, confocal images showed enhanced numbers of B. abortus-GFP after priming macrophages with rIFN-β (Figure 4L with quantification in 4M). Moreover, we infected A549 IFNAR KO human cells or A549 WT with B. abortus for 24 h. Knockout of the type I IFN receptor in human cells also reduced BiP (Supplementary Figure 3A) and XBP1(s) (Supplementary Figure 3B) expression. These findings demonstrate that type I IFN pathway is involved in UPR induction in mouse and human cells. Together, these data reveal that IFN-β is crucial for inducing the UPR during B. abortus infection and affects bacterial replication.

UPR blockade inhibits Brucella replication in vitro

Recent evidence indicates important functions for the UPR in promoting or counteracting intracellular proliferation of bacterial pathogens (23, 32–35). Our data suggest that B. abortus induces the UPR upon infection at least in part through activation of STING. However, it is not clear if this response benefits the host or the pathogen. To address the UPR role in B. abortus replication, we evaluated B. abortus CFU in TUDCA treated cells. Our results revealed that UPR blockade does not interfere with B. abortus initial uptake but decreased recoverable B. abortus CFU in vitro 24 h post infection (Figure 5A). In accordance with these data, confocal images showed reduced number of B. abortus-GFP upon TUDCA treatment (Figure 5B and 5C). These data provide evidence of a critical role for the Brucella-induced UPR in enabling bacterial intracellular replication inside macrophages.

Figure 5. UPR blockade inhibits Brucella replication in vitro.

Figure 5.

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BMDMs derived from C57BL/6 and STING mice were infected with B. abortus or B. abortus-GFP or pre-treated with TUDCA (1 mg/mL) for 30 min and then infected for 2h, 6h or 24 h. (A) CFU in BMDMs untreated or pre-treated with TUDCA (1 mg/mL). (B) Fluorescence microscopy analysis of B. abortus-GFP replication 24 h post-infection. Images are representative of all experiments analyzed. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape evaluation is in red, and DAPI (DNA) is in blue. Scale bar, 30 μm. (C) Estimation of the number of intracellular Brucella by confocal microscopy. The number of GFP-expressing bacteria was counted using 50 cells for each condition. Data are representative of three independent experiments. Significant differences are denoted by an asterisk for p<0.05, two-way ANOVA for each time point on panel A and for panel C.

UPR blockade inhibits Brucella replication and IFN-β expression in vivo

To investigate the role of UPR during infection in vivo, we treated mice with 4-PBA, a chemical chaperone that inhibits the endoplasmic reticulum stress, and evaluated the number of CFU in mouse livers and spleens one week after infection. 4-PBA treatment reduced BiP expression levels in the liver of C57BL/6 infected mice (Figure 6A) and IFN-β expression in the liver of these animals (Figure 6B). Moreover, 4-PBA treated mice showed lower levels of recoverable B. abortus CFU in the livers (Figure 6C) and in the spleens (Figure 6D). Interestingly, the effect of 4-PBA on bacterial growth in the spleens is less prominent in STING KO mice compared to 4-PBA treated C57BL/6 mice, corroborating the crucial role of Brucella-induced UPR in favoring bacterial replication.

Figure 6. UPR blockade inhibits Brucella replication in vivo.

Figure 6.

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C57BL/6 and STING KO mice were untreated or treated ad libitum with 200 mg/dL 4-PBA in their drinking water for one week, starting one day prior to infection, and i.p. inoculated with 106 B. abortus. One-week post-infection the livers and spleens were extracted. (A) BiP and (B) IFN-β gene expression were determined in C57BL/6 mouse livers by qPCR. (C) B. abortus CFU was enumerated in the liver and (D) in the spleens of infected animals. Data are expressed as mean of five animals. Data are representative of three independent experiments. Significant differences are denoted by an asterisk for p<0.05, Student’s t test for panels A-C and one-way ANOVA for panel D.

Discussion

Bacterial pathogens (15, 36) that have an intracellular life cycle have evolved several strategies to subvert host detection to generate niches that ensure their survival and persistence. Viral infections have long been known to both induce and modulate ER stress (37, 38). Similarly, bacterial infection results in ER stress and the induction of the UPR (15, 36). Here we showed that B. abortus induced the UPR in mouse and human macrophages, corroborating previous studies showing that Brucella proliferation results in ER disruption and UPR initiation (22, 23).

In the context of Brucella infection, ER stress plays a critical role in prompting inflammation (22, 30, 31). In this study, we observed that the UPR induced following B. abortus infection governed the induction of several pro-inflammatory mediators, including IL-6 and IL-1β, like previously demonstrated by others (30, 31), and demonstrated that the B. abortus-induced UPR also strongly regulated several type I IFN inducible molecules, including GBPs. Previous data showed that inflammasome activation during B. abortus infection requires functionally active GBPs (10). Therefore, we hypothesize that the reduced IL-1β secretion upon TUDCA treatment could be associated with reduced GBP expression upon UPR blockade. Moreover, UPR induction likewise directly stimulates IFN-β expression. Corroborating these data, previous studies showed that ER stress is linked to enhanced IFN-β production in response to LPS stimulation (21, 27, 39).

Considering the early induction of the UPR, we sought to investigate the role of pathogen detection by PRRs in this activation process. Previous studies have shown that NOD1 and NOD2, members of the NOD-like receptor family of PRRs, link ER stress with inflammation in mouse and human cells upon B. abortus infection (30). Here we demonstrated that B. abortus-induced UPR is STING-dependent. Remarkably, deletion of the Brucella guanylate cyclase significantly reduced the ER stress response, suggesting an upstream role of STING in inducing the UPR that is possibly linked to bacterial CDNs recognition on the onset of the infection. These data are in accordance with the fact that a Brucella VirB-deficient mutant, which does not reach the ER or replicate, is still able to induce an intact UPR signaling (23). Hence, B. abortus second messenger c-di-GMP, a potent STING agonist, supports the STING-induced UPR during B. abortus infection. Our results suggest that this pathway may be more universally employed by other intracellular pathogens. Recently, Moretti et al demonstrated that c-di-AMP from Listeria innocua can induce STING-mediated autophagy that resolves ER stress by removing ER stress membranes (40). Since previous studies showed that the Brucella Δ1520 mutant induced lower levels of IFN-β (10), it is not clear if bacterial CDNs have a direct effect on the UPR through STING activation or an indirect effect, through a STING-dependent type I IFN production. Together, these results reveal the significant relevance of bacterial c-di-GMP in STING-dependent UPR induction during bacterial infections and unravel one possible mechanism by which STING can participate in the UPR.

Despite the relevance of CDNs on UPR induction, the Δ1520 mutant still induces partial BiP expression; this observation raises the hypothesis that other pathways could be linked to the STING-independent UPR activation. Previous studies showed that Brucella could induce the UPR via T4SS secreted effectors like VceC (22) or via the microtubule-modulating Brucella protein TcpB (23) that can help to explain the residual UPR activation induced by the Brucella Δ1520 mutant. Furthermore, it’s possible that some other Brucella-related molecules can also induce the UPR in a STING-dependent manner. It was previously demonstrated that STING and cyclic GMP–AMP synthase (cGAS) are important to engage the type I IFN pathway during B. abortus infection (10), hence the UPR response could be linked to a cGAS-mediated recognition of B. abortus. Unfolding the complete plethora of Brucella molecules that are linked to Brucella-induced UPR will be important to further unravel the link between B. abortus infection and the UPR.

Moreover, we showed an important contribution of IFN-β in inducing the UPR in mouse and human cells. These data are corroborated by the fact that rIFN-β is partially able to restore the UPR in STING KO macrophages and synergistically enhances the XBP1(s) response in infected macrophages. The in vivo data showing less IFN-β in the livers of 4-PBA treated mice are in accordance with previous results showing that the UPR and the ER stress are linked to synergistic IFN-β induction via XBP1 (21). Based on our data showing that TUDCA pre-treatment only partially blocks CXCL10 secretion, we hypothesize that an initial UPR-independent IFN production acts synergistically with B. abortus infection to enhance UPR induction that leads to a robust type I IFN expression. Furthermore, we demonstrated that priming cells with rIFN-β enhanced B. abortus replication, indicating the role of IFN-β in Brucella survival. Recently, we showed that STING KO derived macrophages are more susceptible to B. abortus infection (10). However, taking into account the requirement for STING in the production of several other proinflammatory mediators in vitro (10), it seems possible that other pathways than type I IFN are linked to the increased susceptibility of STING KO macrophages.

During bacterial infections it is not clear whether the ER stress response benefits the host or the pathogen (32). To address this issue, we evaluated B. abortus replication upon UPR blockade. We showed that TUDCA treated cells had a reduced B. abortus intracellular growth, revealing the UPR critical role in supporting B. abortus replication. Consistent with these data, the IRE1 pathway was previously implicated in favoring Brucella replication (41). Indeed, it was similarly shown that activation of the IRE1 pathway is critical to Brucella to stablish a safe replication niche in a Yip1A-dependent way (42). Likewise, during Brucella melitensis infection, TcpB-induced UPR also supports intracellular replication in macrophages (23). Additionally, other bacterial pathogens, like group A streptococcus, exploits the induction of ER stress and UPR as a pathogenic strategy (43).

Remarkably, we showed that during B. abortus infection in vivo, the UPR is also linked to enhanced bacterial replication in livers and spleens of infected mice, mainly in C57BL/6 mice compared to STING KO animals, showing that the UPR favors B. abortus replication both in vitro and in vivo in a STING-dependent manner. Interestingly, it was previously demonstrated by others that TUDCA had no effect on B. abortus replication in spleens (30). However, bacterial replication was evaluated after 3 days post-infection, which may explain the difference between these data.

In summary, these data suggest that B. abortus-induced UPR is triggered in part by bacterial c-di-GMP, in a STING-dependent manner, and that this response supports bacterial replication. These results have extensive impact for other bacteria that manipulate the UPR, particularly those that replicate or traffic within the ER. Furthermore, as the UPR has been targeted therapeutically in both inflammatory and infectious diseases, insights in this area can improve our understanding of the links between ER stress signaling and immune responses not only in the context of infections but also in diseases like obesity, cancer, and neurodegenerative diseases.

Supplementary Material

1

Key points:

  • Brucella abortus induces a STING-dependent UPR

  • Bacterial c-di-GMP recognition by STING triggers B. abortus-induced UPR

  • UPR blockade inhibits B. abortus replication in vitro and in vivo

Acknowledgments

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #402527/2013–5, #406883/2018–1 and #302660/2015–1), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, APQ #837/15, APQ#01945/17 and Rede Mineira de Imunobiologicos #00140–16), and National Institute of Health R01 AI116453.

Abbreviations used in this paper:

UPR

Unfolded Protein Response

MOI

multiplicities of infection

BMDM

bone marrow-derived macrophage

2′3′-cGAMP

2′5–3′5′-cyclic GMP-AMP

GBP

guanylate-binding protein

cGAS

cyclic GMP-AMP synthase

STING

stimulator of interferon genes

c-di-GMP

cyclic dimeric guanosine monophosphate

CDN

cyclic dinucleotide

IFN

interferon

TUDCA

tauroursodeoxycholic acid

4-PBA

4-phenylbutyrate

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