was also found in the human monocytic cell line THP-1 (Figure 1B), as well as in primary murine bone marrow-derived macrophages (BMM) (Figure 1C) infected with F.n. for 24 hours at an MOI of 100 and 50, respectively. In a search for potential mechanisms responsible for this down- regulation, we explored the possibility that microRNAs (miRs) may be involved. We used the online bioinformatics program TargetScan (http://www.targetscan.org) to identify potential miRs that may regulate SHIP. The program identified miR-155 as the sole predicted miR to bind SHIP on its 39 UTR (Figure 1D). To assess the ability of miR-155 to specifically target the 39UTR of SHIP we performed dual-luciferase reporter assays. For this, an f- luc luciferase reporter alone (psiCHECK) or the same reporter with the 39UTR of SHIP (psiCHECK-INPP5D) were separately transfected into chinese hamster ovary (CHO) cells using Lipofectamine 2000. To control for differences in transfection efficiency a pRL-CMV Renilla luciferase construct was co- transfected with the psiCHECK vectors. To test the effect of miR-155 on expression of the transfected SHIP 39 UTR reporter, synthetic miR-155 or scrambled miR were co-transfected into the cells at concentrations ranging from 10 nM to 50 nM. At 48 hours post-transfection cells were washed and lysed. Dual-luciferase activity in the transfectants was then measured. As shown in Figure 1E, the introduction of miR-155 suppressed the luciferase activity of psiCHECK-INPP5D but not of psiCHECK alone, indicating that miR-155 targeted the 39 UTR of SHIP. As an additional control, cells were transfected with psiCHECK- INPP5D followed by a scrambled miR, and these showed no decrease in luciferase production. MiR-155 Is Induced In Vitro and In Vivo in Response to F.n. Infection Having established that SHIP is down-regulated during F.n. infection and that miR-155 is a negative regulator of SHIP, we next examined whether mature miR-155 and BIC, the non-protein coding gene that encodes miR-155 [25], mRNA were induced in MiR155 Regulates Host Response infected cells. For this, human PBM were infected with F.n. and the expression of miR-155 and BIC were measured by qRT-PCR. Results showed a dose-dependent induction of mature miR-155 (Figure 2A) as well as BIC (Figure 2B) in PBM infected at MOI 1, 10, or 50 for 24 hours. We next examined the time course of miR- 155 and BIC induction. Results showed a gradual increase in both mature miR-155 (Figure 2C) and BIC mRNA (Figure 2D) from 4 to 24 hours post-infection when infected at an MOI of 50. This time course of mature miR-155 induction is consistent with previous findings in murine macrophages stimulated with toll-like receptor ligands [24]. Next, we tested whether F.n. infection led to miR-155 induction in vivo. For this, mice were challenged with 200 CFU of F.n. or with PBS, delivered intraperitoneally. The animals were sacrificed at 24, 48, and 72 hours post-infection (n = 6 per time point) and the lungs, liver, and spleen were harvested. RNA from homogenates was assayed for mature miR-155 by qRT-PCR (Figure 2E). Tissue-specific patterns of miR-155 matched those previously reported [26], where basal relative expression was highest in the spleen and lowest in the liver. Results showed a clear F.n.-induced induction of miR-155 in all organs tested, suggesting that F.n. elicits expression of mature miR-155 in vivo. Bacterial Viability Contributes to MiR-155 Induction Since infection with F.n. induces miR-155, we next tested whether bacterial viability was required for this response. PBM were infected with live, paraformaldehyde-killed or heat-killed F.n. at an MOI of 50 for 24 hours. While both forms of killed bacteria were able to induce relative miR-155 levels above that of uninfected cells, the levels were lower than what is induced by live bacteria (Figure 3A). To gain a better understanding of why viability is important, we examined miR-155 levels after 24 hours in PBM either exposed to 50 MOI of F.n. for the full 24 hour period or exposed for 2 hours before removal of extracellular bacteria. The PBM exposed to F.n. for the full 24 hours showed a stronger induction in miR-155 than those exposed for only Figure 2. miR-155 is induced in vitro and in vivo in response to F.n. infection. (A–B) PBM were infected with F.n. for 24 hours at an MOI of 1, 10, or 50. Relative miR-155 (A) and BIC (B) expression were measured by qRT-PCR and then converted to fold change over uninfected. (C–D) PBM were infected with F.n. at an MOI of 50 for 4, 8, or 24 hours. Relative miR-155 (C) and BIC (D) expression were measured by qRT-PCR and then converted to fold change over uninfected. Graphs represent the mean and standard deviation of samples from three independent infections. (E) Mice were injected peritoneally with 200 CFU of F.n. or PBS, then euthanized at 24, 48, and 72 hours post-infection. Relative miR-155 expression was measured by qRT-PCR for the liver, lung and spleen and then converted to fold change over uninfected. An n of 6 was used for each timepoint. Graphs represent the mean and standard deviation. Data were analyzed by a paired Student t test. An asterisk (*) indicates a p-value,0.05. doi:10.1371/journal.pone.0008508.g002 PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8508