Finally, TRAM mediates TLR4 signalling exclusively 7 acting as a bridging adapter to recruit TRIF to the TLR4 complex. Regarding Mal, studies have shown that Mal interacts with MyD88, TRIF and TRAM 7, 8, but not SARM (data not shown). Although the adaptors are believed to participate in the activation of TLR signalling cascades, a number of recent studies highlight the role of TLR adaptors in the negative regulation

of alternative TLR 6, 9. Regarding the IFN-β gene itself, transcriptional activation requires assembly of a multiprotein complex to form the IFN-β “enhanceosome” 10 which is divided into four positive regulatory domains (PRD) whereby ATF-2/c-Jun binds to the PRDIV element within the IFN-β enhancer region and is activated by Tamoxifen supplier JNK. IRF3 and IRF7 are activated by ligand-mediated phosphorylation upon which they are rapidly translocated to the nucleus where they bind the PRDI-III enhancer element within the IFN-β promoter 10. Using gene-targeted mice, recent studies have shown that both IRF3 and IRF7 play essential roles in Type I IFN-β expression 11, 12. Regarding NF-κB (p50:RelA), phosphorylated NF-κB translocates to the nucleus where it binds to the PRDII element within the IFN-β enhancer 10; the role of p50, RelA and c-Rel in IFN-β gene induction is relatively

minor 13. Taken together, these studies suggest that IRF are the master Barasertib regulators of IFN-β gene induction and that NF-κB plays a relatively minor role. Understanding how pro-inflammatory TLR adaptors can modulate non-cognate TLR in certain situations has many implications, not the least of which is a comprehensive understanding of the interplay between various TLR that are likely activated during microbial infections. Although the ability of TLR adaptors to activate specific signalling pathways has been well defined, the ability to negatively regulate non-cognate TLR signalling

cascades requires further investigation 9, 13. Recently, it has been Montelukast Sodium shown that MyD88 negatively regulates TLR3/TRIF-induced corneal inflammation 9. Also, potentiation of poly(I:C)-mediated IL-6 induction and JNK phosphorylation was observed in Mal−/− BM-derived macrophages (BMDM) when compared with WT BMDM 6. Herein, we provide the first detailed mechanistic analysis of how TLR signalling may be counterregulated by non-canonical mechanisms. As shown in Fig. 1A, following quantitative real-time RT-PCR measurements, we demonstrate that although stimulation of WT BMDM, expressing TLR3 endosomally 14, with poly(I:C) resulted in IFN-β gene induction, a significantly greater induction of IFN-β was evident in Mal−/− BMDM. In contrast to poly(I:C), we found comparable levels of IFN-β induction in WT and Mal-deficient BMDM stimulated with the TLR7 ligand, R848 and the TLR9 ligand, CpG (Supporting Information Fig. 1).