sensing via the TLR pathway is the driving force in miR-155 induction. It is important to note that F.t. has been well established to inhibit host immune response. It has been shown in the mouse that F.t. does not elicit any inflammatory cytokine production from dendritic cells [12]. We have previously found that F.t. does elicit some inflammatory cytokine production from monocytes, but to a far lesser degree than F.n. [11]. Just recently it has been reported that the difference in the ability of F.t. to induce inflammatory cytokine production between human dendritic cells and mono- cytes is due to differences in CD14 expression on these cells. So that monocytes which have high CD14 expression are capable of eliciting some response whereas dendritic cells are not [50]. In any case, the ability of host cells to mount an effective immune response to F.t. is impaired by its highly subversive nature. Finding that the less virulent F.n. can strongly induce miR-155 and down-regulate SHIP whereas the highly virulent F.t. elicits a suboptimal miR-155 response and an inability to down-regulate SHIP makes miR-155 of compelling interest for studies of host response to microbial pathogens. We have recently examined the mechanisms by which F.t. subverts host response [11] and found that it preferentially down-regulates both TLR2 and components of the PI3K/Akt pathway, whereas F.n. does not. This suggests a mechanism by which F.t. may prevent miR-155 induction, as TLR2 and PI3K/Akt are components of the miR-155 induction pathway by Francisella. In the current study we found that miR-155 positively regulates TNFa and IL-6 production in response to Francisella. Given that miR-155 has been shown to target the negative regulator SHIP and that SHIP negatively regulates NFkB activation and pro- inflammatory cytokine production, we conclude that miR-155 is pro-inflammatory within the context of Francisella infection. Indeed, SHIP has already been shown to negatively regulate neutrophil responses to TLR2 activation via peptidoglycan [51] and macrophage responses to Francisella infection [17]. Further, miR-155 over-expressing mice [52] develop a similar myelopro- liferative phenotype to that of SHIP2/2 mice [53]. Hence, it appears that as with miR-155 expression itself, numerous specific factors can influence the nature of response to miR-155. In conclusion, this report demonstrates that miR-155 is induced by Francisella and that the host response against this pathogen is enhanced through the targeting of SHIP by miR-155. Further, the differential induction of miR-155 by virulent versus less virulent Francisella subspecies suggests that differences in miR responses may at least partially explain the highly pathogenic nature of F.t. versus F.n. Further studies are required to determine the exact cause(s) of this differential response, such as whether it is completely attributable to TLR2 and PI3K/Akt regulation and if so, how these are down-regulated by the virulent Francisella. To our knowledge, this is the first study to describe differential regulation of miR-155 by two bacteria of the same subspecies. We find that miR-155 induction inversely correlates with virulence in human monocytes. Materials and Methods Cell and Reagents THP-1 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in RPMI-1640 (Gibco-BRL, Rockville, MD) supplemented with 5% heat- inactivated fetal bovine serum (FBS) (HyClone, Logan, UT), L- glutamine, penicillin (10,000 U/ml) and streptomycin (10,000 mg/ ml) (Invitrogen, Carlsbad, CA). CHO cells were obtained from the ATCC and cultured as previously described by [26]. The MiR155 Regulates Host Response BAY7085 IKK inhibitor and Sn50 NFkB peptide inhibitor were a generous gift from Dr. Denis Guttridge (The Ohio State University). LY294002 (20 mM), U0126 (2.5 mM) and SP00125 (5 mM) were obtained from Calbiochem (San Diego, CA). SB203580 (5 mM) and DMSO vehicle control (0.2%) was obtained from Sigma-Aldrich (St. Louis, MO). Cytochalasin-D was purchased from Biosource (Camarillo, CA) and used at a concentration of 5 mg/ml. Transgenic and Knockout Mice The C57BL/6J wild-type and TLR2 signaling mutant mice (TLR2tm1kir/J) were obtained from The Jackson Laboratory (Bar Harbor, MA). Wild-type and miR-1552/2 (B6.129S7- Mirn155tm1Brd) mice were obtained from Mutant Mouse Regional Resource Centers (MMRRC) (Columbia, MO). Bone Marrow-Derived Macrophages Mice were sacrificed and the femurs were removed. Bone marrow was flushed from the femurs and the cells were cultured in DMEM (Invitrogen, Carlsbad, CA) with 10% heat-inactivated FBS, 30% sterile filtered L-cell conditioned media, 0.1% b- mercaptoethanol (BioRad, Hercules, CA), and penicillin/strepto- mycin for six to seven days. The murine fibroblast cell line L929 was a generous gift from Dr. Ste´phanie Seveau (The Ohio State University). L929 cells were grown to confluence in minimum essential media (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), non-essential amino acids, sodium pyruvate and penicillin/streptomycin (Invitrogen, Carlsbad, CA). Conditioned media from the L929 cells was collected, passed through a 0.2 mm filter and used to supplement the murine macrophage cultures. After 6 to 7 days of culture, BMM were washed 3 times in sterile PBS, scraped, then plated overnight in 12-well tissue culture plates. Purity of harvested macrophages was verified though CD11b+ staining by flow cytometry. Peripheral Blood Monocyte Isolation Human peripheral blood monocytes (PBM) were isolated by centrifugation through a Ficoll gradient followed by CD14-positive Magnet-Assisted Cell Sorting (MACS, Miltenyi Biotec, Auburn, CA) according to manufacturer instructions as previously described [11]. Flow cytometry using CD14 antibody showed a minimum of 98% purity for each sample. PBM were maintained in RPMI-1640 containing 10% heat-inactivated FBS and L-glutamine. Bacterial Infections All infections were conducted in 5% or 10% FBS-containing RPMI-1640 without antibiotic. F. novicida U112 (JSG1819) and F. tularensis subspecies tularensis (SCHU S4) were generously provided by Dr. John Gunn (OSU), and grown on Chocolate II agar plates (Becton, Dickinson and Company, Sparks, MD) at 37uC. SCHU S4 infections were conducted by CDC approved select agent users at The Ohio State University BSL3 Select Agent facility in accordance with the BSL3 bio-safety plan. All SCHU S4 strain infected samples and matched reference samples were decontam- inated in accordance with approved protocols by the BSL3 advisory committee to ensure effective killing of microbes before removal from the facility. MOI was determined by optical density at 600 nm and verified by plating the inoculum overnight and counting CFU as done previously [11]. Heat-killed F.t. novicida were prepared by heating at 98uC for 10 minutes. Paraformalde- hyde (PFA)-killed F.n. were prepared by treating with 4% PFA for 30 minutes and then washing with PBS three times to remove PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8508