examined SHIP mRNA expression in wild-type and miR-1552/2 macrophages. The loss of miR-155 was sufficient to lead to enhanced SHIP mRNA expression as compared to wild-type macrophages, further supporting the finding that miR-155 targets SHIP (Figure 5F). We then tested the pro-inflammatory response between wild-type and miR-1552/2 macrophages infected with F.n. MiR-1552/2 macrophages had significantly lower IL-6 production as compared to wild-type macrophages (Figure 5G). Collectively these data show that miR-155 negatively regulates SHIP and promotes inflammatory cytokine response to F.n. Virulent F.t. Induces Suboptimal MiR-155 and Pro-Inflammatory Cytokine Responses Since we have established that the less virulent F.n. induces robust miR-155 induction in monocytes/macrophages and that it can regulate TNFa and IL-6, we next examined whether the miR- 155 response was similar with the highly virulent F.t. isolate SCHU S4. PBM were infected with F.n. or F.t. for 24 hours. Relative expression of miR-155 (Figure 6A) and BIC (Figure 6B) were determined by qRT-PCR. While F.t. SCHU S4 led to MiR155 Regulates Host Response modest miR-155 and BIC expression, it was significantly lower than that elicited by F.n. Since miR-155 negatively regulates SHIP, and miR-155 induction is minimal following F.t. infection, we tested the ability of F.t. to down-regulate SHIP expression. We infected PBM with F.n. or F.t. for 24 hours. F.n. elicited a stronger miR-155 induction than F.t. and consistent with this, SHIP down-regulation was seen with infection by F.n. but not by F.t. (Figure 6C). In light of these findings, we examined the production of TNFa and IL-6 in response to F.n. and F.t., which we have now shown to be regulated by miR-155 and thus should be produced at lower levels in response to F.t. than to F.n. PBM were infected at an MOI of 50 for 24 hours and the levels of TNFa (Figure 6D) and IL-6 (Figure 6E) were determined by ELISA. Concordantly, it was found that the production of both cytokines was significantly lower in F.t.-infected samples than F.n.-infected samples. It is well established that Francisella bypasses or subverts host responses [11,12], and the lack of miR-155 response after infection with the virulent strain may be a key contributing factor. Figure 6. Virulent F.t. elicits suboptimal miR-155 and pro-inflammatory cytokine responses. (A–B) PBM (n = 7) were infected with F.n. or F.t. at an MOI of 100 for 24 hours. Relative miR-155 (A) and BIC (B) expression were measured by qRT-PCR. (C) PBM were infected with F.n. or F.t. at an MOI of 50 for 24 hours, then cell lysates probed for SHIP by Western blotting (top panel). The lower panel is a reprobe of the same membrane with anti-actin antibody. (D–E) PBM were infected with F.n. or F.t. at an MOI of 50 for 24 hours. TNFa (D) and (E) IL-6 in supernatants were measured by ELISA. Graphs represent the mean and standard deviation of samples 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.g006 PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8508