to be activated during Francisella infection [17]. PBM were pretreated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SP00125 (JNK inhibitor), SB203580 (p38 inhibitor) or DMSO vehicle control for 30 minutes and then infected with F.n. for 6 hours at an MOI of 50. Inhibition of PI3K completely blocked miR-155 induction (Figure 3G). An earlier report in B cells showed that the MAPKs ERK and JNK but not p38 were necessary for miR-155 induction by B-cell receptor activation [27]. Consistent with this, we found that inhibition of both ERK and JNK resulted in reduced miR-155 induction but inhibition of p38 showed no effect. Given these results we can conclude that miR-155 induction in response to F.n. requires the downstream activation of PI3K, ERK, and JNK. NFkB is a critical mediator of TLR signaling as well as a myriad of other cellular responses [28,29]. However, its role in miR-155 induction is unclear with one report showing that it was essential [30] but another concluding that it was not [27]. We surmised that its involvement could be dependent upon the nature of the stimulus, so we examined the role of NFkB within the context of Francisella infection. Here, PBM were pretreated with either the IkB kinase (IKK) inhibitor BAY7085 or with DMSO vehicle control for 90 minutes, followed by infection with F.n. at 50 MOI for 6 hours. As shown in Figure 3H, pretreatment with BAY7085 completely blocked the Francisella-induced miR-155 response. To verify the efficacy of this NFkB inhibition, TNFa secretion was measured after Francisella infection with or without pretreatment with BAY7085. As expected, there was no detectable TNFa release with BAY7085 pretreatment (Figure 3I). A lactate dehydrogenase (LDH) assay showed cytotoxicity of less than 10% after incubation with BAY7085 for 8 hours, again confirming that the cells were viable (data not shown). To confirm the requirement for NFkB, the peptide inhibitor SN50 was also used and found to block miR-155 induction (data not shown). Collectively, these results suggest that Francisella-induced miR- MiR155 Regulates Host Response 155 induction requires NFkB activation. This finding helps to explain why bacterial viability was required for maximal miR-155 induction (Figure 3A&B). A pervious report showed that maximal NFkB activation in response to F.t. LVS required bacterial viability and de novo bacterial protein synthesis [14]. Thus killed bacteria will have suboptimal NFkB activation as compared to live bacteria, and therefore suboptimal miR-155 induction. Bacterial Internalization and MiR-155 Induction Having found that TLR activity through MyD88 was critical for miR-155 induction, we hypothesized that cell surface contact with the bacteria would be sufficient to elicit miR-155. To test this, we pretreated PBM with an actin polymerization inhibitor, cytocha- lasin-D, or DMSO vehicle control for 30 minutes [31]. PBM were then infected with an MOI of 50 for 6 hours and miR-155 expression was measured by qRT-PCR (Figure 4A). MiR-155 expression was measured at 6 hours post-infection since the effectiveness of cytochalasin-D is reduced at later time points. MiR-155 induction in infected cells was not significantly different between vehicle control and cytochalasin-D treatments, suggesting that phagocytosis and internalization of Francisella may not be required during the early stages of infection for a miR-155 response. To verify the effectiveness of cytochalasin-D, CFU assays were performed in parallel with the same samples. After removal of viable extracellular bacteria with gentamicin, host cell lysates were plated and bacteria counted. Results showed approximately a 3-log reduction in uptake following cytochalasin-D pretreatment (Figure 4B). F.n.-Mediated MiR-155 Induction Is Independent of Caspase-1 Inflammasome Activation One hallmark of Francisella infection is that the bacteria can escape from the phagosome into the cytosol within an hour after internalization [3,32], which leads to inflammasome and caspase-1 Figure 4. Effects of host-cell entry and inflammasome activation on miR-155 induction. (A) PBM were pretreated for 30 minutes with 5 mg/ml cytochalasin D (CytoD), infected with F.n. at an MOI of 50 for 6 hours, then relative miR-155 expression measured by qRT-PCR. (B) CFU assays were conducted in parallel with the samples from Figure 4A. (C) Wild-type (WT) and caspase-12/2 bone marrow-derived macrophages were infected with F.n. at an MOI of 50 for 8 hours and miR-155 was assayed by qRT-PCR. (D) ELISAs were done to measure IL-1b in supernatants from the samples in Figure 4C. Graphs represent the mean and standard deviation from three independent infections. Data were analyzed by a paired Student t test. An asterisk (*) indicates a p-value,0.05. doi:10.1371/journal.pone.0008508.g004 PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8508