IFN-b Induction by Brucella Requires MyD88/STING Figure 5. Brucella DNA is the major component responsible for IFN-b and TRAIL expression in macrophages. BMDM from 129Sv/Ev or IFN-abR KO mice were infected with B. abortus strain S2308 (103 bact/cell), stimulated with Brucella DNA (1 mg/56105 cells), Brucella DNA treated with DNAse (0.2 U/mg DNA), poly I:C (10 mg/ml) or medium alone. After that, total RNA was harvested and mRNA levels of IFN-b (A) and TRAIL (B) were determined by real-time RT-PCR and normalized to b-actin. Error bars represent the mean 6SD of samples assayed in triplicate. Similar results were obtained in two-independent experiments. Statistically significant difference of IFN-b or TRAIL levels from stimulated macrophages versus non- stimulated cells is denoted by an asterisk, from IFN-abR KO mouse macrophages compared to wild type is denoted by two asterisks and from Brucella DNA compared to Brucella DNA treated with DNAse with three asterisks (p,0.05). doi:10.1371/journal.pone.0023135.g005 were found in IFN-abR KO mice when compared to the wild type control animals. In contrast to our findings, Roux et al [31] have shown that Brucella cfu recovered from IFN-abR KO mice compared to parental BALB/c mice showed no significant differences during the first 4 weeks of infection. The differences observed in these studies may be related to the mouse strain used, since we have worked with 129Sv/Ev background and Roux et al [31] have used BALB/c mice. Further, the virB T4SS plays a Figure 6. STAT1 phosphorylation induced by B. abortus or its DNA is impaired in mice lacking IFN-ab receptor. BMDMs from wild type (129Sv/Ev) and IFN-abR KO mice were stimulated for 6 and 12 hrs with B. abortus S2308 (103 bact/cell) or Brucella DNA (1 mg/56105 cells). Cell lysates were analyzed by immunoblotting using anti-STAT1 and anti-phospho-STAT1 at tyrosine 701 residue. One experiment is shown representative of three. doi:10.1371/journal.pone.0023135.g006 crucial role in Brucella intracellular replication [32]. Since the virB T4SS system secretes effector proteins able to modify host cell functions, we also investigated whether virB was involved in Brucella host control in IFN-abR KO mice. As demonstrated in Figure 2, similarly to observed to virulent strain 2308, IFN-abR KO mice infected with Brucella DvirB mutant showed reduced bacterial burden compared to wild type mice. The in vivo mechanisms by which type I IFN signaling increases host susceptibility to bacterial infection remain unclear. One possible mechanism is that induction of type I IFN by Listeria was shown to suppress macrophage activation by reducing the ability to respond to IFN-c down-regulating IFN-cR expression [44]. Mice lacking IFN-abR have increased expression of IFN-cR and their reduced susceptibility to L. monocytogenes infection is dependent on IFN-c. Herein, we observed that splenocytes from Brucella-infected IFN-abR KO mice produced higher amounts of IFN-c and NO, two critical components to host control of this bacterial infection [45]. Therefore, this mechanism of antagonistic cross-talk between type I and II IFNs might be an important aspect involved in IFN-abR KO mice resistance to Brucella infection. Additionally, abundant type I IFN predisposes lympho- cytes to apoptosis during Listeria infection and it has a negative effect on bacterial handling in the mouse [46]. In this study, we have observed that Brucella-infected 129Sv/Ev mice induced higher percentage of apoptotic spleen cells as demonstrated by PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23135