MyD88 and STING Signaling Pathways Are Required for IRF3-Mediated IFN-b Induction in Response to Brucella abortus Infection Leonardo A. de Almeida1, Natalia B. Carvalho1, Fernanda S. Oliveira1, Thais L. S. Lacerda1, Anilton C. Vasconcelos2, Lucas Nogueira4, Andre Bafica4, Aristo´ bolo M. Silva3, Sergio C. Oliveira1* 1 Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte-Minas Gerais, Brazil, 2 Department of Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte-Minas Gerais, Brazil, 3 Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte-Minas Gerais, Brazil, 4 Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis-Santa Catarina, Brazil Abstract Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-b in macrophages and splenocytes. Further, IFN-b induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-b expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-b and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-abR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-c and NO production by IFN-abR KO spleen cells and reduced apoptosis. Citation: de Almeida LA, Carvalho NB, Oliveira FS, Lacerda TLS, Vasconcelos AC, et al. (2011) MyD88 and STING Signaling Pathways Are Required for IRF3- Mediated IFN-b Induction in Response to Brucella abortus Infection. PLoS ONE 6(8): e23135. doi:10.1371/journal.pone.0023135 Editor: Dario S. Zamboni, University of Sa˜o Paulo, Brazil Received March 28, 2011; Accepted July 7, 2011; Published August 2, 2011 Copyright: 2011 de Almeida et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Brazilian Council of Science and Technology (CNPq), CNPq/ANPCyT (# 490528/2008-2), Research Foundation of the State of Minas Gerais (FAPEMIG), Brazilian Agency for Development of Graduate Courses (CAPES/PNPD), CNPq/MAPA, CNPq/REPENSA and National Institute of Science and Technology on Vaccines (INCTV) grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: scozeus@icb.ufmg.br Introduction Brucella abortus is a Gram-negative, facultative intracellular coccobacillus which causes brucellosis in humans and in cattle. In humans B. abortus causes undulant fever, endocarditis, arthritis and osteomyelitis and, in animals, it leads to abortion and infertility resulting in serious economic losses [1,2]. The protective response against B. abortus infection requires CD4+ and CD8+ T lymphocytes, Th1-type cytokines such as interferon-gamma (IFN- c) and tumor necrosis factor (TNF-a), and activated macrophages and dendritic cells [3–5]. The innate immune system is the first line of defense mechanisms that protect hosts from invading Brucella. It begins with the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The best characterized PRRs known to recognize Brucella PAMPs are the Toll-like receptors (TLRs), which are transmem- brane receptors that sense lipids, lipoproteins, proteins and nucleic acids [6–8]. This recognition activates intracellular signaling pathways that culminate in the induction of inflammatory cytokines, chemokines, interferons and upregulation of co- stimulatory molecules [9]. It has been shown that Brucella is recognized by several TLR-associated pathways triggering proin- flammatory responses that impact both the nature and the intensity of the immune response [10]. Since their discovery nearly 50 years ago, IFNs have been described as products of virus-infected cells capable of inducing an antiviral state in neighbouring cells through a paracrine signaling [11]. Conversely, type I IFNs have been traditionally assigned a minor role in antibacterial host defenses [12]. Recently, however, conserved bacterial products, such as LPS and DNA, were found to activate distinctive signal transduction pathways compared to those activated by viruses and lead to high levels of type I IFN production [13]. Moreover, the role of type I IFN in antibacterial defenses is still controversial. The induction of type I IFN during some intracellular Gram-positive bacterial pathogens infection appears to be detrimental to the host. In IFN- a/b-receptor KO mice infected with Listeria monocytogenes the titer of bacteria recovered from the liver and spleen was 102–103-fold lower than in the wild type mice [14,15]. Another intracellular bacterium that induces high levels of type I IFN is a hypervirulent PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23135