Toll-like receptor (TLR) signalling is a critical part of the body’s innate immune response and the first line of defence against invading pathogens. TLR3 and TLR4 signalling in particular, in response to viruses and bacteria respectively, lead to the induction of anti-viral cytokines such as IFN-β and pro-inflammatory cytokines. Interleukin-27 (IL-27) is a heterodimeric cytokine consisting of an Epstein-Barr virus�induced gene 3 (EBI3) and a p28 subunit and is rapidly produced by APCs upon exposure to TLR ligands (Yoshida & Hunter 2015). IL-27 synthesis induced by TLR ligation critically depends on IRF3 and transcriptional amplification requires a two-step process including the assembly of the ISGF3 complex downstream of IFNAR (Molle et al. 2007; Molle et al. 2010). IL-27 exerts pleiotropic immunomodulatory effects on a broad spectrum of immune cells, having both pro-inflammatory and anti-inflammatory properties. IL-27 has been seen to have a role in many autoimmune diseases such as Multiple sclerosis (MS) and has been seen to suppress pathogenic Th17 cells in these diseases. We identified a novel role for the serine/threonine kinase IRAK1 in the negative regulation of IL-27 production downstream of TLR3 and TLR4. We have seen IRAK1 negatively regulate key molecules in this signalling pathway, such as STAT1 and IRFs, at both the mRNA and protein level, as well as effecting the nuclear translocation of key transcription factors. We identified a role for TBK1 in the positive regulation of TLR3/4- induced IL-27 production and saw effects of the kinase activities of TBK1/IKKi on the transcription of IL-27 subunits, STATs and IRFs and roles for these kinases in the translocation of key STATs and IRFs to the nucleus. We also observed that PBMCs from treatment naïve MS patients showed a reduced ability to produce IL-27 in response to TLR3 stimulation, compared to controls. This suggests that in MS patients the TLR3-IL-27 axis may be deregulated. Furthermore we demonstrated that this novel regulator IRAK1 is hyper-activated in MS patients compared to controls and that this correlates with reduced SHP1 levels. SHP1 is a phosphatase known to be a negative regulator of cytokine signalling in the innate and adaptive immune responses (Lorenz 2009). We identified SHP1 as a novel substrate of the non-canonical kinases, IKKi and TBK1. We showed novel interactions of SHP1 with IKKi, TBK1 and IRF3, all key molecules in TLR signalling. Furthermore we showed that phosphorylation of SHP1 by IKKi, and potentially TBK1, is involved in TRIF�IFN-β and TRIF-p65 signalling. We demonstrated this by overexpressing a SHP1 mutant which cannot be phosphorylated by IKKi and saw that it acted as a dominant negative in these TRIF-mediated pathways. We identified that SHP1 is K63-linked polyubiquitinated downstream of TLR3 and that this is impaired following inhibition of the kinase activities of IKKi/TBK1. We showed novel interactions of SHP1 with TRAF3 and present preliminary evidence that TRAF and Pellino family members lead to the modification of SHP1, while no such modification is seen with mutant SHP1. This will be explored in future studies in the lab in an attempt to identify the E3 ubiquitin ligase that leads to the polyubiquitination of SHP1 in TLR signalling. We have furthered the molecular insight into SHP1 regulation with relevance to TLR signalling and beyond.