PI3K/Akt pathway is subject to negative regulation and one of the key regulators is SHIP, which we have also found to be important during Franciesella infection. Murine bone marrow-derived macro- phages (BMM) lacking SHIP display increased NFkB activity and enhanced cytokine production [17], similar to the responses seen with constitutively active Akt [18]. More recently we have found that the loss of SHIP or constitutive activation of Akt promotes phagosome-lysosome fusion of Francisella in macrophages. Thus this pathway is important for intracellular control [19,20]. Collectively, these findings implicate the PI3K/Akt pathway and SHIP as critical regulators of the host-response to Francisella. While studying the role of SHIP during Francisella infection, we found that infection with the less virulent F.n. leads to SHIP down- regulation. In a search for mechanisms to explain this, we entertained the possibility that microRNAs may play a role. In fact during the preparation of this manuscript it was reported that SHIP is targeted by a microRNA, miR-155 [21]. MiRs are a recently-discovered means of regulating both transcript and protein levels of specific genes [22]. They are processed yet non- translated RNA molecules of approximately 22 base pairs in length that primarily target the 39untranslated region (UTR) of mRNA transcripts in a sequence-specific manner. Recent reports show that treatment with TLR ligands can induce miR-146 and miR-155 expression within macrophages [23,24]. Yet the significance of these findings within the context of a bacterial infection remains largely unknown; however, is of great interest [20]. MiR155 Regulates Host Response Here, we show that Francisella infection induces miR-155 expression in a TLR-dependent manner and that this leads to down-regulation of SHIP. Notably, the less virulent F.n. subspecies strongly induces miR-155 while the virulent F.t. isolate SCHU S4 does not. Hence, expression of miR-155 is a component of host defense against Francisella and the difference in miR-155 response to these two subspecies may help explain the success of F.t. as an infectious agent in humans. Results SHIP Expression Is Down-Regulated in Response to F.n. Infection We have previously demonstrated that the pro-inflammatory cytokine response to Francisella infection requires the activation of the PI3K/Akt pathway and that this response is down-regulated by SHIP, an established negative regulator of the PI3K/Akt pathway [17,18]. To follow up these findings, we examined the expression of SHIP during F.n. infection. Here, human PBM were infected with F.n. (MOI 50) for 24 hours and SHIP protein levels were analyzed by Western blotting. Results, shown in Figure 1A, demonstrate a dramatic down-regulation of SHIP in infected cells versus uninfected controls. The lower panel is a reprobe of the same membrane with actin antibody to ensure equal loading of protein in both lanes. Of note, viability of PBM at 24 hours post infection was greater than 90%. Similar down-regulation of SHIP Figure 1. SHIP expression is down-regulated in response to F.n. infection. (A) PBM were infected with F.n. at an MOI of 50 for 24 hours. Cell lysates were analyzed by Western blotting use an anti-SHIP antibody in the top panel. ‘R’ designates resting/uninfected cells and ‘24 h’ designates infected with F.n. The lower panel is a reprobe of the same membrane with an anti-actin antibody. (B) THP-1 were infected with F.n. at an MOI of 100 for 24 hours. Cell lysates were resolved as in Figure 1A. (C) Western blots for SHIP in murine bone marrow-derived macrophages infected with F.n. at an MOI of 50 for 24 hours. Cell lysates were resolved as in Figure 1A. (D) Predicted interaction between miR-155 and the 39UTR of SHIP (INPP5D) mRNA. (E) Normalized luciferase activity in cells transfected with the 39UTR of SHIP (psiCHECK_INPP5D) or with vector alone (psiCHECK), and cotransfected with a control Renilla luciferase vector. Synthetic miR-155 or non-specific (scrambled) miRs were subsequently transfected at concentrations of 0, 10, 25 and 50 nM. Luminometer readings were taken 48 hours post-transfection. The graph represents f-luc expression normalized to r-luc expression, then normalized to percent maximal response. doi:10.1371/journal.pone.0008508.g001 PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8508