probably downstream of the RIG-I pathway or of an unknown receptor. Since STING was found to basally reside in the endoplasmic reticulum, similarly to Brucella, this cell compartment would be an important site that facilitates STING to signal during this bacterial infection. A recent study demonstrated that the IFN- b response to L. monocytogenes was lost in STING KO mouse embryonic fibroblasts [26], and in mouse oviduct epithelial cells and human cells during C. muridarum infection when siRNA was used [27]. These findings reinforce STING as an important component of the TLR-independent interferon response during bacterial infection. In summary, our study demonstrated that Brucella induces IFN- b through IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. Additionally, IFN-b expression induced by Brucella is independent of TLRs but MyD88- dependent, pathway not yet described for Gram-negative bacteria. Further, being Brucella DNA a major bacterial component to induce IFN-b, our study suggests that this molecule is transcribed by RNA polymerase III to be sensed by an unknown receptor that signals via STING. To the best of our knowledge, this is the first report demonstrating that bacterial DNA can be sensed by RNA polymerase III/STING pathway to coordinate type I IFN production. Finally, type I IFN signaling enhances host suscepti- bility to B. abortus infection independently of bacterial T4SS. The mechanism involved in IFN-abR KO mice resistance is unknown but it seems to involves enhanced IFN-c and NO production and reduced apoptosis. Materials and Methods Ethics Statement This study was carried out in strict accordance with the Brazilian laws 6638 and 9605 in Animal Experimentation. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Federal University of Minas Gerais (Permit Number: CETEA89/2008). Mice, cell culture and bacteria IFN-abR KO mice were gifted by Dr. Luiz F. Reis (Ludwig Institute, Sa˜o Paulo, Brazil). TLR2, TLR4, TLR9 or TRIF KO mice were kindly provided by Shizuo Akira from Osaka University in Japan. The wild-type strains 129Sv/Ev or C57BL/6 mice were purchased from the Federal University of Minas Gerais animal facility (UFMG, Belo Horizonte, Brazil). Genetically deficient and control mice were maintained at UFMG and used at 6–8 week of age. Mouse RAW 264.7 macrophages (American Type Culture Collection, ATCC Nu TIB-71) were grown in Dulbecco’s Modified Eagle Medium (DMEM) high glucose containing 10% FCS and 1% penicillin G sodium (100 U/ml), and streptomycin sulfate (100 mg/ml). Bone marrow cells were obtained from femora and tibia of mice and they were derived in macrophages (BMDMs) as previously described [57]. B. abortus virulent strain 2308 was obtained from our own laboratory collection. The virB mutant strain was kindly provided by Dr. Renato de Lima Santos (UFMG, Belo Horizonte, Brazil). They were grown in Brucella broth medium (BD-Pharmingen, San Diego, CA) for 3 days at 37uC. Purification of genomic DNA from bacterial culture To purify Brucella genomic DNA, a modification of the method described by Wilson [58] was used. Briefly, cells were resuspended in 0.5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), heat killed at 80uC for 15 min, and incubated at 37uC for 1 h with 0.5% sodium dodecyl sulfate and proteinase K (200 mg/ml). Cell IFN-b Induction by Brucella Requires MyD88/STING wall debris, denatured proteins, and polysaccharides were removed by precipitation of the lysate with 5 M NaCl and CTAB-NaCl solutions at 65uC for 10 min. DNA was extracted by a standard protocol with phenol-chloroform-isoamyl alcohol, precipitated with isopropanol, washed with 70% ethanol, and dried. The DNA-containing pellet was reconstituted in 100 ml of nuclease-free water containing RNAse (50 mg/mL). The concen- tration and purity of the DNA were determined spectrophoto- metrically. Infection and Brucella counts in spleens Mice were infected i.p. with 106 CFU of B. abortus strain 2308. To determine residual Brucella CFU in the spleens of mice, five animals from each group were examined at 2 weeks after infection. Spleens from individual animals were homogenized in PBS, 10- fold serially diluted, and plated on Brucella broth agar (Difco, BD- Pharmingen, San Diego, CA). Plates were incubated at 37uC and the number of CFU was counted after 3 days as previously described [57]. IFN-c, IFN-b, IL-17 and NO production by splenocytes Splenocyte cultures from 129Sv/Ev or IFN-abR KO mice were stimulated by addition of 102 live B. abortus strain S2308 per cell or 1 mg/ml Escherichia coli LPS in a total volume of 200 ml of medium/well. Unstimulated cells were used as a negative control, and cells stimulated with Con A (5 mg/ml; Sigma-Aldrich, St. Louis, MO) were used as a T cell-activating control. Spleen cells were incubated at 37uC with 5% CO2. Levels of IFN-c or IL-17 in the supernatants were measured using a commercially available ELISA Duoset kit (R&D Systems, Minnesota, MN). IFN-b levels were measured by Mouse Interferon-b ELISA Kit (PBL Biomedical Laboratories, Piscataway, NJ). To assess the amount of NO produced, spleen cell culture supernatants from IFN-abR KO or 129 Sv/Ev mice stimulated with the same antigens described above were assayed for accumulation of the stable final product of NO, NO2 which was determined by the Griess reaction. Briefly, culture supernatants (50 ml) were mixed with 50 ml of Griess reagent (1% sulfanilamide, 0.1% naphthy- lethyline diamine dihydrochloride and 2.5% phosphoric acid) in triplicate in 96-well plates at room temperature for 5 min. The OD at 550 nm was then measured. NO2 concentration was determined by comparison with NaNO2 (Sigma, St. Louis, MO) as a standard. IFN-b production by BMDM and immunoblotting BMDM cells from 129Sv/Ev, IFN-abR or TRIF KO and C57BL/6, MyD88 or TLR9 KO animals completely differenti- ated into macrophages on day 10 of culture were stimulated with 103 B. abortus strain S2308 bacteria per macrophage. The levels of IFN-b in the supernatants of 24 hrs stimulated BMDMs were measured by Mouse Interferon Beta ELISA Kit (PBL Biomedical Laboratories, Piscataway, NJ). For immunoblotting analysis, BMDM cells from IFN-abR KO or 129 Sv/Ev mice were stimulated either with live B. abortus at 103 bacteria/cell or 1 mg of purified Brucella DNA (1 mg/56105 cells). After 6 or 12 hours, cells were lysed with M-PER extraction buffer (Pierce, Rockford, IL) containing a phosphatase inhibitor mixture (Calbiochem, Darm- stadt, Germany). Protein extracts (30 mg) were then separated by SDS-PAGE/NuPage system (Invitrogen Life Technologies, Carls- bad, CA), electroblotted onto a nitrocellulose membrane, and immunoblotted with specific antibodies for phospho-STAT1 (Y701) (1:1000; Cell Signaling, Boston, MA) or for total STAT1 (1:1000; Cell Signaling, Boston, MA). Antibody binding was detected with HRP-conjugated anti-IgG (1:2000; Cell Signaling, PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23135