IFN-b Induction by Brucella Requires MyD88/STING Figure 9. IFN-b expression induced by B. abortus or its DNA requires RNA polymerase III and the adaptor molecule STING. Raw 264.7 macrophages were treated with ML-60218 (RNA polymerase III inhibitor) for 10 hours and stimulated with B. abortus strain S2308 (103 bact/cell), with Brucella DNA (1 mg/56105 cells), Brucella DNA treated with DNAse (0.2 U/mg DNA), poly I:C (10 mg/ml) and medium alone. For STING knockdown, macrophages were treated with non-targeting control siRNA, a specif-targeting STING or non-treated control. After three hours of stimulation total RNA was harvested and mRNA levels of IFN-b were determined by real-time RT-PCR and normalized to b-actin. Error bars represent the mean 6SD of samples assayed in duplicate. Similar results were obtained in three-independent experiments. Statistically significant difference of IFN-b levels from Brucella- or its DNA-stimulated cells compared to unstimulated cells is denoted by an asterisk and from non-treated (NT) macrophages compared to siSTING or ML-60218 treated cells is denoted by two asterisks (p,0.05). doi:10.1371/journal.pone.0023135.g009 Furthermore, we have compared the IFN-b expression induced by Brucella DNA, the lipoprotein Omp19 and its LPS. Our data supports that Brucella DNA is a major bacterial component to induce IFN-b in BMDM. Regarding TLR recognition of Brucella, we and others have shown previously that TLR2, TLR4 and TLR9 recognize bacterial components and induce pro-inflamma- tory cytokine production [6–8]. More recently, we have shown that the MyD88 adaptor molecule is crucial to host control against Brucella infection [9]. In this study, we aimed to examine the role of TLRs and their contribution to the Brucella-induced IFN-b response. Overall, the fact that expression of IFN-b was unimpaired in TLR2, TLR4 and TLR9 KO BMDM during infection strongly indicated that Brucella- or its DNA-induced IFN- b expression occurs mostly independent of TLRs. Surprisingly, type I IFN expression by Brucella or its DNA was dependent on MyD88 in BMDM. A pathway involving MyD88 and an unknown PRR recognizing various Gram-positive bacteria such as Streptococcus and Bacillus anthracis has been previously reported [51,52]. However, to the best of our knowledge, activation of a MyD88-dependent, TLR2/TLR4/TLR9-independent signaling pathway is quite unique in Gram-negative bacteria. Recently, Lapaque et al [53] have reported that Brucella-induced IRGs expression (Irga6 and Irgm3) requires type I IFN production. Similarly to our findings, these authors revealed that Irga6 and Irgm3 induction by B. abortus is MyD88-dependent but TLR2/ TLR4/TLR5/TLR9-independent. Since the adaptor molecule TRIF is involved in type I IFN production, we also decided to investigate whether Brucella could induce IFN-b expression through TRIF [54]. As shown in Figure 8A, Brucella or its DNA induces IFN-b expression in a TRIF-independent manner. Additionally, we addressed whether the adaptor molecule TRIF was involved in Brucella infection in vivo. Our results demonstrated that TRIF played no role in host control of B. abortus. Brucella enters the host cell, prevents fusion of the phagosome with the lysosome by altering the intracellular traffic of the early phagosome vesicle [55] being located in structures that resemble the endoplasmic reticulum [56]. Therefore, DNA from dead Brucella is available in this endoplasmic reticulum-like organelle and/or escape to the cytosol compartment being available to bind to a cytosolic DNA sensor. Additionally, Brucella abortus expresses the virB T4SS that plays a crucial role in the bacterial replication [32]. Bacteria use type IV secretion systems for genetic exchange and to deliver effector molecules to eukaryotic target cells. In addition to DNA from dead Brucella that can be released in the BCV (Brucella-containing vacuole) and/or escape to the cytosol compartment being available to bind to a cytosolic DNA sensor, there is the possibility of the Brucella DNA be released by the translocation through the type IV secretion system. Even though, the virB was not involved in host control of Brucella in IFN-abR KO mice, the role of the type IV secretion system in releasing bacterial DNA in the cytosol remains to be fully characterized during B. abortus infection. Furthermore, the cellular sensors that detect DNA and triggers type I IFN production have remained largely unknown. The cytosolic receptors which are capable of sensing pathogen nucleic acids released from lysed bacteria or from bacterial secretion system during infection are now under intensive investigation [13]. Regarding that, retinoic acid inducible gene I (RIG-I), melanoma differentiation 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]. In this study, our findings are consistent with the hypothesis that RNA polymerase III detects some segments of Brucella DNA and transcribes them into RNA ligands that induce IFN-b possibly through the RIG-I pathway. Since, inhibition of RNA polymerase III enzymatic activity totally abrogated IFN-b expression induced by Brucella DNA. These results strongly suggest that RNA polymerase III plays a key role in sensing bacterial DNA during B. abortus infection. Furthermore, an endoplasmic reticulum resident transmembrane protein termed STING (stimulator of interferon genes) has been identified as an adaptor required by RIG-I to induce type I IFN in response to L. monocytogenes and C. muridarum [26,27]. In order to determine whether STING plays any role in Brucella sensing, we have used siRNA technology. siRNA silencing demonstrated that STING is also an important mediator of IFN-b induced by Brucella or its DNA functioning PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23135