IFN-b Induction by Brucella Requires MyD88/STING Figure 8. TRIF-mediated signaling is dispensable for IFN-b expression induced by B. abortus or its DNA. (A) BMDMs from TRIF KO and wild type (129Sv/Ev) 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 PCR and normalized to b-actin. Error bars represent the mean 6SD of samples assayed in triplicate. Similar results were obtained in three-independent experiments. Statistically significant difference of IFN-b levels from stimulated macrophages versus non-stimulated cells is denoted by an asterisk, from wild type mice to TRIF KO with two asterisks and from Brucella DNA compared to Brucella DNA treated with DNAse with three asterisks (p,0.05). (B) BMDM cells from TRIF KO and wild type (129Sv/Ev) 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 TRIF KO compared to the wild type mice by two asterisks. Error bars represent the mean 6SD of samples assayed in triplicate. (C) Five 129Sv/Ev and TRIF KO mice were infected i.p. with a dose of 106 CFU of B. abortus strain S2308. 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. Similar results were obtained in three-independent experiments. doi:10.1371/journal.pone.0023135.g008 Brucella-infected macrophages. Next, we reasoned whether B. abortus induces IFN-inducible genes via a type I IFN autocrine loop after engagement of the type I receptor. Thus, we monitored STAT1 activation, one outcome of IFN secretion and type I IFN receptor engagement. Brucella-infected wild type BMDM showed an increased in STAT1 tyrosine 701 at 6 hrs post-infection. In contrast, STAT1 tyrosine 701 phosphorylation was totally abrogated in IFN-abR KO macrophages infected with Brucella. Collectively, these findings demonstrate that B. abortus induction of IFN-b occurs through an IRF3-dependent pathway and that subsequent STAT1 activation via IFN-abR leads to a positive feedback loop involved in BMDM response to Brucella. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23135