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Toll-like Receptors (TLR)s are an essential class of Pattern Recognition Receptors (PRR)s used by the innate immune system to recognise conserved microbial motifs which are characteristic of invading pathogens. Activation of TLRs initiates signalling cascades resulting in the activation of transcription factors such as NF-κB and members of the interferon regulatory factor (IRF) family which regulate gene expression and mediate an immune response to the pathogen. Dysregulation of TLR-signalling can result in inflammatory and autoimmune diseases and for this reason they are subject to tight regulation. This study demonstrates Cactin to be a novel negative regulator of TLR-signalling pathways. Overexpression of Cactin inhibits activation of NF-κB whilst suppressed expression of Cactin leads to stronger and more prolonged activation of NF-κB in response to LPS. The expression of Cactin is regulated by NF-κB, thus representing a novel negative feedback mechanism for suppression of the inflammatory response. Cactin protein is stabilised due to phosphorylation in cells treated with the pro-inflammatory cytokine IL-1β. Similarly, the stabilisation of Cactin was observed in response to the TLR ligands LPS and Poly(I:C). Cactin also negatively regulates the activation of IRF3 and IRF7 and suppression of endogenous Cactin augments Poly(I:C)-mediated IFNβ induction. Cactin localises to the nucleus by virtue of two nuclear localisation sequences (NLS)s and its nuclear expression is essential for manifesting its inhibitory effects. Within the nucleus Cactin inhibits the DNA-binding activity of the NF-κB subunit p65. Suppression of endogenous Cactin leads to increased binding of IRF3 to the IFNβ promoter, though Cactin had no effect on IRF3 phosphorylation or translocation. This indicates that Cactin also targets IRF3 at the level of DNA binding. Furthermore, Cactin negatively regulates IRF7-mediated IFNα induction and affects the protein stability of IRF7. Notably, these inhibitory effects are transcription factor specific as Cactin positively regulates the activation of AP-1 subunit c-Jun, demonstrating that Cactin can differentially regulate transcription factor activation. A number of Cactin-interacting proteins were also identified in this study. Interestingly Cactin interacts with a nuclear member of the IκB family, IκBL. Cactin and IκBL show very similar nuclear expression patterns and exhibit similar negative regulatory effects on NF-κB, IRF3 and IRF7. However, these two proteins function independently to inhibit the activation of these transcription factors. In summary this study highlights Cactin as a novel regulator of TLR signalling pathways by regulating the ability of specific transcription factors to bind to their cognate DNA sequences and induce transcription.