MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response Thomas J. Cremer1, David H. Ravneberg2, Corey D. Clay2, Melissa G. Piper-Hunter3, Clay B. Marsh3, Terry S. Elton4, John S. Gunn 5 , Amal Amer2,3,5, Thirumala-Devi Kanneganti6, Larry S. Schlesinger2,3,5, Jonathan P. Butchar3, Susheela Tridandapani1,2,3,5* 1 Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America, 2 Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, Ohio, United States of America, 3 Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America, 4 College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America, 5 Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, United States of America, 6 St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America Abstract The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/ macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 39UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro- inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro- inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down- regulation and pro-inflammatory response and may account for the virulence of Type A Francisella. Citation: Cremer TJ, Ravneberg DH, Clay CD, Piper-Hunter MG, Marsh CB, et al. (2009) MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response. PLoS ONE 4(12): e8508. doi:10.1371/journal.pone.0008508 Editor: Robin Charles May, University of Birmingham, United Kingdom Received May 22, 2009; Accepted December 1, 2009; Published December 30, 2009 Copyright: 2009 Cremer 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 sponsored by the National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE) Program. The authors wish to acknowledge membership within and support from the Region V ‘Great Lakes’ RCE (NIH award 1-U54-AI-057153). This work was also supported by RO1 AI059406 and P01 CA095426. J.P.B. is supported by the American Cancer Society Ohio Division Postdoctoral Fellowship. T.J.C. is sponsored by NIH/NIAID award # 1-T32-AI-065411, a National Research Service Award (NRSA) training grant administered by the Center for Microbial Interface Biology (CMIB), at The Ohio State University. 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: tridandapani.2@osu.edu Introduction Francisella tularensis is a highly infectious Gram-negative bacterium that infects phagocytic cells of the immune system [1–3]. Exposure to this pathogen causes the disease known as tularemia and a dose of as few as ten colony forming units can be lethal to humans [4,5]. Thus, the CDC has classified this pathogen as a Category A select agent [6]. There are five known subspecies of Francisella [5,7], and the subspecies novicida (F.n.) is less infectious to humans than the Type A F. tularensis subspecies tularensis (F.t.). However, F.n. provides an excellent model organism because it shares a similar intracellular lifecycle to F.t. and leads to tularemia- like pathologies in mouse models [4]. Effective host cell defense against Francisella is subverted by numerous mechanisms. Indeed, interferon response [8], toll-like receptor (TLR) signaling [9–11], and antigen presentation [11,12] are all found to be compromised by Francisella. Although Gram- negative, Francisella presents a modified form of lipopolysaccharide (LPS) that only minimally activates TLR4 [10,13]. Instead, most of the cell surface-driven host response is through TLR2 [14,15] and finding ways to enhance the response are of interest for novel therapeutics [16]. Understanding these host responses and how Francisella undermines them is critical for our ability to successfully prevent and treat infection. One important response downstream of TLR stimulation is activation of the PI3K/Akt pathway [17,18]. Our laboratory has shown that PI3K/Akt activation is host-protective against Francisella, as mice expressing a macrophage-specific, constitutive- ly-active form of Akt are protected from an otherwise-lethal challenge compared to wild-type littermates [18]. Expectedly, the PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8508