Figure 1. Brucella induces IFN-b in macrophages and spleen cells. (A) BMDM from 129Sv/Ev or IFN-abR KO mice were infected with B. abortus strain S2308 (103 bact/cell) and IFN-b was measured by ELISA at 24, 48 and 72 hours. (B) Spleen cells from B. abortus infected mice at fifteen days post-infection were isolated and total RNA was harvested and mRNA levels of IFN-b were determined by real-time RT-PCR and normalized to b-actin. (C) Spleen cells from B. abortus infected mice at fifteen days post-infection were cultured with 102 bacteria/cell for 48 hrs and IFN-b was detected by ELISA. Error bars represent the mean 6SD. Similar results were obtained in two-independent experiments. Statistically significant difference of IFN-b levels from IFN-abR KO mice compared to wild type is denoted by an asterisk (p,0.05). doi:10.1371/journal.pone.0023135.g001 infected mice compared to uninfected controls as indicated by TUNEL+ cells (Figure 3A and 3B). However, Brucella-infected IFN-abR KO mouse spleens exhibited a drastic reduction in splenic apoptosis compared to wild type controls. The results presented in Figure 3B as % of TUNEL-positive cells were determined by counting TUNEL+ cells in mouse spleen sections. These results were also confirmed by the percentage of Annexin V+/7AAD2 cells detected in spleens. IFN-abR KO splenocytes infected with Brucella did not show enhancement in the number of IFN-b Induction by Brucella Requires MyD88/STING Annexin V+/7-AAD2 cells compared to uninfected controls; however, it was observed in 129Sv/Ev mice (Figure 3C). In order to detect the type I IFN-stimulated genes (ISGs) up-regulated in spleens during Brucella infection, we measured the expression of TRAIL, IP-10 and IRF7 by real-time RT-PCR. Figure 3D shows that only the proapoptotic gene TRAIL was significantly up- regulated in Brucella-infected splenocytes from wild type mice and it was highly suppressed in IFN-abR KO mouse spleens. For IP-10 and IRF7, we did not observe statistically significant difference in gene expression in receptor-deficient mice compared to the wild type (data not shown). These results demonstrate that B. abortus- induced apoptosis in vivo is largely dependent on type I IFN signaling. Induction of IFN-b and TRAIL expression requires IRF3 The transcription factor IRF3 has been shown to mediate the induction of IFN-b gene in response to pathogens and TLR signaling [33]. To determine whether induction of IFN-b by B. abortus also involves IRF3, Raw 264.7 macrophages were treated with siRNA 1075 specific for IRF3 and the cells infected with Brucella S2308 or stimulated with poly I:C as positive control. Transfection of IRF3-specific siRNA in Raw 264.7 cells resulted in a dramatic reduction of IFN-b expression levels (90%) as determined by real-time RT-PCR (Figure 4A). Inhibition of IRF3 led to a profound reduction of IFN-b transcripts following B. abortus or poly I:C stimulation, compared to siRNA non-treated cells. This result identifies IRF3 as a component of the signaling pathway leading to type I IFN induction by B. abortus-infected macrophages. Similarly to what was observed for IFN-b, IRF3 inhibition resulted in a dramatic reduction in TRAIL expression (95%) in Brucella-infected macrophages (Figure 4B). Taken together, these findings demonstrated that IRF3 is a critical transcriptional regulator of IFN-b and TRAIL gene expression during B. abortus infection. Brucella DNA induces IFN-b and TRAIL expression in macrophages Recent evidences suggest that induction of type I IFN by bacteria is mediated by nucleic acids from different pathogens [34]. In order to identify specific Brucella components involved in IFN-b expression, we have tested Brucella DNA, Omp19 (outer- membrane lipoprotein) or LPS. As observed in Figures 5A and 5B, Brucella DNA induces high levels of IFN-b and TRAIL expression in 129Sv/Ev BMDM. In contrast, reduced transcripts for TRAIL but not for IFN-b were observed in DNA-exposed macrophages from IFN-abR KO mice. These results lead us to conclude that IFN-b expression induced by Brucella or its DNA occurs independently of type I IFN receptor signaling. Brucella Omp19 did not induce the expression of IFN-b and Brucella LPS induced very low levels of this cytokine mRNA (Figure S2). Treatment of Brucella DNA with DNase I abolished the stimulatory activity, a result suggesting that the DNA is the main ligand for an unknown receptor responsible for triggering the IFN-b response. STAT1 phosphorylation induced by Brucella or its DNA is dependent on type I IFN signaling Once produced type I IFNs can signal in an autocrine and paracrine manner leading to the phosphorylation-dependent activation of STAT1 through type I IFN receptor [35]. To determine whether B. abortus or its DNA induces STAT1 activation, we performed western blot analysis using specific antibodies targeting STAT1 phosphorylated at tyrosine 701 in PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23135