strain of Mycobacterium tuberculosis. It was showed that mice infected with this M. tuberculosis strain died at faster rate due to failure to induce Th1 cellular mediated immunity [16]. The lack of development of Th1 immunity in response to this bacterium appears to be associated with increased induction of type I IFNs. In the Chlamydia muridarum infection model, IFN-a/b-receptor KO mice were found to be more resistant to infection than wild type mice [17]. The observed susceptibility of the wild type mice to C. muridarum was due to the induction of type I IFNs resulting in enhanced apoptosis of lung infiltrated macrophages. However, there are other bacterial infection models such as Legionella pneumophila, Bacillus anthracis and Group B Streptococcus where the function of type I IFNs are protective for host suppressing bacterial invasion due the enhancement of IFN-c/NO produc- tion [18–20]. Although TLRs are the best characterized innate receptors related to recognize and to trigger a signaling for an efficient response against pathogens, they are only a piece of a broad repertoire of molecules capable to induce cellular signaling to fight against a microbial infection. Among them, the cytosolic receptors which are capable of sensing pathogen nucleic acids released from lysed bacteria or from bacterial secretion system during infection have been intensively studied [13]. Regarding that, retinoic acid inducible gene I (RIG-I), melanoma differen- tiation associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), members of RLR family were reported to recognize viral RNA and trigger an anti-viral response inducing type I IFN [21]. Few reports are available describing the role of RLR receptors recognizing bacterial RNAs. Since bacterial mRNAs are not capped and can contain 59 triphosphate, they can induce type I interferon throughout RIG-I signaling [22]. In independent studies, Ablasser and colleagues [23], and Chiu et al. [24] demonstrated that RNA polymerase III transcribes highly AT-rich bacterial DNA present in the host cell cytosol to 59- triphosphate RNA which is preferentially recognized by RIG-I [25]. The downstream signaling mediated by RIG-I requires the protein adaptor IPS-1 (MAVS, Cardiff or VISA), which coordinates the activation of IRF3/7, NF-kB and MAP kinases [21]. In addition to IPS-1, another molecule termed stimulator of interferon genes (STING) has been identified as an adaptor required by RIG-I to induce type I IFN in response to Listeria monocytogenes and C. muridarum [26,27]. Furthermore, other groups have attempted to find a yet undefined bacterial DNA sensor that can signal through a TLR9-independent and RLR-independent pathways [28,29]. In this study, we have shown that B. abortus induced IFN-b in macrophages and splenocytes. To gain insights into the molecules involved in IFN-b synthesis we defined that Brucella DNA is a potent stimulus for this cytokine expression. In addition, IFN-b induced by Brucella requires IRF3 and STAT1 phosphorylarion at tyrosine 701. Further, Brucella or its DNA induces IFN-b independent of TLRs and TRIF signaling but dependent on MyD88. Regarding, the putative cytosolic sensors involved in Brucella DNA recognition, we demonstrated here that RNA polymerase III and the adaptor molecule STING are critical components required for IFN-b signaling pathway. Additionally, we have demonstrated that IFN-abR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype seems to be related to increased IFN-c and NO production by IFN-abR KO spleen cells and reduced apoptotic profile. In summary, these findings propose a novel IFN-b pathway induced by a Gram- negative bacterial DNA that requires the engagement of RNA polymerase III and STING. IFN-b Induction by Brucella Requires MyD88/STING Results Brucella induces IFN-b in macrophages and splenocytes To investigate whether B. abortus induces type I IFN, macrophages or spleen cells were exposed to bacteria and IFN-b was measured at several time points. Wild type (129Sv/Ev) or IFN-abR KO macrophages as well as splenocytes infected with B. abortus strain 2308 displayed increased expression of IFN-b as measured by ELISA or real-time RT-PCR, respectively. In infected BMDM, maximal IFN-b responses were observed at 72 hrs post-infection (Fig. 1A). Curiously, IFN-abR KO macro- phages produced greater amounts of IFN-b compared to the wild type cells. This process is probably a compensatory mechanism due to the lack of the type I IFN receptor. Furthermore, fifteen days after infection IFN-b transcripts were measured in splenocytes by real-time RT-PCR (Fig. 1B) and IFN-b production by ELISA (Fig. 1C). Similarly to what was observed in macrophages, IFN-b was higher in IFN-abR KO spleen cells compared to 129Sv/Ev mice. These findings demonstrate that B. abortus induces IFN-b production in immune cells. Absence of type I IFN signaling enhances host protection to Brucella infection To study the role of type I IFN in host response against Brucella infection, we infected IFN-abR KO and 129Sv/Ev mice with 16106 B. abortus strain 2308. Comparison of colony-forming units (cfu) recovered from the spleens of IFN-abR KO and 129Sv/Ev mice fifteen days post-infection showed reduced bacterial numbers in type I IFN receptor deficient mice (Figure 2A). Similar findings demonstrating the detrimental role of type I IFN signaling to host control of bacterial infection were also observed for L. monocytogenes, Tropheryma whipplei and C. muridarum [14,15,17,30]. Since Brucella mutant to the type IV secretion system (T4SS, virB mutant) fails to elicit IFN-induced gene expression during early infection [31], we decided to also evaluate the role of T4SS during Brucella infection in IFN-abR KO mice. As observed in Brucella strain S2308 expressing T4SS infection, virB mutant showed reduced cfu counts in type I IFN receptor deficient mice compared to the wild type (Figure 2A). However, the reduced numbers of virB mutant cfu found in both mouse strains compared to parental strain 2308 is probably due to already described reduced virulence of this mutant [32]. These results demonstrated that T4SS plays no role in resistance of IFN-abR KO mice to B. abortus infection in vivo. To address whether increased resistance observed in IFN-abR KO mice was associated with pro-inflammatory mediators, we measured IFN-c, IL-17 and NO in spleen cells of type I receptor deficient mice and control animals. Elevated production of IFN-c and NO in Brucella-primed splenocytes in vitro stimulated with live strain 2308 was observed in receptor-deficient mice compared to the wild type (Figure 2B and 2C). Additionally, no difference in IL- 17 production was observed in IFN-abR KO compared to 129Sv/ Ev mice (Figure S1). Together, these findings suggest that type I IFN signaling induced by Brucella is detrimental to infection and regulate inflammatory components required for host protection. Brucella abortus-induced splenic apoptosis is dependent on type I IFN signaling One of the hallmark effects mediated by type I IFNs is the induction of apoptosis in infected cells which has been shown in the L. monocytogenes model to be associated with enhanced infection [14]. To assess whether B. abortus can also induce apoptosis, mice were infected and splenocyte apoptosis was determined by TUNEL assay and by flow cytometry using annexin V. We have observed increased apoptosis in the splenic tissue of 129Sv/Ev PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23135