IFN-b Induction by Brucella Requires MyD88/STING Figure 3. B. abortus induced-apoptosis depends on type I IFN signaling. (A) Spleens from 129Sv/Ev or IFN-abR KO mice after two-weeks of infection with B. abortus were removed and tissue sections were analyzed to determine the apoptotic cells by TUNEL assay as described in material and methods. (B) This graph demonstrates the % of TUNEL-positive cells shown in panel A. (C) Annexin V+/7-AAD2 analysis of spleen cells of Brucella- infected 129Sv/Ev or IFN-abR KO mice performed by flow cytometry. (D) The expression levels of TRAIL were measured by real-time PCR in spleens of Brucella-infected 129Sv/Ev or IFN-abR KO mice. Similar results were obtained in two-independent experiments. Error bars represent the mean 6SD. Statistically significant difference from Brucella-infected and uninfected mice is denoted by an asterisk and between IFN-abR KO mice compared to wild type is denoted by two asterisks (p,0.05). doi:10.1371/journal.pone.0023135.g003 pharmacological inhibitor of RNA polymerase III or siRNA technology for STING was used. As demonstrated in Figure 9, the use of RNA polymerase III inhibitior (ML-60218) at 0.1 or 1.0 mM of concentration dramatically reduced IFN-b mRNA during Brucella infection of macrophages (83%) or during stimulation with its DNA (100%). This finding demonstrates that RNA polymerase III-dependent pathway plays an important role in IFN-b induction by Brucella, probably transcribing bacterial DNA to be recognized by a yet unknown receptor. Furthermore, we tested the role of STING in Brucella-induced IFN-b expression. By real-time RT-PCR analysis, it can be observed that by silencing STING resulted in a significant decrease in IFN-b mRNA levels during B. abortus infection of macrophages (55%) or upon macrophage stimulation with bacterial DNA (93%) compared to siRNA non-treated cells. This result demonstrates that STING is a crucial adaptor molecule of host cell involved in IFN-b induction by Brucella. Discussion Although type I IFNs are well known to induce a robust antiviral host response, the role of type I IFNs in response to bacterial infection is variable and even it is sometimes detrimental to the host. For instance, IFN-abR KO mice exhibit lower L. monocytogenes burdens in the liver and spleen as compared with wild type mice [14,15,36]. Type I IFN signaling is also detrimental to the clearance of M. tuberculosis infection from the spleen [37] and the lung [38]. Furthermore, type I IFN impairs clearance of Chlamydia from the genital tract and lungs [17,39], and it is detrimental to the host during infection with T. whipplei [30]. In contrast, type I IFN is crucial for host resistance to some bacterial infections. For example, IFN-abR KO mice exhibit decreased survival and increased bacterial burdens upon infection with Group B Streptococcus, Streptococcus pneumoniae and Escherichia coli [20]. Additionally, type I IFN also plays a role in restricting L. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23135