IFN-b Induction by Brucella Requires MyD88/STING Figure 7. Brucella-induced IFN-b expression requires MyD88 signaling independently of TLR2, TLR4, and TLR9. (A) BMDM from TLR2, TLR4, TLR9 and MyD88 KO and wild type (C57BL/6) 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 24 hours, total RNA was harvested and mRNA levels of IFN-b were determined by real-time RT-PCR and normalized to b-actin. Statistically significant difference of IFN-b levels from stimulated macrophages versus non-stimulated cells is denoted by an asterisk, from MyD88KO compared to the other mice by two asterisks and from Brucella DNA compared to Brucella DNA treated with DNAse with three asterisks (p,0.05). (B) BMDM cells from TLR9 KO, MyD88 KO and wild type (C57BL/6) were infected with B. abortus strain S2308 (103 bact/cell), stimulated with Brucella DNA (1 mg/56105 cells), poly I:C (10 mg/ml) or medium alone were cultured for 24 hrs and IFN-b was detected by ELISA. Statistically significant difference of IFN-b levels from stimulated macrophages versus non-stimulated cells is denoted by an asterisk, from MyD88KO compared to the other mice by two asterisks. Error bars represent the mean 6SD of samples assayed in triplicate. Similar results were obtained in three-independent experiments. doi:10.1371/journal.pone.0023135.g007 TUNEL assay and by flow cytometry using annexinV+/7-AAD 2 . Conversely, mice lacking IFN-abR were more resistant to Brucella infection and displayed less apoptotic lesions as well as reduced expression of pro-apoptotic gene TRAIL than their wild type counterparts. We speculate here that type I IFN signaling enhances immune cells apoptosis; therefore, causing the increased susceptibility to Brucella. However, further experiments are required to support this hypothesis. Another possible mechanism by which type I IFN enhances host susceptibility to bacterial infection, relates to the observation that these cytokines stimulate the production of IL-27, a molecule that strongly suppresses IL- 17A production [47]. IL-17A produced by cd T cells appears to play an important role in restricting Listeria replication by orchestrating neutrophil responses [48]. In fact, IFN-abR KO mice induced more IL-17A in response to Francisella and Listeria infections [49]. We have investigated here whether Brucella regulates IL-17A production and we found that in bacteria- infected splenocytes the levels of IL-17A in IFN-abR KO mice were similar to wild type spleen cells (Figure S1). This result ruled out the possibility that lack of type I IFN signaling could enhance IL-17A production and host resistance to Brucella infection. Following stimulation with bacterial components, the constitu- tively expressed IRF3 is phosphorylated in the cytoplasm, dimerizes and then translocates into the nucleus to induce the transcription of type I IFN [50]. We therefore, determined the role of IRF3 in IFN-b and TRAIL expression in Raw 264.7 macrophages using siRNA technology. IFN-b and TRAIL expression were dramatically reduced when IRF3 transcription factor was silenced, identifying IRF3 as an important component of the signaling pathway leading to type I IFN induction by PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23135