activation as well as IL-1b release [33,34]. To test whether inflammasome activation was required for the induction of miR- 155, we infected wild-type or caspase-1 knockout bone marrow- derived macrophages with F.n. at an MOI of 50 for 8 hours. MiR- 155 expression was measured by qRT-PCR and it was found that both the wild-type and caspase-12/2 macrophages responded nearly identical (Figure 4C). Thus, F.n.-induced miR-155 expression is caspase-1-independent, suggesting that the caspase- 1 inflammasome is not required. To verify lack of caspase-1 function in these BMM, IL-1b release was measured. Results (Figure 4D) showed that, as expected, IL-1b secretion by caspase- 12/2macrophages was impaired. MiR-155 Promotes Pro-Inflammatory Cytokine Production during F.n. Infection Activation of the PI3K/Akt pathway promotes the pro- inflammatory response to F.n., and SHIP is an established negative regulator of this pathway [35]. We have previously demonstrated that SHIP2/2 macrophages display enhanced pro-inflammatory cytokine production in comparison to wild-type macrophages infected with F.n. [17]. Given the observations that miR-155 is strongly induced in response F.n. infection and that miR-155 can negatively regulate SHIP expression, we hypothesized that miR- 155 exerts its pro-inflammatory function against Francisella by MiR155 Regulates Host Response repressing SHIP. To test the functional consequence of miR-155 expression, PBM were transfected with either exon 3 of BIC (encoding the mature miR-155) or an empty vector. RNA was extracted 22 hours after transfection and qRT-PCR for miR-155 expression was performed. Over-expression resulted in an approximately 3-fold increase in miR-155 compared to vector- only transfection (Figure 5A). SHIP mRNA was then measured in the same samples by qRT-PCR, and results showed that miR-155 over-expression alone was sufficient to significantly decrease SHIP mRNA (Figure 5B). To determine the functional consequence for F.n. infection, PBM were infected 14 hours after transfection with F.n. at an MOI of 50 for 8 hours and the secretion of TNFa (Figure 5C) and IL-6 (Figure 5D) quantified by ELISA. Levels of both cytokines were significantly enhanced in PBM over- expressing miR-155 compared to those with vector control. Results from this over-expression of miR-155 are reflective of the results seen with SHIP2/2 macrophages infected with F.n. by Parsa et al. Collectively, these results are consistent with the hypothesis that miR-155 promotes pro-inflammatory cytokine production largely by downregulating SHIP. To complement the data obtained from miR-155 over- expression, we tested the function of miR-155 in wild-type versus miR-1552/2 macrophages. The loss of miR-155 was verified by qRT-PCR (Figure 5E). Given that miR-155 targets SHIP, we Figure 5. miR-155 promotes pro-inflammatory cytokine production during F.n. infection. (A) PBM were transfected with vector control or miR-155 overexpression construct. 22 hours post-transfection, RNA was extracted and miR-155 over-expression was verified by qRT-PCR. (B) SHIP expression was assayed from the samples in Figure 5A. (C–D) 14 hours post-transfection PBM were infected with F.n. at an MOI of 50 for 8 hours and (C) TNFa and (D) IL-6 measured in supernatants. (E) Wild-type and miR-1552/2 macrophages were assayed for miR-155 expression. (F) SHIP expression was assayed by qRT-PCR from the samples in Figure 5E. (G) Wild-type and miR-1552/2 macrophages were infected with F.n. at an MOI of 50 for eight hours. IL-6 in the supernatant was measured by ELISA. All experiments were performed in triplicate. Graphs represent the mean and standard deviation of samples from three independent experiments. Data were analyzed by a paired Student t test. An asterisk (*) indicates a p-value,0.05. doi:10.1371/journal.pone.0008508.g005 PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8508