In summary we find that miR-155 is strongly induced in vitro and in vivo by F.n. through the TLR signaling pathway and NFkB activation; however, this does not require activation of the inflammasome. Intriguingly we found that the highly virulent F.t. leads to minimal induction of miR-155 as well as a lack of ability to downregulate SHIP expression and lower pro-inflam- matory cytokine production than F.n., which may contribute to the highly virulent nature of F.t. (Figure 7). Discussion In this study we demonstrated that miR-155 is highly induced by F.n. infection and that it targets SHIP to regulate the immune response against this pathogen. Specifically we have found a novel role for miR-155 as a positive regulator of pro-inflammatory cytokine response to Francisella. Over a short period of time, miR- 155 has emerged as an important player in B-cell lymphomas [36], immune response [24], and other functions [26,37,38]. The earliest studies of pathogen-induced miR-155 expression examined the avian leukosis virus (ALV). It was found that ALV induced high levels of BIC expression in infected chickens and that this was highly correlated with B-cell lymphoma [39,40]. However, the most intriguing relationship between miR-155 and viruses is that both the Kaposi’s-scarcoma-associated herpes virus [41] and the Figure 7. Model of miR-155 induction and function during Francisella infection. Francisella is recognized on the host cell surface by TLR2. The signal is transmitted through the adaptor protein MyD88. Subsequently MAPKs, PI3K and Akt are activated, which leads to enhanced NFkB activity, inflammatory cytokine production, and effective host response. SHIP negatively regulates the activation of Akt to prevent effective host response. During Francisella infection miR- 155 is induced through the TLR signaling pathway, PI3K/Akt, ERK, JNK, and NFkB. MiR-155 induction in turn down-regulates SHIP to promote the activation of the PI3K/Akt pathway and inflammatory cytokine production. The highly virulent F. tularensis suppresses or subverts the induction of miR-155 in human monocytes, while the relatively avirulent F. novicida does not. doi:10.1371/journal.pone.0008508.g007 MiR155 Regulates Host Response oncogenic Marek’s disease virus [42] encode functional orthologs of miR-155. This suggests that miR-155 expression may actually benefit certain viruses, and one report has supported a role for miR-155 induction to maintain latent viral genomes through the negative regulation of an established miR-155 target, IKKe [43]. Deletion of miR-155 leads to system-wide changes in immune function. For example, B and T cell responses are compromised in mice genetically deleted for miR-155 [44,45]. MiR-1552/2 mice vaccinated with tetanus toxin fragment C showed overall reduced IgM and switched antigen-specific antibody production from defects in B-cell function. MiR-1552/2 T cells also showed impaired IL-2 and IFNc production after immunization. The T cells also exhibited a bias toward a TH2 response, possibly due to the targeting of c-Maf. Antigen presentation is also affected, as miR-1552/2 dendritic cells were less able to activate T cells expressing a transgenic receptor for ovalbumin in the presence of that protein. Thus, miR-155 serves an essential role in multiple immune cell types. Here, we examined the role of miR-155 in monocytes and macrophages within the context of host response to bacterial pathogens. Previous studies demonstrated that PI3K/Akt pathway is host-protective against Francisella infection [18] and the finding that SHIP negatively regulates this pathway [17] led us to study the intricacies of SHIP regulation during Francsiella infection. As the currently-known regulators of SHIP did not seem to account for all of the changes seen in SHIP expression [46], we examined the possibility that microRNAs may play a role. We found that monocytes and macrophages express miR- 155 in response to Francisella infection and that this down- regulated SHIP. During preparation of this manuscript it was reported by O’Connell et al. that miR-155 can directly target SHIP [21]. Our data are consistent with their findings, so the importance of miR-155 induction in response to Francisella is well supported. Multiple TLR ligands are capable of inducing miR-155 [24], so it is logical that miR-155 induction in response to F.n. requires TLR2 and MyD88. This finding is of considerable interest because while there are additional receptors that are engaged and are of importance in the host response to Francisella, such as the complement receptor and mannose receptor [47–49], it appears that MyD88 and the TLR pathway is of critical importance for miR-155 induction. Downstream of MyD88 activation we found that ERK and JNK were partly required for miR-155 induction, which supports the finding by Yiu et al that found ERK- and JNK- dependent BCR induction of miR-155. In contrast to Yiu et al. but in agreement with Gatto et al. 2008, who used a viral protein to induce miR-155 in B-cells, we found that activation of NFkB was absolutely required for miR-155 induction by Francisella. It is then fitting that inhibition of PI3K blocked miR-155 induction, as PI3K controls NFkB activation in response to Francisella. Hence, it appears as though cell type, cellular context and/or the nature of stimulus may affect the intracellular machinery involved with miR- 155 expression. TNFa and IFNb are at least two cytokines produced during Francisella infection that may contribute to miR-155 induction [24]. However, it has already been shown that these cytokines work through the TNFa receptor, while direct TLR stimulation can induce miR-155 independently of TNFR [24]. This is likely the case with Francisella, as it has been shown to activate TLR2 [14]. Cytosolic sensing of Francisella by the inflammasome triggers multiple host response events [33,34]. Given that neither bacterial internalization nor caspase-1 was clearly required for miR-155 induction, we are lead to conclude that the cytosolic sensing of Francisella is of marginal importance and that the extracellular PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8508