IFN-b Induction by Brucella Requires MyD88/STING Figure 2. Bacterial burden, IFN-c and NO production in Brucella-infected 129Sv/Ev and IFN-abR KO mice. Five 129Sv/Ev or IFN-abR KO mice were infected i.p. with a dose of 106 CFU of B. abortus strain S2308 or DvirB mutant. (A) Spleens were harvested at fifteen days post-infection, and the number of CFU in disrupted tissue was determined by 10-fold serial dilution and plating. (B) Spleen cells from B. abortus S2308 infected mice were cultured with 102 bacteria/cell, ConA (5 mg/ml) or medium alone for 72 hrs and IFN-c was detected by ELISA. (C) Regarding nitric oxide, nitrite concentration was measured by Griess reaction (mM) using the same stimuli described for IFN-c measurements except the use of E. coli LPS (1 mg/ml) as positive control. Error bars represent the mean 6SD. Similar results were obtained in three-independent experiments. Statistically significant difference from IFN-abR KO mice compared to wild type is denoted by an asterisk (p,0.05). doi:10.1371/journal.pone.0023135.g002 BMDM. As shown in Figure 6, an increased STAT1 Tyr701 phosphorylation after B. abortus or its DNA stimulation was evidenced at 6 hrs but not at 12 hrs in 129Sv/Ev mice. In contrast, STAT1 phosphorylation was completely absent when IFN-abR KO BMDM were infected with Brucella or activated with its DNA. Brucella-induced IFN-b expression is TLR- and TRIF- independent but MyD88-dependent To determine the role of TLRs, TRIF and MyD88 signaling in IFN-b expression elicited by Brucella or its DNA, real-time RT- PCR was performed in TLR2, TLR4, TLR9, TRIF and MyD88 KO BMDM. As shown in Figure 7A and Figure 8A, IFN-b expression by B. abortus strain 2308 or its DNA is independent of TLR recognition and the adaptor molecule TRIF but dependent on MyD88 signaling. Our results demonstrated that in contrast to BMDM from wild type, TLRs KO or TRIF KO, IFN-b expression was totally abrogated in MyD88 BMDM infected with Brucella or stimulated with its DNA. To confirm these results we performed ELISA to measure IFN-b and we obtained similar results as observed for real-time PCR (Figure 7B and 8B). These findings suggest that other receptor signaling through MyD88 different from TLR2, TLR4 or TLR9 is involved in type I IFN response during B. abortus infection. Since the role of TRIF signaling during B. abortus infection in vivo has not been reported, we decided to determine the function of this adaptor molecule in host control of this bacterial infection. As shown in Figure 8C, fifteen days following infection with 16106 cfu of B. abortus strain 2308, bacterial numbers were similar in spleens of wild type and TRIF KO mice. Therefore, these results suggest that TRIF is not involved in the host control of B. abortus infection. Brucella-induced IFN-b is dependent on RNA Polymerase III and STING In addition to surface- or endosome-localized innate receptors, host cells also express several cytosolic sensors that induce type I IFN in response to nucleic acid ligands such as RNA and DNA [13]. However, the mechanisms by which bacteria stimulate cytosolic sensors are under investigation. Recently, Chiu et al. [24] reported that DNA could stimulate a RNA sensor. In this mechanism, RNA polymerase III transcribes cytosolic DNA thereby generating RNA ligands for RIG-I like receptors (RLR). In addition to the RNA polymerase III pathway, mouse and human cells have other cytosolic DNA sensors driving the transcriptional activation of type I IFN [29]. Further, STING (stimulator of interferon genes) was recently identified as a downstream signaling adaptor required for IFN-b induction in response to cytosolic DNA [26]. In order to address whether B. abortus- or bacterial DNA-induced IFN-b expression in Raw 264.7 macrophages was dependent on RNA polymerase III or STING, a PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23135