TI - MiR-155 Induction by F.n . Requires Activation of the TLR Signaling Pathway . . AB - 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 MyD88-/- 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 MyD88-/- macrophages was drastically reduced ( Figure 3C ) . Confirming the absence of MyD88 , we found that TNFalpha was not secreted by the MyD88-/- macrophages following infection ( Figure 3D ) as should be expected given that TNFalpha 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 NFkappaB 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 NFkappaBp65 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 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 . NFkappaB 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 NFkappaB within the context of Francisella infection . Here , PBM were pretreated with either the IkappaB 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 NFkappaB inhibition , TNFalpha secretion was measured after Francisella infection with or without pretreatment with BAY7085 . As expected , there was no detectable TNFalpha 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 NFkappaB , 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-155 induction requires NFkappaB 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 NFkappaB activation in response to F.t . LVS required bacterial viability and de novo bacterial protein synthesis [14] . Thus killed bacteria will have suboptimal NFkappaB activation as compared to live bacteria , and therefore suboptimal miR-155 induction .