However, Tween is routinely added to liquid media to reduce cell clumping, therefore Protopanaxtriol enabling homogeneous cell suspensions of mycobacteria to be obtained for in vitro macrophage challenge experiments. The repression of MyD88-dependent signalling may represent a transcriptional signature of M. bovis-mediated subversion of host immune responses. Indeed, inhibition of IFN-c-induced MHC class II expression in M. tuberculosis-infected macrophages has been shown to involve MyD88-dependent signalling mediated through TLR2. Thus, the suppression of TLR2-MyD88-dependent signalling may represent one mechanism for immuno-evasion by the mycobacterial pathogen enabling persistence within the host macrophage Quinine hydrochloride Dihydrate during infection. In contrast, the activation of MyD88-independent signalling may present an alternative route used by host macrophages to circumvent suppressed MyD88dependent signalling and induce downstream transcription factors which promote chemokine and cytokine production during infection. It is also important to note that as this is a transcriptomics study, the functional role of various pre-existing TLR adaptor proteins in macrophage cellular pathways during M. bovis infection cannot be assessed at the RNA level; consequently, their involvement in these pathways cannot be fully excluded. Further work involving both transcriptomic and proteomic platforms is required to investigate fully a role for macrophage TLR2-dependent/MyD88-independent signalling pathways in response to mycobacterial infection. Genes encoding members of the TLR3 signalling pathway also displayed differential expression in the M. bovis-challenged MDM at the 6 hour and 24 hour time points. TLR3 encodes a membrane-bound intracellular PRR that localises to endosomes and is involved in the recognition of viral PAMPs, such as double stranded reoviral RNA. TLR3-mediated signalling occurs through the TICAM1 adaptor which recruits TRAF3 to activate IRF3-mediated transcription of type I IFN. In the current study, TLR3 was upregulated in the M. bovis-challenged MDM samples at 6 hours and 24 hours post-challenge, while upregulation of TRAF3, IRF3 and INFB1 was also observed at the 24 hour time point. TICAM1 was not differentially expressed at either the 6 hour or 24 hour time points. The involvement of RLRs and TLR3 in mediating the macrophage response to M. bovis infection is intriguing given their well-documented role in the detection of viral PAMPs. However, transfection experiments have shown that murine bone marrowderived macrophage production of type I IFN in response to RNA isolated from Legionella pneumophila is mediated via RIG-I ; while RNA from Helicobacter pylori was also shown to induce type I IFN production via a RIG-I-mediated signalling pathway in mouse dendritic cells. Moreover, increased TLR3 RNA and protein expression has been reported in human leukocytes following in vitro stimulation with bacterial lipopolysaccharide, while increased relative TLR3 expression has been reported by us in the transcriptome of peripheral blood leukocytes from cattle displaying active BTB compared to the PBL transcriptome of non-infected control animals. Collectively, these findings support a role for cytosolic RLRs and TLR3 during host infection with intracellular bacterial pathogens. It has been proposed that activation of intracellular PRRs by bacterial PAMPs is due to the translocation of bacterial RNA into host cells, or is a consequence of the generation of hostderived RNA ligands caused by the pathogen-mediated disruption of host cellular pathways during infection.