MiR155 Regulates Host Response Figure 3. Bacterial viability contributes to miR-155 induction through the TLR pathway. (A) PBM were infected with live, heat-killed (HK), or paraformaldehyde-fixed (PFA) F.n. at an MOI of 50 for 24 hours, then miR-155 measured by qRT-PCR. (B) PBM were infected with live F.n. or heat- killed F.n. at an MOI of 50, then with or without gentamicin after 2 hours, then miR-155 was assayed by qRT-PCR. (C) Wild-type (WT) or MyD882/2 bone marrow-derived macrophages were infected with F.n. at an MOI of 50 for 24 hours, then relative miR-155 expression measured by qRT-PCR and then converted to fold change over uninfected. (D) ELISA for TNFa secretion in supernatants from the samples in Figure 3C. (E) Wild-type (WT) and TLR2 (TLR2 Mut) signaling mutant bone marrow-derived macrophages were infected with F.n. at an MOI of 50 for eight hours and miR-155 was assayed by qRT-PCR. (F) WT and TLR2 Mut macrophages were infected at an MOI of 50 of F.n. for 5, 20, 40, and 60 minutes. Cell lysates were analyzed by Western blotting for pIKKa, pNFkBp65, and Actin. (G) PBM were pretreated for 30 minutes with inhibitors of PI3K (LY), ERK (UO), JNK (SP), p38 (SB), or with DMSO vehicle control, infected with F.n. at an MOI of 50 for 6 hours, then miR-155 was assayed by qRT-PCR. (H) PBM were pretreated with the IKK inhibitor BAY7085 (BAY) or DMSO vehicle control for 90 minutes, infected with F.n. at an MOI of 50 for 6 hours, then miR-155 was assayed by qRT- PCR. (I) ELISA for TNFa in supernatants from the samples in Figure 3F. All experiments were performed in triplicate, and each experiment performed three times. Graphs represent 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.g003 2 hours (Figure 3B). Conversely, levels of miR-155 induced by killed bacteria were comparable between the continuous exposure and the 2-hour exposure. These results suggest that a single exposure to F.n. induces miR-155, but that continuous infection elicits higher levels. These results also help explain Figures 2C and 2D, where the highest level of miR-155 was seen at 24 hours. MiR-155 Induction by F.n. Requires Activation of the TLR Signaling Pathway Previous studies have determined that TLR2 is critical for the recognition of Francisella and induction of the pro-inflammatory response. To test the involvement of TLR signaling in miR-155 induction we infected bone marrow-derived macrophages (BMM) from either wild-type or MyD882/2 mice with F.n. at an MOI of 50 for 24 hours. Wild-type macrophages showed strong induction of miR-155, whereas the miR-155 response in MyD882/2 macrophages was drastically reduced (Figure 3C). Confirming the absence of MyD88, we found that TNFa was not secreted by the MyD882/2 macrophages following infection (Figure 3D) as should be expected given that TNFa induction is entirely TLR2- and MyD88-dependent [14]. Hence, miR-155 induction by F.n. is MyD88-dependent. To further test the role of TLR signaling in the induction of miR-155 we infected wild-type and TLR2 signaling mutant macrophages with F.n. at an MOI of 50 for eight hours. The results show that TLR2 is required for the induction of miR-155 (Figure 3E). As a control for the TLR2 signaling mutant we tested for signaling activation of the NFkB pathway. It is clear that the TLR2 signaling mutant functions as expected since these macrophages do not display activation of the IKK complex or lead to NFkBp65 phosphorylation whereas the wild-type do (Figure 3F). We next tested the involvement of PI3K and the mitogen- activated protein kinases (MAPKs), downstream mediators known PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8508