Brucella Abortus Triggers a Cgas-Independent STING Pathway

Brucella abortus Triggers a cGAS-Independent STING Pathway To Induce Host Protection That Involves Guanylate-Binding Proteins and This information is current as Inflammasome Activation of September 28, 2021. Miriam M. Costa Franco, Fernanda Marim, Erika S. Guimarães, Natan R. G. Assis, Daiane M. Cerqueira, Juliana Alves-Silva, Jerome Harms, Gary Splitter, Judith Smith,

Thirumala-Devi Kanneganti, Nina M. G. P. de Queiroz, Downloaded from Delia Gutman, Glen N. Barber and Sergio C. Oliveira J Immunol published online 4 December 2017 http://www.jimmunol.org/content/early/2017/12/03/jimmun ol.1700725 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 4, 2017, doi:10.4049/jimmunol.1700725 The Journal of Immunology

Brucella abortus Triggers a cGAS-Independent STING Pathway To Induce Host Protection That Involves Guanylate- Binding Proteins and Inﬂammasome Activation

Miriam M. Costa Franco,*,1 Fernanda Marim,*,1 Erika S. Guimara˜es,* Natan R. G. Assis,* Daiane M. Cerqueira,* Juliana Alves-Silva,* Jerome Harms,† Gary Splitter,† Judith Smith,‡ Thirumala-Devi Kanneganti,x Nina M. G. P. de Queiroz,{ Delia Gutman,{ Glen N. Barber,{ and Sergio C. Oliveira*,‖

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by Downloaded from microarray analysis that several type I IFN–associated genes, such as IFN-b and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP–AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1b secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS- independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN http://www.jimmunol.org/ regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated–macrophages showed reduction in IL-1b secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these ﬁndings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2. The Journal of Immunology, 2018, 200: 000–000.

he innate immune system is important as the first line of Ddx41 (8), LSm14A (9), and AIM2 (10), among others. The large by guest on September 28, 2021 defense to sense invading pathogens (1). Nucleic acids numbers of DNA sensors identified in the host suggest that they T represent critical pathogen signatures that trigger a host may play redundant roles during infection. proinflammatory immune response (2). Much progress has been One important DNA sensor is stimulator of IFN genes (STING), a made in understanding how DNA and RNA trigger host defense signaling molecule associated with the endoplasmic reticulum that is countermeasures; however, several aspects of how cytosolic critical to control the transcription of numerous host defense genes, nucleic acids are sensed remain unclear. Microbial nucleic acids including type I IFNs in response to invading DNAviruses, bacteria, or commonly find their way into subcellular compartments of the transfected DNA (11). STING leads to activation of TBK1, which infected cells to be sensed by innate receptors. Several DNA phosphorylates IFN regulatory factor (IRF)-3, a transcription factor sensors have been identified that recognize cytosolic DNA. required for the induction of IFN-b expression (12). Actually, STING These include RNA polymerase III (3), DNA-dependent activator functions as a direct sensor of bacterial-derived cyclic dinucleotides of IFN-regulatory factors (4), Lrrfip1 (5), Ifi204 (6), Mre11 (7), (CDNs) as well as an adaptor molecule in DNA recognition (13).

*Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, The microarray data presented in this article have been submitted to the Gene Belo Horizonte, 31270-901 Minas Gerais, Brazil; †Department of Pathobiological Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number Sciences, University of Wisconsin–Madison, Madison, WI 53706; ‡Department of GSE96071. Pediatrics, University of Wisconsin, Madison, WI 53792; xDepartment of Immunol- { Address correspondence and reprint requests to Dr. Sergio C. Oliveira, Laborato´rio ogy, St. Jude Children’s Research Hospital, Memphis, TN 38105; Department of ‖ de Imunologia de Doenças Infecciosas, Departamento de Bioquı´mica e Imunologia, Cell Biology, University of Miami, Miami, FL 33136; and Instituto Nacional de Universidade Federal de Minas Gerais, Avenida Antoˆnio Carlos 6627, Pampulha, Cieˆncia e Tecnologia em Doenças Tropicais, Salvador, 40110-160 Bahia, Brazil Belo Horizonte, Minas Gerais, Brazil. E-mail address: [email protected] 1M.M.C.F. and F.M. contributed equally to this work. The online version of this article contains supplemental material. ORCIDs: 0000-0002-1893-3592 (E.S.G.); 0000-0002-8905-1159 (N.R.G.A.); 0000- Abbreviations used in this article: BCV, Brucella-containing vacuole; BMDM, bone 0002-8051-3396 (D.M.C.); 0000-0002-7627-3000 (J.A.-S.); 0000-0003-1343- marrow–derived macrophage; c-di-GMP, cyclic dimeric GMP; CDN, cyclic dinucleotide; 0977 (J.H.); 0000-0003-4158-8253 (J.S.); 0000-0002-6395-6443 (T.-D.K.); 0000- cGAMP, cyclic GMP–AMP; cGAS, cGAMP synthase; EdU, 5-ethynyl-2-deoxyuridine; 0002-9789-1035 (N.M.G.P.d.Q.); 0000-0003-4062-5577 (S.C.O.). GBP, guanylate-binding protein; IRF, IFN regulatory factor; KO, knockout; MEF, murine Received for publication May 17, 2017. Accepted for publication November 5, 2017. embryonic fibroblast; MOI, multiplicity of infection; qPCR, quantitative PCR; siRNA, small interfering RNA; STING, stimulator of IFN genes; TEM, transmission electron This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e microscopy; WT, wild-type. Tecnolo´gico Grants 464711/2014-2, 402527/2013-5, 443662/2014-2, and 302660/ ˜ 2015-1, Fundaçao de Amparo a Pesquisa do Estado de Minas Gerais Grant APQ Ó 837/15 and Rede Mineira de Imunobiologicos Grant 00140-16, as well as by Coor- Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 denaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior Grant 030448/2013-1 and National Institutes of Health Grant R01 AI116453.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700725 2 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA

Recent studies have demonstrated that cytosolic DNA species trigger demonstrated (28). Briefly, the c-di-GMP–responsive riboswitch was STING activation after binding to the enzyme cyclic GMP–AMP cloned from 348 bp upstream of the Vibrio cholerae VC1722 gene synthase (cGAS) generating the second-messenger cyclic GMP– through the VC1722 start codon and inserted into pBBR1MCS-4 (GenBank U25060), upstream of a promoterless luxCDABE operon. To AMP (cGAMP) (14, 15). CDNs produced by cGAS bind to STING confirm the levels of c-di-GMP produced, the parental WT strain and in the endoplasmic reticulum, inducing a conformational change in D1520 mutant were transformed with this plasmid containing the c-di- the molecule that leads to relocation of STING and TBK1 to the GMP–responsive riboswitch driving lux expression. Bioluminescence perinuclear region of the cell, resulting in activation (16). was assayed on a Veritas microplate luminometer 100 (Promega) 24 h after transfection. During intracellular bacterial infections, activation of STING can Additionally, we used a chemical c-di-GMP inhibitor, termed Ebselen. be accomplished via two different mechanisms. First, STING can Mouse (C57BL/6) bone marrow–derived macrophages (BMDMs) were directly recognize bacterial CDNs and thus function as a primary plated on six-well tissue culture plates at 5 3 105 per well in 2 ml per well pattern recognition receptor (13). Alternatively, DNA sensing via culture media (RPMI 1640 plus 10% FBS) and cultured overnight at 37˚C cGAS triggers the synthesis of cGAMP, which then engages STING in a 5% CO2 incubator. The next day, cells were pretreated with Ebselen (50 mM) for 30 min, then they were uninfected (medium) or infected with as a secondary receptor (15). Listeria monocytogenes actively se- a multiplicity of infection (MOI) of 100 of B. abortus (strain 2308) for 24 h cretes the bacterial second messenger c-di-AMP that binds directly to in the presence or absence of Ebselen. Supernatant was then harvested and STING and activates the production of IFN-b in mice (17). However, assayed for mouse IFN-b using the Legend Max mouse IFN-b ELISA kit in contrast to L. monocytogenes, Mycobacterium tuberculosis, (BioLegend) following the manufacturer’s instructions. Legionella pneumophila,andChlamydia trachomatis appear to ac- Cell culture tivate this same STING-dependent pathway but via the DNA sensor cGAS (18, 19). BMDMs were generated and cultured in DMEM medium as described (22). Downloaded from Briefly, bone marrow cells were differentiated for 10 d in DMEM (Life Brucella abortus is a Gram-negative facultative intracellular Technologies, Carlsbad, CA) containing 10% FBS (HyClone), 1% HEPES bacterium that causes brucellosis, with pathological manifestations (Life Technologies), 1% penicillin G sodium (100 U/ml), and streptomycin of arthritis, endocarditis, and meningitis in humans, and in cattle it sulfate (100 mg/ml), 10% L929 cell–conditioned medium, as the source of leads to abortion and infertility, resulting in serious economic losses M-CSF for macrophages at 37˚C in 5% CO2. A day prior to stimulation of infection, macrophages were harvested and seeded onto 24-well plates at to the livestock industry (20). This pathogen resists killing by the density of 5 3 105 cells per well (for cytokine and Western blot neutrophils and replicates inside macrophages and dendritic cells, analysis) or 1 3 105 cells per well over a sterile coverslip (for microscopy http://www.jimmunol.org/ maintaining a long-lasting interaction with host cells. Recently, we analysis). BMDMs were infected with B. abortus virulent strain 2308 Brucella or B. abortus D1520 with the indicated MOIs (see figure legends). WT have identified DNA as a major bacterial component that 2/2 induces type I IFN and IL-1b. Our study revealed that Brucella and STING murine embryonic fibroblasts (MEFs) were provided by Dr. G.N. Barber (University of Miami). Cells were maintained in high- DNA operates through a mechanism dependent on STING and glucose DMEM (Life Technologies) supplemented with 10% FBS (Life AIM2 (21, 22). However, currently it is unclear whether cGAS Technologies), 10 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml participates as a Brucella DNA sensor and what the contribution of streptomycin (Life Technologies/Invitrogen) at 37˚C in 5% CO2/95% air in cGAS and STING are in protective immunity and in possible co- a humidified incubator. MEFs were seeded on 24-well plates containing sterile coverslips at 1 3 104 cells per well a day before the experiment and operation with AIM2. kept on normal growth medium. In this study, we demonstrate that the DNA sensor STING detects B. by guest on September 28, 2021 abortus infection and triggers a type I IFN response and IRF-1– Purification of B. abortus DNA and transfection experiments dependent signaling cascade, leading to the upregulation of several B. abortus was grown for 3 d at 37˚C under constant agitation and the genes, including the guanylate-binding proteins (GBPs). Further- DNA was purified using the Illustra bacteria genomic Prep Mini Spin kit more, GBPs were critical to induce inflammasome activation and (GE Healthcare) according to the manufacturer’s instructions. Transient IL-1b secretion, and GBPchr3 knockout (KO) mice were more sus- transfections of BMDMs and MEFs were carried out using a Lipofect- ceptible to Brucella infection compared with wild-type (WT) ani- amine 2000 (Invitrogen) ratio (in milliliters) of 1:0.25, following the manufacturer’s directions. Cells were cultured in DMEM and transfected mals. Importantly, cGAS and STING were required to induce type I with Brucella DNA (1 mg per well), dsDNA90 (3 mg/ml), and 29,39- IFN responses; however, STING but not cGAS played the major role cGAMP (3 mg per well) (InvivoGen). in controlling bacterial infection in macrophages and in vivo. In vivo and in vitro infection with B. abortus Materials and Methods Mice were infected via i.p. injection of 1 3 106 CFU of virulent B. abortus strain S2308. Animals were kept for different periods of time and then Mice killed for CFU counting and pathology. Cultured cells were infected Wild-type C57BL/6 and 129 Sv/Ev mice were purchased from the Federal in vitro with virulent B. abortus strain 2308, B. abortus–GFP, or B. abortus University of Minas Gerais. STING2/2,cGAS2/2, AIM22/2,MAVS2/2, D1520 in DMEM supplemented with 1% FBS (macrophages) or 10% FBS GBP22/2,GBPchr32/2,IRF-12/2, and IFNAR2/2 mice were described (MEFs). Different MOIs of bacteria were used as challenge for different previously (10, 11, 23–26). The animals were maintained at the Federal analysis, as described below. University of Minas Gerais and used at 6–8 wk of age. All animal experiments were preapproved by the Institutional Animal Care and Use Com- B. abortus counts in spleens mittee of the Federal University of Minas Gerais (CETEA no. 128/2014). Five mice from each group of C57BL/6, STING2/2, cGAS2/2, AIM22/2, 2/2 chr32/2 Bacterial strains GBP2 , and GBP were infected with B. abortus as described above and killed at the mentioned time intervals. To count residual Bru- Bacteria used in this study included B. abortus virulent strain S2308 cella CFU, the spleen collected from each animal was macerated in 10 ml obtained from our laboratory collection and a variant that constitutively ex- of saline, serially diluted, and plated in duplicate on Brucella broth agar. presses GFP (Brucella-GFP). The Brucella cyclic dimeric GMP (c-di-GMP) After 3 d of incubation at 37˚C, the number of CFU was determined as guanylate cyclase mutant strain (D1520) was generated in our laboratory described previously (29). using the constructs previously described (27). Before being used for cell infection or DNA extraction, bacteria were grown in Brucella broth medium Cytokine analysis (BD Pharmingen, San Diego, CA) for 3 d at 37˚C under constant agitation. BMDMs were stimulated by Brucella infection (MOI of 100:1) or DNA Measurement of c-di-GMP in Brucella WT and mutant strains transfection as described above. Where indicated, cells were treated with 100 U/ml IFN-b 18 h prior the course of infection or were untreated. After To measure relative levels of intracellular c-di-GMP, a luciferase (lux) 17 h, supernatants from cell culture were harvested and assayed for the reporter vector containing the Vc2 riboswitch was used as previously production of murine IL-1b, CXCL10/IP-10, IL-6, and TNF-a by ELISA The Journal of Immunology 3

(R&D Systems), in accordance with the manufacturer’s instructions. Hu- All data are presented as relative expression units after normalization to the man CXCL10 was measured in the supernatants of hTERT transfected b-actin gene, and measurements were conducted in triplicate. cells by ELISA (R&D Systems), in accordance with the manufacturer’s instructions. Knockdown via siRNA Western blot analysis BMDMs were transfected with siRNA from siGENOME SMARTpools (Dharmacon) with the GenMute siRNA transfection reagent according to BMDMs were lysed with M-PER protein extraction reagent (Thermo the manufacturer’s instructions (SignaGen). siGENOME SMARTpool Scientific) supplemented with 1:100 protease inhibitor mixture siRNAs specific for mouse GBP2 (M-040199-00-0005), GBP3 (M- (Sigma-Aldrich). Equal amounts of protein were loaded onto 12% SDS 063076-01-0005), and GBP5 (M-054703-01-0005), were used in this polyacrylamide gel, transferred to nitrocellulose membranes (Amersham study. A control siRNA pool was used (D-001206-14-05). Forty-eight Biosciences), and blocked for 1 h at room temperature with TBS containing hours after transfection, cells were infected with B. abortus (MOI of 0.1% Tween 20 and 5% nonfat dry milk. The following primary Abs were 100:1) for 24 h as described above. hTERT cells were transfected with incubated overnight at 4˚C: rabbit mAb IRF-1 (no. 8478l; Cell Signaling siRNA from siGENOME SMARTpools siRNA STING (J-024333-20- Technology), rabbit mAb b-actin (no. 4970; Cell Signaling Technology), 0002), siRNA cGAS (L-015607-02-0005), or siRNA control (D-001810- anti–Caspase-1 (p20, mouse mAb no. AG-20B-0042; Adipogen, and IL-1b 10-05) at 80 mM siRNA and 1 ml of Lipofectamine RNAiMAX (Thermo (mouse mAb no. 3A6; Cell Signaling Technology). For testing small in- Fisher) in 100 ml of Opti-MEM media. Culture medium was replaced 48 h terfering RNA (siRNA) knockdown efficiency on hTERT cells, a similar after transfection with DMEM supplemented with 10% heat-inactivated procedure was performed using rabbit anti-STING polyclonal Ab at 1:5000 FBS, penicillin-streptomycin, 20% 199 media, sodium pyruvate, and and rabbit anti-cGAS (no. D1D3G; Cell Signaling Technology) at 1:1000. L-glutamine solution and the cells were incubated for an additional 24 h. Subsequently, membranes were incubated for 1 h at room temperature with After 72 h of the transfection process, hTERT cells were infected with B. anti-rabbit IgG HRP-conjugated (no. 7074; Cell Signaling Technology) or abortus or mutant strain D1520 (MOI of 100) or transfected with bacterial anti-mouse IgG HRP-conjugated (Cell Signaling Technology) Abs. Pro- DNA (1 mg) and the supernatant was collected after 17 h to measure Downloaded from teins were visualized using Luminol chemiluminescent HRP substrate human CXCL10 by ELISA. (EMD Millipore) in an Amersham Imager 600 (GE Healthcare). Labeling of Brucella DNA in macrophages Gene array analysis A Click-iT 5-ethynyl-2-deoxyuridine (EdU) imaging kit (Molecular Transcripts were profiled for Brucella-infected and bacterial DNA– Probes) was used to provide specific labeling of bacterial DNA in infected transfected BMDMs from STING KO and C57BL/6 mice. Total RNA was macrophages. Brucella was grown as described above and EdU solution isolated from BMDMs with the RNeasy RNA extraction kit (Qiagen) and was added (20 mM) to the growth medium for 6 h before macrophage http://www.jimmunol.org/ analyzed by Bioanalyzer RNA 6000 Nano (Agilent Technologies). Gene infection. Bacteria were then washed three times in PBS before being array analysis was examined by Illumina Sentrix BeadChip array (mouse added to macrophages to prevent carryover of unbound EdU. Detection of WG6 version 2 for RNA extracted from BMDMs) (Affymetrix) at the incorporated EdU with Alexa Fluor 488 was performed in 24-h-infected Oncogenomics Core Facility, University of Miami. Microarray data based macrophages following the manufacturer’s instructions (see below). Ad- on the Affymetrix mouse 2.0 ST platform were normalized using the ro- ditionally, ProLong Gold with DAPI mounting medium (Invitrogen) was bust multichip averaging algorithm as implemented in the Bioconductor used to label eukaryotic as well bacterial DNA in all slides prepared for package Affy. The probes were annotated using the Bioconductor annota- microscopy. tion package mogene20sttranscriptcluster.db. Fold change was used to compare each pair of microarray samples. The heat map was generated by Immunofluorescence and microscopy analysis R package ggplot2. Microarray analysis was performed at the Center of Intracellular localization of AIM2, STING, GBP2, IRF-3, and NF-kB–p65 Computational Science, University of Miami. Gene Expression Omnibus by guest on September 28, 2021 was analyzed by immunofluorescence in macrophages or MEFs infected accession number is GSE96071 (https://www.ncbi.nlm.nih.gov/geo/query/ Brucella Brucella acc.cgi?token=uvcjkweipvyzhyb&acc=GSE96071). with -GFP, or transfected with DNA as follows: 1) localization of STING and nuclear translocation of transcription factors was Real-time RT-PCR analyzed in MEFs infected as described above with Brucella-GFP (MOI of 1000:1) or transfected with Brucella DNA for 1, 2, 3, and 4 h; 2) intra- RNA was extracted from BMDMs with TRIzol reagent (Invitrogen) to cellular localization of AIM2 was analyzed in macrophages infected with isolate total RNA in accordance with the manufacturer’s instructions. EdU-incorporated Brucella (MOI of 100:1) for 24 h; 3) intracellular lo- Reverse transcription of 2 mg of total RNA was performed using Illustra calization of GBP2 was analyzed in macrophages infected with Brucella- Ready-To-Go RT-PCR Beads (GE Healthcare) according to the manu- GFP (MOI of 100:1) for 24 h. At specific times, cells were washed twice facturer’s directions. Real-time RT-PCR was performed using 23 SYBR with PBS and fixed in 4% paraformaldehyde (pH 7.4) at room temperature Green PCR master mix (Applied Biosystems) on an ABI 7900 real-time for 30 min. After fixation, coverslips were washed three times with PBS PCR instrument (Applied Biosystems). The appropriate primers were used and kept at 4˚C until immunofluorescence was performed. Permeabiliza- to amplify a specific fragment corresponding to specific gene targets as tion was done in PBS containing 0.3% Triton X-100 for 15 min, and cells follows: b-actin, forward, 59-GGC TGT ATT CCC CTC CAT CG-39, re- were subsequently blocked for 1 h with 1% BSA in PBS at room tem- verse, 59-CCA GTT GGT AAC AAT GCC ATG T-39; IFN-b, forward, perature prior to incubation with anti–IRF-3, anti–NF-kB-p65, anti-GBP2, 59-GCC TTT GCC ATC CAA GAG ATG C-39, reverse, 59-ACA CTG TCT or anti-AIM2 primary Abs at 4˚C overnight. Anti-rabbit conjugated with GCT GGT GGA GTT C-39; CXCL10, forward, 59-CCTGCCCACGTG- Alexa Fluor 546 was used for detection of primary Abs. Coverslips were TTGAGAT-39, reverse, 59-TGATGGTCTTAGATTCCGGATTC-39; GBP2, mounted in slides using ProLong Gold with DAPI mounting medium forward, 59-CTG CAC TAT GTG ACG GAG CTA-39, reverse, 59-CGG (Invitrogen). Confocal microscopy analysis was performed in a Zeiss 880 AAT CGT CTA CCC CAC TC-39; GBP3, forward, 59-CTG ACA GTA confocal system. Three coverslips were analyzed per sample and images AAT CTG GAA GCC AT-39, reverse, 59-CCG TCC TGC AAG ACG ATT were taken using a 340 objective for six random areas of each coverslip CA-39; GBP4, forward, 59-GGA GAA GCT AAC GAA GGA ACA A-39, providing information for an average of 100 cells per coverslip. The dif- reverse, 59-TTC CAC AAG GGA ATC ACC ATT TT-39; GBP5, forward, ferences in intensity of anti-GBP2 labeling in infected versus uninfected 59-CTG AAC TCA GAT TTT GTG CAG GA-39, reverse, 59-CAT CGA macrophages were quantified to assess alterations in GBP2 intracellular CAT AAG TCA GCA CCA G-39; IRGB10, forward, 59-TAA TGC CCT localization. Briefly, the intensity of gray levels (pseudocolored in red in TCG GGG AAT AGG-39, reverse, 59-CTG GTT TGA AGT TAG TTG the image) was measured using ImageJ from anti-GBP2 images in three TCC CA-39; MX1, forward, 59-GGGGAGGAAATAGAGAAAATGAT-39, circular areas of 3 mm diameter for each cell. This was done to obtain an reverse, 59-GTTTACAAAGGGCTTGCTTGCT-39; PYDC3, forward, 59- estimation of mean intensity of GBP2 staining in at least 9 mm2 of each GCCTGATGGAAGCTTGGGAA-39, reverse, 59-CTGGGGAGTCAG- cell. Regions of brightest Ab signal on each cell (perinuclear regions) were TGGTTCAC-39; PYHIN1, forward, 59-TCTGGACCCTCCAGTGTCTT- preferentially chosen. Abs were purchased from the following sources: 39, reverse, 59-ACCTTGCTGGTGACCATTTT-39; CXCL11, forward, anti-GBP2 (Proteintech), anti–IRF-3 (FL-425) (Santa Cruz Biotechnol- 59-AGGAAGGTCACAGCCATAGC-39, reverse, 59-CGATCTCTGCCA- ogy), anti–NF-kB-p65 (Cell Signaling Technology), anti-AIM2 (Santa TTTTGACG-39; TNFSF10, forward, 59-CAACGAGCTGAAGCAGAT-39, Cruz Biotechnology). Anti-mouse and anti-rabbit secondary Abs conju- reverse, 59-GGGTCCCAATAACTGTCATC-39; NOS2, forward, 59-AGC gated with Alexa Fluor 488 or Alexa Fluor 546 were purchased from ACT TTG GGT GAC CAC CAG GA-39, reverse, 59-AGC TAA GTA TTA Jackson ImmunoResearch Laboratories. Rabbit polyclonal Ab against GAG CGG CGG CA-39; ARG1, forward, 59-TGA CAT CAA CAC TCC STING was described previously (11). For evaluation of STING activation CCT GAC AAC-39,reverse,59-GCC TTT TCT TCC TTC CCA GCA G-39. by B. abortus mutant strain D1520, MEFs or hTERT cells were infected 4 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA with B. abortus S2308 or B. abortus D1520 (MOI of 1000:1) or transfected 4˚C. Finally, the cells were washed three times, suspended in PBS buffer, with dsDNA90 (3 mg/ml) or cGAMP (1 mg per well) for 4 h. Cells were and evaluated using Attune acoustic focusing equipment (Life Tech- processed for immunofluorescence as described above and incubated with nologies). The results were analyzed using FlowJo software (Tree Star, anti-STING or anti-Brucella LPS (1:100) Abs for bacterial staining. Ashland, OR). Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen) and microscopy analysis was performed as Statistical analysis described above. The results of this study were analyzed using ANOVA, as indicated, with Estimation of intracellular Brucella numbers by confocal GraphPad Prism 5 computer software (GraphPad Software). A p value , 0.05 was considered significant (*p , 0.05, **p , 0.01, ***p , 0.001). microscopy BMDMs were infected as described above with Brucella-GFP (MOI of Results 10:1). Such lower bacterial load at the beginning of infection ensured that macrophages from more susceptible mouse strains would not have an STING is critical for Brucella DNA–mediated innate immune overgrowth of intracellular Brucella and could be analyzed. Bacteria signaling number was assessed in cells infected for 24, 48, and 72 h. Cells infected To test the role of STING during B. abortus infection in vitro, for only 24 h were kept in the absence of antibiotics during the whole experiment. After the first 24 h, gentamicin (10 mg/ml) was added to the primary murine BMDMs were isolated from STING KO and medium of cells infected for longer periods to prevent secondary infec- C57BL/6 mice. These cells were infected with virulent B. abortus tions. Fixation and permeabilization of the cells were performed as for S2308 or transfected with B. abortus S2308 DNA and microarray immunofluorescence. Staining of the actin cytoskeleton with rhodamine- analysis was performed. Microarray analysis revealed that several phalloidin (0.04 mM in 0.3% Triton X-100 in PBS; Thermo Fisher) was type I IFN–related genes are downregulated in STING KO cells performed to visualize cell shape. Preparation of slides and acquisition of Downloaded from microscopy data were performed as described for immunofluorescence. either infected with Brucella or transfected with bacterial DNA as Counts of intracellular bacteria were performed manually by visualization observed in Fig. 1A and 1B. To confirm that the induction of these of individual GFP-expressing Brucella. type I IFN–related mRNAs were STING-dependent genes, we Measurement of Brucella CFU in macrophages performed quantitative PCR (qPCR) analysis. Macrophages from C57BL/6 mice transfected with Brucella DNA robustly activate an For the measurement of viable intracellular bacteria using CFU, after array of genes, including IFN-b, GBP3, GBP4, and GBP5

transfection with siRNA and infection with B. abortus strain 2308 or http://www.jimmunol.org/ B. abortus D1520 at different MOIs (see figure legends), cells were washed (Fig. 1C–F). However, the expression of these genes was dra- twice with PBS and then lysed for 10 min at room temperature in 800 mlof matically reduced in STING KO cells. GBPs are known to PBS containing 0.1% Triton X-100 under manual agitation. Lysates were colocalize with vacuolar bacterial pathogens such as Salmonella diluted from 10 to 1000 times in PBS and plated on petri dishes containing typhimurium and Mycobacterium bovis and to recruit antimicro- Brucella broth agar. Petri dishes were incubated for 3–4 d at 37˚C before CFU counting. bial effector mechanisms (30, 31). Additionally, other STING- dependent genes were also validated by qPCR such as CXCL11, Transmission electron microscopy of infected macrophages Mx1, TNFSF10, PYDC3, and PYHIN1 (Supplemental Fig. 1). BMDMs (1 3 106) from C57BL/6 and GPBcrh3 KO mice were derived as Taken together, our data demonstrated that both Brucella infection described previously, infected with B. abortus (MOI of 100:1) for 6 h at and bacterial DNA induce the expression of several innate im- by guest on September 28, 2021 37˚C, and washed three times with phosphate buffer (1 M). The cells were mune genes in a STING-dependent fashion. then fixed with glutaraldehyde (2.5% in 1 M phosphate buffer) for 24 h at 4˚C, washed three times with phosphate buffer (1 M), and the samples Brucella and its DNA induce STING translocation and were sent to the Microscopy Center at the Federal University of Minas activation of IRF-3 and NF-kB Gerais for dehydration, treatment with osmium tetroxide and uranyl ace- tate, and transmission electron microscopy (TEM) analysis. To evaluate the Given the importance of STING in regulating cytoplasmic DNA percentage of ruptured Brucella-containing vacuole (BCV) membranes, signaling events, we infected MEFs with Brucella-GFP or trans- we evaluated 30 macrophages per group. Each macrophage was evaluated fected with Brucella DNA and observed STING translocation in relationship to the total number of bacteria in 314,500 magnification. Then, the membrane integrity of each BCV was carefully evaluated in a through confocal microscopy. After 4 h of infection with Brucella higher magnification (360,000). After analysis, we calculated the per- and 2 h of transfection with bacterial DNA, STING rapidly un- centage of ruptured BCVs in relationship to the total number of bacteria derwent trafficking from the endoplasmic reticulum to the counted. perinuclear-associated endosomal regions of the cell (Fig. 2A). Flow cytometry analysis This event usually accompanies STING phosphorylation and degradation, likely to avoid sustained STING-activated cytokine 2/2 2/2 C57BL/6, STING , and cGAS mice were infected with B. abortus production (32). Additionally, after transfection of WT MEFs with (1 3 106 CFU). Seven days after infection, the spleen cells were harvested and washed twice with sterile PBS. After washing, the cells were adjusted Brucella DNA, IRF-3 and the p65 subunit of NF-kB became to 1 3 106 cells in RPMI 1640 medium supplemented with 10% FBS, 150 phosphorylated and translocated into the nucleus; however, these U of penicillin G sodium, and 150 mg of streptomycin sulfate per well in a events did not occur in STING KO cells (Fig. 2B, 2C). These data 96-well plate. All cells were stimulated with Con A (5 mg/ml), 1 mgof indicate that the pathway of IRF-3 and NF-kB activation induced brefeldin A was added per well, and the cultures were incubated at 37˚C for 4 h. After the incubation period, the cells were centrifuged at 1500 rpm by bacterial DNA is STING-dependent. for 7 min at 4˚C and washed with PBS containing 1% BSA (PBS/BSA). STING is required for production of proinflammatory Then, the cells were incubated with anti-CD16/CD32 (FcBlock) (1:30 diluted in PBS/BSA) for 20 min at 4˚C, washed in PBS/BSA, and incu- mediators in vitro bated for 20 min at 4˚C with a mixture of the following Abs: hamster IgG To investigate the roles of STING and cGAS during cytosolic anti-murine CD3 conjugated to biotin (clone 500A2; 1:200) and rat IgG2b sensing of Brucella DNA or bacterial infection in inducing anti-murine CD4 conjugated to allophycocyanin-Cy7 (clone GK 1.5; 1:200). All Abs were obtained from BD Biosciences (San Diego, CA). proinflammatory cytokine production, BMDMs were isolated After that, splenocytes were washed again with PBS/BSA and incubated from STING and cGAS KO mice. Brucella DNA or bacterial with streptavidin conjugated to PE-Cy5.5 (1:30) for 20 min at 4˚C and infection–triggered IFN-b expression was partially dependent on fixed and permeabilized using BD Cytofix/Cytoperm reagent (BD Bio- STING and cGAS (Fig. 3A). However, the level of IFN-b ex- sciences) according to the manufacturer’s instructions. The cells were then incubated with rat IgG1 anti-murine IFN-g conjugated to PE (clone pression induced by transfected bacterial DNA was much higher XMG1.2; 1:30; BD Biosciences) or with rat IgG1 anti-murine IL-4 than Brucella infection. Delivery of the STING ligand cGAMP via conjugated to PE (clone 11B11; 1:30; BD Biosciences) for 30 min at transfection bypassed the cGAS requirement for IFN-b expression The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. STING-mediated Brucella-induced innate immune activation gene proﬁle. BMDM C57BL/6 and STING2/2 were transfected with bacterial genomic DNA (1 mg per well) or infected with B. abortus strain 2308 (MOI of 100:1) for 17 h. (A) Total RNA was puriﬁed and examined for gene expression by Illumina Sentrix BeadChip array (Mouse WG6 version 2). Highest variable genes were selected. Rows represent individual genes; columns represent individual samples. Pseudocolors indicate transcript levelsbelow,equalto,orabovethemean(green,black,and red, respectively). The scale represents the intensity of gene expression (log10 scale ranges between 21 and 1). (B) Fold change values of the highest variable genes analyzed by microarray are shown. qPCR analysis is shown of BMDMs from STING2/2 transfected with Brucella DNA compared with WT macrophages for the following genes: (C)IFN-b,(D)GBP3,(E)GBP4, and (F) GBP5. Data are representative of at least three independent experiments. *p , 0.05 comparing WT versus STING (two-way ANOVA). 6 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA

IL-6. However, other Brucella ligands or bacterial DNA may activate NF-kB in a cGAS/STING-independent pathway, for example through the TLR pathway. We have previously demonstrated that IFN-b can be partially produced by innate cells activated with Brucella components via TLR7 (33). Furthermore, we have shown in this study using MAVS KO macrophages that type I IFN expression depends in part on the RIG-I pathway indepen- dently of the STING/cGAS axis (Fig. 3I). Additionally, we have demonstrated that inflammasome activation is important to induce protective immunity against Brucella infection (22). However, the molecular mechanisms that govern assembly of the DNA sensor AIM2 are less clear than those described for the NLRP3 inflammasome. Because type I IFN con- tributes to activation of the AIM2 inflammasome in response to Francisella novocida (34), we determined the role of STING in IL-1b secretion and caspase-1 activation during B. abortus infection. Macrophages from STING KO mice transfected with bacterial genomic DNA or infected with Brucella showed a re-

duction in IL-1b secretion and caspase-1 activation when com- Downloaded from pared with cGAS KO or WT cells (Fig. 3E, 3F). Therefore, only STING deﬁciency leads to a reduction in IL-1b and caspase-1 activation when compared with cGAS KO macrophages. These ﬁndings are consistent with a reduction of IL-1b but not pro– IL-1b observed in STING KO cells in the Western blot (Fig. 3F),

suggesting that a lack of STING affects caspase-1 processing. http://www.jimmunol.org/ Furthermore, addition of exogenous rIFN-b increased the levels of CXCL10, TNF-a, IL-6, and IL-1b in Brucella-infected macrophages, demonstrating the role of this molecule in modulating proinflammatory cytokine production. Finally, NOS2 and Arg1 have been identified as markers for M1 and M2 macrophages, respectively (35). In this study, we observed that STING KO macrophages had an increase in Arg1 expression and a decrease in NOS2 expression compared with WT and cGAS KO cells (Fig. 3G, 3H). This profile is suggestive of M2-type by guest on September 28, 2021 FIGURE 2. B. abortus DNA induces STING activation and transloca- macrophages (alternatively activated macrophages) that are typi- tion of NF-kB and IRF-3. MEFs from WT or STING KO were transfected cally associated with bacterial persistence. with B. abortus DNA (1 mg per well, for 2 or 4 h as indicated) and cells Taken together, these results demonstrate that even though cGAS + from WT mice were infected with B. abortus S2308-GFP for 4 h (MOI of and STING are important sensors involved in the production of 1000:1), fixed, and subjected to immunofluorescence microscopy analysis inflammatory cytokines, STING plays the predominant role during A k B C of STING ( ), NF- B(), or IRF-3 ( ). Pronounced translocation of Brucella infection. STING was observed as aggregated speck formation in the perinuclear region 2 h after cells were transfected with bacterial DNA or 4 h after Brucella produces c-di-GMP that induces STING-dependent B. abortus S2308-GFP infection. STING-dependent NF-kB and IRF-3 type I IFN responses activation was induced in WT MEFs by Brucella DNA transfection but not in KO cells. Ab staining is shown in middle panels for STING (A), NF-kB Bacterial CDN levels are regulated by the opposing activities of (B), and IRF-3 (C), and nuclei staining (DAPI) is shown in blue on the right cyclases and phosphodiesterases. To determine the levels of c-di- panels. The left panels are merged images from those shown in the middle GMP produced by Brucella WT strain versus c-di-GMP guanylate and right panels. Data are representative of three independent experiments cyclase mutant (D1520), we transformed all strains with a plasmid and three replicates in each experimental group. Scale bar, 30 mm (per- containing a c-di-GMP–responsive riboswitch that drives lux ex- taining to all panels). pression as previously demonstrated (28). The Brucella D1520 mutant displayed a phenotype showing lower levels of c-di-GMP and CXCL10 production in agreement with previous reports when compared with WT strain S2308 (Fig. 4B). Furthermore, we establishing that cGAS functions upstream of STING (15) performed confocal microscopy analysis in WT MEFs infected (Supplemental Fig. 2). We also observed that CXCL10 produc- with Brucella D1520 mutant and WT S2308 strains. Cells infected tion, a surrogate cytokine for type I IFN expression, was reduced with WT bacteria or transfected with DNA or cGAMP showed a in STING and cGAS KO BMDMs infected or transfected with specific STING-activation profile characterized by an aggregate Brucella DNA (Fig. 3B). Because STING activation is required speck formation in the perinuclear region of the cell; however, we for NF-kB translocation induced by bacterial DNA (Fig. 2), we did not observe this activation phenotype in Brucella D1520 also measured TNF-a and IL-6 production by STING and cGAS mutant–infected MEFs (Fig. 4A). Additionally, we infected KO BMDMs transfected with bacterial DNA or infected and BMDMs from STING KO and C57BL/6 mice with Brucella compared with WT cells. Either Brucella-infected or DNA- D1520 mutant or WT S2308 strains and measured IFN-b expression transfected macrophages from cGAS and STING KO mice pro- and IL-1b production. Our results demonstrated that the Brucella duced a reduction in TNF-a and a modest decrease in IL-6 D1520 mutant, which produces lower levels of c-di-GMP, induced compared with WT cells (Fig. 3C, 3D). These data suggest that lower expression levels of IFN-b and IL-1b secretion compared with the STING pathway is required for full production of TNF-a and the virulent strain S2308 (Fig. 4C, 4D) and equivalent to production The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Proinflammatory cytokine production induced by Brucella is partially dependent on the STING pathway. BMDMs derived from C57BL/6, STING2/2,andcGAS2/2 mice were transfected with DNA purified from B. abortus (1 mg per well) encapsulated with Lipofectamine or infected with B. abortus (MOI of 100:1). Total RNA was extracted and qPCR was performed to measure IFN-b (A) expression. Culture supernatants were harvested 17 h after treatment to measure CXCL10 (B), TNF-a (C), IL-6 (D), and IL-1b (E) by ELISA assay. Where indicated, cells were treated with 100 U/ml IFN-b 18 h before the course of infection or were untreated. (F) The same culture supernatants or cell lysates were harvested 17 h postinfection and pro–IL-1b (cell lysates), IL-1b (supernatant), and caspase-1 processing were determined by Western blot. Equal loading was controlled by measuring b-actin in the corresponding cell lysates. (G) NOS2 and (H) Arg1 gene expression was determined in macrophages infected with B. abortus (MOI of 100:1) for 24 h by qPCR. (I) Macrophages from C57BL/6 and MAVS2/2 mice were stimulated with B. abortus S2308 (MOI of 100:1) for 24 h and qPCR analysis was performed for IFN-b expression. Data are representative of at least three independent experiments and three replicates in each experimental group. *p , 0.05 comparing WT versus STING, #p , 0.05 comparing WT versus cGAS, &p , 0.05 comparing STING versus cGAS (all by two-way ANOVA), ***p , 0.0001 comparing MAVS2/2 with C57BL/6. 8 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA in the STING KO macrophages. To confirm the Brucella D1520 observed in Fig. 5A were correlated to CXCL10 production that mutant findings, we tested a chemical inhibitor of c-di-GMP, termed was reduced in siRNA control cells infected with Brucella D1520 Ebselen (36). Macrophages treated with Ebselen and infected with mutant compared with WT bacteria (Fig. 5C). Additionally, cells WT Brucella produced much less IFN-b compared with cells untreated with cGAS or STING siRNA produced diminished levels treated and infected (Fig. 4E). The greater difference observed in of CXCL10 compared with siRNA control when they were in- type I IFN responses between the use of Ebselen compared with the fected with either Brucella strain or with bacterial DNA. D1520 mutant strain might be related to the fact that Ebselen inhibits Collectively, these results suggest that Brucella is able to pro- the binding of c-di-GMP to receptors containing an RxxD domain duce its own second messenger to activate the STING pathway including PelD and diguanylate cyclases with a broader action and directly. In the present study, we provide strong evidence that the potential off-target effects (36). In contrast, the D1520 mutant strain bacterial c-di-GMP is important in activating STING in mouse has a deletion on a single Brucella diguanylate cyclase. Taken to- macrophages and human cells. gether, these results suggest that c-di-GMP produced by Brucella is a key metabolite to induce type I IFN responses. Furthermore, we STING but not cGAS is required for in vitro and in vivo control measured CFU counts of Brucella D1520 mutant and WT S2308 of Brucella infection strains after 48 h of infection in macrophages. Brucella D1520 To determine whether STING or cGAS restricts Brucella growth in mutant showed reduced bacterial numbers compared with WT macrophages and in vivo, we infected WT, STING, and cGAS KO bacteria in either C57BL/6 or STING KO cells (Fig. 4F). BMDMs and mice and quantified Brucella-GFP+ in vitro at 24, 48, To confirm our findings in the human system, we used human and 72 h by confocal microscopy and in vivo at 1 and 3 wk post-

fibroblast cells (hTERT). After infection of hTERT with Brucella infection. WT macrophages and cGAS KO infected with an MOI of Downloaded from D1520 mutant or WT S2308 strain, we observed STING activation 100:1 efficiently controlled intracellular replication, whereas STING by confocal microscopy in the cells infected with WT bacteria but KO BMDMs contained larger numbers of B. abortus at all time not with the mutant strain (Fig. 5A). Additionally, we performed intervals studied (Fig. 6A, 6B). Because the cGAS/STING axis is siRNA silencing of STING and cGAS in hTERT cells. The siRNA important for stimulating type I IFN, we investigated whether these knockdown efficiency is demonstrated in Fig. 5B. The results DNA sensors also play a role in host defense in vivo. We infected http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Deletion of the Brucella guanylate cyclase reduces cellular cyclic di-GMP levels and IFN-b expression. (A) MEFs from WT mice were transfected with dsDNA90 (3 mg/ml) or cGAMP (1 mg per well) or infected with B. abortus S2308 or B. abortus D1520 (MOI of 1000:1) for 4 h, fixed, and subjected to immunofluorescence microscopy analysis of STING. Pronounced translocation of STING was observed as aggregated speck formation in the perinuclear region 4 h after cells were transfected with dsDNA90, cGAMP, or infected with B. abortus S2308, but not after infection with B. abortus D1520. Ab staining is shown in middle panels for STING, and nuclei staining (DAPI) is shown in blue on the right panels. Left panels are merged images from those shown on middle and right panels including staining with anti-Brucella LPS in green. Data are representative of three independent experiments. Scale bar, 25 mm. (B) Wild-type Brucella or D1520 mutant strains were transfected with a c-di-GMP–regulated lux reporter system. Bacteria were grown to stationary phase and relative luminescence units (RLU) were measured (n = 4 replicates). *p , 0.05 comparing WT virulent Brucella with D1520 mutant. Macrophages derived from C57BL/6 and STING2/2 mice were infected with B. abortus WT or D1520 mutant strain (MOI of 100:1). After 17 h, total RNA was purified and the gene expression of IFN-b (C) was analyzed by qPCR, and IL-1b (D) was measured in the supernatant by ELISA. (E) BMDMs from WT mice were treated or not treated with Ebselen. Cells were either not infected (medium) or infected (MOI of 100:1) with B. abortus for 24 h with or without Ebselen (50 mM). Supernatants were then harvested, and IFN-b production was measured by ELISA. (F) BMDMs from C57BL/6 and STING2/2 mice were infected with B. abortus WT or D1520 mutant strain (MOI of 10:1) for 48 h and bacterial CFU counts were analyzed. Data are representative of three independent experiments and three replicates in each experimental group. *p , 0.05 comparing C57BL/6 mice versus STING KO mice, $p , 0.05 comparing WT Brucella strain 2308 with the D1520 mutant (two-way ANOVA). The Journal of Immunology 9 Downloaded from

FIGURE 5. STING activation in human ﬁbroblasts following B. abortus infection. hTERT cells were transfected with dsDNA90 (3 mg/ml) or infected

with B. abortus strain 2308 or B. abortus D1520 mutant (MOI of 1000:1) for 4 h, fixed, and subjected to immunofluorescence microscopy analysis of http://www.jimmunol.org/ STING. (A) Pronounced translocation of STING was observed as aggregated speck formation in the perinuclear region 4 h after cells were transfected with dsDNA90 or infected with B. abortus strain 2308 but not after infection with the B. abortus D1520 mutant. Ab staining is shown in the middle panels for STING (red) and nuclei staining (DAPI) is shown in blue on the right panels. Left panels are merged images from those shown on middle and right panels including staining with anti-Brucella LPS in green. Scale bar, 25 mm. (B) hTERT cells were transfected with mock, control siRNA (nonspecific), STING siRNA, or cGAS siRNA for 3 d. The efficiency of cGAS and STING silencing was demonstrated by immunoblotting, with b-actin serving as a loading control. (C) hTERT cells were transfected with B. abortus DNA or infected with B. abortus strain 2308 or the B. abortus D1520 mutant for 24 h and supernatants were collected for CXCL10 measurement by ELISA. Data are representative of three independent experiments and three replicates in each experimental group. *p , 0.05 compared with untreated cells, #p , 0.05 compared with siRNA control plus Brucella, &p , 0.05 compared with siRNA STING plus Brucella (two-way ANOVA). by guest on September 28, 2021 age- and sex-matched WT, cGAS, and STING KO mice i.p. with Fig. 3). These data suggest that STING modulates host liver pa- 1 3 106 CFU of B. abortus virulent strain 2308. STING KO dis- thology at early and late stages of Brucella infection. played a significantly higher bacterial burden at 1 and 3 wk postin- IRF-1 and type I IFN expression are required to control fection compared with WT animals. However, cGAS KO had no Brucella replication in macrophages defect in overall resistance to B. abortus, as we observed similar bacterial numbers in spleens of these mice compared with WT The transcription factor IRF-1 impacts adaptive immune responses by (Fig. 6C). Because Th1 responses are crucial for efficient control of regulating MHC class I expression and influencing development of B. abortus, we measured the frequency of CD4+ T cells producing NK and T cells (37). Additionally, IRF-1 was identified by its binding IFN-g or IL-4 in WT, cGAS, and STING KO mice after infection. As to DNA sequences that are common to the promoters of IFN-a/b demonstrated in Fig. 6D, the frequency of CD4+ T cells producing genes (38). Thus, IRFs were proposed to be the regulators of type I IL-4 is higher in STING KO animals compared with WT and cGAS IFN responses. In this study, we evaluated the level of IRF-1 protein KO mice. Regarding the percentage of CD4+ T cells producing expression in macrophages of WT, STING, cGAS, and AIM2 KO IFN-g, similar levels were detected in all mouse strains tested (data animals following infection with Brucella or transfection with bac- not shown). Furthermore, the presence of markers associated with terial DNA. We observed a major increase in IRF-1 expression in M2 macrophages shown previously also coincided with enhanced WT cells after bacterial infection or DNA transfection compared frequency of CD4+ T cells producing IL-4 and bacterial loads in with the negative control (Fig. 7A). However, this upregulation was STING KO mice compared with cGAS KO and WT control. partially dependent on the STING/cGAS pathway, as we observed Taken together, these findings suggest that cGAS appears to be reduced IRF-1 expression in cGAS and STING KO macrophages. dispensable for bacterial resistance in vivo and in vitro. These Conversely, lack of AIM2 robustly augmented IRF-1 expression in results confirmed that STING is critical to host defense against macrophages infected with Brucella or DNA transfected, demon- B. abortus in vitro and in vivo. strating that AIM2 regulates type I IFN responses. Furthermore, we tested whether IRF-1 expression was dependent on the IFNAR STING regulates liver granuloma during B. abortus infection signaling pathway. As shown in Fig. 7B, IFNAR KO cells trans- Infection with B. abortus results in the formation of liver and fected with bacterial DNA or infected with Brucella had diminished spleen granulomas, where inflammatory cells aggregate to restrain levels of IRF-1 when compared with WT macrophages. bacterial growth. Because only STING KO mice were more sus- Because others demonstrated that IRF-1 KO animals are more ceptible to infection in vivo, we analyzed the role of STING in susceptible to Brucella infection in vivo (39), we investigated the regulating liver pathology. At 1 and 6 wk postinfection, STING hypothesis that type I IFN signaling and IRF-1 affect the ability of KO mice displayed a significant reduction in granuloma number macrophages to control Brucella replication. Therefore, we infected and size when compared with WT counterparts (Supplemental WT, IFNAR KO, and IRF-1 KO BMDMs with Brucella-GFP+ and 10 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA Downloaded from http://www.jimmunol.org/ FIGURE 6. STING but not cGAS is required for Brucella control in macrophages and in mice. BMDMs derived from C57BL/6, cGAS, and STING KO mice were infected with Brucella-GFP+ (MOI of 10:1) for 24, 48, or 72 h and processed for fluorescence microscopy analysis. The number of GFP- expressing bacteria per cell was counted on 200 cells for each mouse strain, for all three times points assessed. Results are shown in (A) as the average number of Brucella per macrophage. Images shown in (B) were taken from macrophages infected with Brucella-GFP+ for 72 h and are representative of all experiments analyzed. Mouse strains are indicated on the left. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape determination is shown in red, and DAPI (DNA) is shown in blue. Scale bar, 10 mm. (C) Residual B. abortus CFU in the spleen of WT, STING, and cGAS KO mice (n = 5) were determined at 1 and 3 wk after infection. (D) C57BL/6, cGAS, and STING KO mice were infected with B. abortus, and 1 wk postinfection, splenocytes were submitted to flow cytometry analysis. Cells were assessed for CD3+CD4+ producing IL-4. Data are the mean 6 SD of five mice per group. The graphs are representative of three independent experiments and three replicates in each experimental group. *p , 0.05 comparing STING KO versus WT mice, &p , 0.05 comparing STING versus cGAS KO (two-way ANOVA). by guest on September 28, 2021 quantified bacteria over time (Fig. 7C). Single-cell analysis on STING and not cGAS. Furthermore, we observed by confocal revealed that WT macrophages restricted bacterial replication microscopy that Brucella infection induces the formation of GBP2 after 24 h of infection, whereas IFNAR and IRF-1 KO cells failed aggregates (37% increase compared with uninfected cells) located to control bacterial replication and harbored a significantly larger in close proximity to bacterial-containing compartments in mac- number of bacteria compared with WT BMDMs (Fig. 7D). The rophages (Fig. 8C, 8D). Additionally, we performed electron mi- greater susceptibility to Brucella replication observed in IRF-1 croscopy analysis and we detected that 74.2% of BCV was KO cells suggests that IRF-1 is important in multiple antimicro- disrupted in C57BL/6 macrophages when compared with 38.5% in bial defense mechanisms. GBPchr3 KO cells (Fig. 8E, 8F). This result suggests that the GBP machinery is important to target the BCV to release bacterial STING and type I IFN signaling are required for GBP components to host cell cytosol. expression in Brucella-infected macrophages Taken together, these findings also provide evidence that GBP2 GBPs are IFN-inducible GTPases that exert antimicrobial effects and GBP3 are produced in response to B. abortus infection, and (24). Additionally, GBPs encoded by genes on mouse chromo- that GBP2 and GBP3 are under the control of type I IFN signaling some 3 (GBP1, GBP2, GBP3, GBP5, and GBP7) promote rec- at least partially via the STING pathway. ognition of the vacuolar bacterium S. typhimurium, leading to the escape of the bacteria into the cytosol (31). In this study, we de- GBPs control B. abortus replication in vitro and in vivo tected by qPCR analysis the downregulation of GBP2 and GBP3 GBPs can target vacuolar bacteria such as Salmonella and Fran- genes in the absence of IRF-1 and IFNAR in macrophages in- cisella and induce the recruitment of antimicrobial peptides to kill fected with Brucella (Fig. 8A). However, the residual expression the bacteria (25, 31). To investigate whether GBPs contained on of GBP2 and GBP3 in IRF-1 KO macrophages indicates that an mouse chromosome 3 (GBPchr3) or GBP2 alone directly affected alternative pathway independent of IRF-1 exists to induce GBP the viability of bacteria in macrophages, we treated WT BMDMs expression. To address that, we infected IFNAR KO macrophages with GBP2 and GBP pool (containing GBP2, GBP3, and GBP5) with B. abortus and found that lack of type I IFN signaling siRNA and infected them with B. abortus, and bacteria were robustly reduced GBP2 and GBP3 expression (Fig. 8A). Addi- quantified at 24 h postinfection. Analysis of CFU counts revealed tionally, we observed that GBP2 and GBP3 expression is partially that WT macrophages restricted bacterial replication whereas GBP dependent on the STING pathway, probably via IFN-b production pool– and GBP2 siRNA–treated cells failed to control Brucella (Fig. 8B). Interestingly, reduction of GBP2 and GBP3 expression growth intracellularly and harbored a significantly larger number of following Brucella infection in macrophages was only dependent bacteria (Fig. 8G). We further analyzed the role of GBP2 and The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 7. IRF-1 and type I IFN signaling are required to restrict Brucella replication in macrophages. Macrophages derived from (A) C57BL/6, by guest on September 28, 2021 STING, cGAS, and AIM2 KO mice or (B) 129Sv/Ev and IFNAR KO mice were infected with B. abortus (at MOI of 100:1) or transfected with bacterial DNA (1 mg per well) or poly(dA:dT) (1 mg per well) encapsulated with Lipofectamine or Lipofectamine alone as control. Cell lysates were harvested 17 h after treatment and processing by Western blot to determine the levels of IRF-1. (C) BMDMs derived from WT (129 Sv/Ev), IFNAR2/2, or IRF-12/2 mice were infected with Brucella-GFP (MOI of 10:1) for 24 h and processed for ﬂuorescence microscopy analysis. GFP-expressing bacteria are shown in green, phalloidin staining of the actin cytoskeleton for cell shape determination is shown in red, and DAPI (DNA) is shown in blue. Images show infected 129Sv/ Ev, IFNAR2/2, or IRF-12/2 macrophages on the top, middle, and lower panels, respectively, as indicated on the left. Scale bar, 30 mm. (D) The number of GFP-expressing bacteria was assessed for each cell, and 200 cells were analyzed for each mouse strain. Data are representative of three independent experiments and three replicates in each experimental group. ***p , 0.001, number of Brucella per cell comparing WT versus IFNAR and IRF-1 KO. **p , 0.01, number of Brucella per cell comparing IRF-1 versus IFNAR KO (two-way ANOVA).

GBPchr3 in controlling bacterial infection in vivo by infecting GBP2 pathogen recognition receptor and NF-kB responses were normal and GBPchr3 KO and WT mice and measuring CFU in spleen at 1 but an inflammasome activatory signal was affected. To confirm wk postinfection. As shown in Fig. 8H, only GBPchr3 KO mice were these findings, we used macrophages from GBP2 and GBPchr3 KO more susceptible to Brucella infection when compared with GBP2 mice and transfected them with bacterial DNA or infected them with KO or WT animals. These findings demonstrated that GBPs mediate Brucella and measured IL-1b secretion. The results demonstrated Brucella control in vitro and in vivo. that GBP2 and GBPchr3 KO cells also showed a reduced secretion of IL-1b when macrophages were either transfected with Activation of inflammasome during Brucella infection partially DNA or infected with the bacteria, corroborating our siRNA data requires GBPs (Supplemental Fig. 4). Taken together, these findings are consistent Because GBPs play a role in releasing bacteria from vacuoles and with a role for GBPs in releasing Brucella components into the thus enable greater access to cytosolic sensors, we sought to cytosol for inflammasome detection. address the role of GBPs in inflammasome activation during Brucella infection. Brucella-infected macrophages from AIM2 is required for Brucella control in mice C57BL/6 mice treated with siRNA for GBP2 or a GBP pool AIM2 was found to recognize cytoplasmic dsDNA through its were assessed for pro–IL-1b in cell lysates, IL-1b secretion, and HIN-200 domain and ASC via its pyrin domain (10). Franciscella caspase-1 activation. GBP2 and GBP pool–treated macrophages tularensis and L. monocytogenes DNA activate the AIM2 displayed a significant reduction in cytokine release and caspase-1 inflammasome by its interaction with ASC to induce caspase-1 activation compared with siRNA control-treated cells (Fig. 9A, 9B). (34, 40). For AIM2 to detect dsDNA, Brucella or its DNA must Although GBP2 and GBP pool siRNA-treated cells had a reduced escape the BCV and enter the cytoplasm. To determine whether IL-1b secretion, the amount of pro–IL-1b was intact, indicating that AIM2 inflammasome directly senses Brucella DNA intracellularly, 12 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Type I IFN induced by STING activation is required for GBP expression that controls bacterial replication. BMDMs from C57BL/6, 129/ SvEv and STING, cGAS, IRF-1, and IFNAR KO mice were infected with B. abortus (MOI of 100:1) and total RNA was extracted at 17 h postinfection. Analysis of GBP2 and GBP3 expression by qPCR of macrophages from (A) 129Sv/Ev, IRF-1, and IFNAR KO or (B) C57BL/6, STING, and cGAS KO. The results are shown as mean 6 SD of fold induction and normalized to b-actin gene. Data are representative of at least three independent experiments. *p , 0.05 comparing WT versus STING or WT versus IFNAR, #p , 0.05 comparing WT versus IRF-1, &p , 0.05 comparing STING versus cGAS or IFNAR versus IRF-1 (two-way ANOVA). (C) B. abortus infection induces aggregation of GBP2 protein. Macrophages derived from C57BL/6 mice were infected with B. abortus–GFP (green) for 24 h, fixed, and subjected to immunofluorescence of GBP2 (in red). GBP2 localization in an uninfected cell is shown on the left panel and evident clustering of GBP2 can be observed in cells infected with Brucella-GFP (right panel). Scale bar, 10 mm. (D) Quantification of gray levels (pseudocolored in red in this image) from anti-GBP2 signal was performed on an area of ∼9 mm2 of each cell and mean values were plotted in. Data are representative of three independent experiments. *p , 0.05 comparing uninfected versus infected cells. (E) BMDMs from C57BL/ 6 and GBPcrh3 mice were infected with B. abortus for 6 h and the integrity of BCV membranes were evaluated by TEM. The red arrows indicate regions of BCV membrane rupture. The percentage of disrupted BCV membranes was evaluated and is represented in (F). Scale bar, 500 nm. B, B. abortus; n, nucleus. (G) BMDMs from C57BL/6 mice were transfected with siRNA from siGENOME SMARTpools (Dharmacon) for GBP2 and GBPpool (GBP2, GBP3, and GBP5) for 48 h and infected with B. abortus (MOI of 100:1) for 24 h and bacterial CFU counts were analyzed. ***p , 0.001 in relationship to siRNA control. (H) C57BL/6 and GBP2 and GBPchr3 KO were i.p. inoculated with 106 CFU of B. abortus strain S2308 and 1 wk postinfection the CFU were determined in spleens. Data are expressed as mean 6 SD of five animals. Data are representative of three independent experiments and three replicates in each experimental group. ***p , 0.001 comparing GBPchr3 KO in relationship to C57BL/6 and GBP2 KO. we infected C57BL/6 macrophages and observed that labeled the formation of an inflammasome aggregation of AIM2 and AIM2 colocalized with Brucella DNA that was previously stained Brucella DNA specks as result of cell activation (Fig. 9C). We using the Click-iT EdU imaging kit. Indeed, costaining of AIM2 also determined the role of AIM2 in regulating Brucella growth and bacterial DNA from Brucella-infected macrophages revealed in vivo. Consistent with our previous in vitro findings using The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/

FIGURE 9. Inflammasome activation by Brucella partially requires functionally active GBPs. BMDMs from C57BL/6 mice were transfected with siRNA from siGENOME SMARTpools (Dharmacon) for GBP2 and GBP pool (GBP2, GBP3, and GBP5) for 48 h and infected with B. abortus (MOI of 100:1) for 17 h and IL-1b (A) secretion was measured by ELISA and pro–IL-1b (cell lysates), mature IL-1b (supernatant), and caspase-1 activation by Western blot (B). Data are representative of at least three independent experiments and three replicates in each experimental group.*p , 0.05 from GBP2 and GBP pool siRNA in relationship to siRNA control (two-way ANOVA). (C) Anti-AIM2 staining of Brucella-infected macrophages by confocal microscopy reveal aggregated speck formation of AIM2 in association with bacterial DNA. Brucella DNA was specifically labeled with Click-iT kit and can be seen in green D in the upper panel. DAPI staining allows visualization of bacterial DNA. Scale bar, 20 mm. ( ) C57BL/6 and AIM2 KO mice (n = 5) were i.p. inoculated by guest on September 28, 2021 with 106 CFU of B. abortus strain S2308. Residual B. abortus CFU in the spleen of WT and AIM2 KO mice were determined at 1, 3, and 6 wk postinfection. Data are expressed as mean 6 SD of five animals per time point and are representative of three independent experiments. *p , 0.05, ***p , 0.001 in relationship to C57BL/6. macrophages identifying a critical role for AIM2 and ASC to infection by macrophages is so critical to the innate immune re- trigger caspase-1 activation and IL-1b secretion (22), AIM2 KO sponse, we sought to define the receptors that sense Brucella DNA mice showed a reduced resistance to Brucella at 1, 3, and 6 wk in the cytosol and determine their role in the host response to postinfection as determined by bacterial numbers in the spleens infection. Previously, we have shown that AIM2 is important for (Fig. 9D). Bacterial load recovery was 0.7, 1.6, and 2.3 logs higher sensing Brucella DNA and triggering IL-1b secretion and at respective weeks in AIM2 KO mice when compared with caspase-1 activation (22), but the role of the cGAS/STING axis C57BL/6 animals. Taken together, these results strongly suggest during this bacterial infection remained to be determined. that inflammasomes are important to induce resistance to B. abortus In this study, we observed that Brucella infection or bacteria infection and implicate AIM2 receptors in this process. Additionally, DNA transfection triggers the expression of innate immune genes we proposed a schematic model of how STING pathway con- such as IFN-b and GBPs in a STING-dependent manner. Fur- nects with AIM2 inflammasome activation (Fig. 10). thermore, bacterial infection or DNA transfection induces STING activation in MEFs as observed by aggregated speck formation in Discussion the perinuclear region of the cell. IRF-3 and NF-kB translocation Pathogenic bacteria can use many different strategies to enter and to the nucleus was also observed in DNA-transfected MEFs and establish infection inside the host, and the immune system has shown to be STING-dependent. Given the potential positive and mechanisms to detect and eliminate a broad range of these bacteria. negative roles of type I IFN during bacterial infections, we sought Cytosolic detection leads to activation of potent antimicrobial to determine the overall role of cGAS and STING in B. abortus effector pathways such as the inflammasome and the cytosolic pathogenesis. Infection of cGAS and STING KO macrophages led surveillance pathway. The cGAS/STING axis is an important to a partial decrease in type I IFN expression when compared with cytosolic surveillance pathway by which innate immune cells WT cells. Additionally, lack of STING had a partial effect on recognize both viral and bacterial pathogens that access the cytosol, production of NF-kB–dependent cytokines, such as TNF-a and but different bacteria have evolved different mechanisms to initiate IL-6, suggesting that Brucella partially activates NF-kB through a this response (11, 23). Additionally, bacterial ligands must secure STING-dependent pathway. However, STING but not cGAS entry into the cytoplasm to activate cytosolic sensors; however, the control bacterial replication in vitro. The same phenotype was mechanisms by which concealed ligands are liberated in the cy- observed in infected KO mice in vivo, demonstrating that lack of toplasm have remained unclear. Because the detection of Brucella cGAS had no defect in overall resistance to B. abortus. Higher 14 cGAS-INDEPENDENT STING-MEDIATED PROTECTION AGAINST BRUCELLA Downloaded from

FIGURE 10. Working model. The intracellular bacteria B. abortus enters the host cell and ensures its survival by forming the BCV. Initially, the recognition of bacterial c-di-GMP activates STING and triggers type I IFN response and upregulation of NOS2 and GBPs expression. GBPs promote lysis of http://www.jimmunol.org/ the BCV by exposing bacterial components, such as bacterial DNA in the cytosol, thus enabling activation of AIM2 and IL-1b secretion. Additionally, release of Brucella genomic DNA in the cytosol may activate cGAS generating cGAMP (dotted line), resulting in further amplification of the type I IFN signaling pathway. bacterial burdens were detected in spleens of STING KO mice I IFN signaling events. Furthermore, STING activation leads to compared with cGAS KO and WT animals. Additionally, STING GBP expression and inflammasome activation. altered the formation of hepatic granulomas in vivo, which is an IRF-1 has been identified as a transactivator of IFN-b (44); important host strategy to restrain bacterial growth. Furthermore, therefore, we used BMDMs from IFNAR and IRF-1 KO mice to by guest on September 28, 2021 we observed a reduced secretion of IL-1b and caspase-1 activation determine their relative roles in restricting Brucella replication in and GBP2 and GBP3 expression in STING KO cells infected with macrophages. By confocal microscopy, we observed more intra- B. abortus when compared with cGAS-deficient macrophages. cellular Brucella-GFP+ per macrophage in both IFNAR and IRF-1 Collectively, these findings suggest lack of cGAS is less critical to KO cells compared with WT cells. In the present study, STING the activation of innate immune effector mechanisms related to was required for full expression of IRF-1 after Brucella infection protective immunity against Brucella. in macrophages. STING activation, type I IFN signaling, and the In the context of animal models of bacterial infection, M1-type transcription factor IRF-1 were also required for robust expression macrophages (classically activated macrophages) and NO produc- of GBP2 and GBP3 following Brucella infection. Because we tion are often, but not exclusively, associated with host protection observed that GBP siRNA-treated macrophages produced a major (41). Conversely, M2-type macrophages are typically associated reduction in IL-1b secretion, we therefore concluded that there is with bacterial persistence. In this study, we observed that the a specific requirement of STING and GBPs in Brucella-induced presence of markers associated with M2 macrophages in STING inflammasome activation. KO mice compared with cGAS KO and WT controls, which also Members of the IFN-inducible GTPase family are executioners coincided with enhanced frequency of CD4+ T cells producing IL-4. of cell-autonomous immunity and have the ability to target the Previously, Xavier et al. (42) demonstrated that M2 macrophages vacuolar membrane encapsulating intracellular parasites and support increased levels of intracellular Brucella replication during bacteria (45, 46). To determine whether GBPs were directly in- chronic infection. Taken together, these findings suggest that volved in the activation of inflammasomes, we knocked down by STING activation seems to be involved in inhibiting the differen- siRNA GBP2 and the pool of GBP2, GBP3, and GBP5 in C57BL/ tiation of M2 macrophages in Brucella-infected cells that parallels 6 macrophages infected with Brucella. In this study, cells not with enhanced bacterial replication in STING KO cells. expressing GBP2 or the pool of GBPs produced much less IL-1b L. monocytogenes effectively short-circuits the cGAS cytosolic than did the cells transfected with control siRNA. Additionally, surveillance pathway by providing its own second messenger, GBP2 and GBP pool knocked-down cells had a reduced ability to whereas M. tuberculosis and L. pneumophila generate the type I control Brucella replication intracellularly. However, Brucella IFN signal via DNA binding to cGAS. Interestingly, Watson et al. infection observed in cells treated with the GBP pool was mark- (18) demonstrated that cGAS also plays no role in control- edly augmented compared with cells treated GBP2 only siRNA, ling M. tuberculosis infection. Additionally, S. typhimurium suggesting the GBP proteins work together nonredundantly to predominantly activates type I IFN via a cGAS/STING- control Brucella. 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