The role of STATs in TLR signalling

Innate immunity is crucial to living beings as it allows both multi-cellular and single cellular organisms to detect and eradicate invading pathogens. The Toll-like receptors (TLRs) are pivotal to the innate immune response as they bind microbial components and activate the prototypic pro-inflammatory molecule, Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB). The activation of NFκB is critical to initiating an anti-pathogenic state enabling the clearance of pathogens and the instigation of the adaptive immune response. Whilst the downstream signalling mechanisms of TLR signalling have been studied in great detail, more focus on cross-talk between signalling pathways is required. The main aim of this thesis is the examination of cross-talk between Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) and TLR pathways, and the role STAT1 and STAT3 have in TLR-induced inflammatory responses. Following stimulation with TLR ligands, STAT1 and STAT3 were demonstrated to undergo a rapid Serine-727 (Ser-727) phosphorylation independent of tyrosine phosphorylation. The Ser-727 phosphorylation of STAT1 and STAT3 was found to be dependent on Myeloid Differentiation Primary Response Gene 88 (MyD88) and TIR-Domain-Containing Adapter-Inducing Interferon-β (TRIF) signalling but not secretion of Interferon-β (IFN-β). Bioinformatic analyses of STAT1 and STAT3 identified putative TNF Receptor-Associated Factor-6 (TRAF6) Binding Motifs (T6BMs), a well-documented site important for association with TRAF6, a central molecule in TLR signalling. STAT1 and STAT3 were shown to interact with TRAF6 through over-expression assays and endogenous immunoprecipitations. TLR stimulation was found to induce mitochondrial reactive oxygen species (ROS) production thought to be a result of STAT3 mitochondria localisation. STAT3 and TRAF6 were found to localise to mitochondria separately, though the two proteins co-localised in the cytoplasm. STAT3 S727A mutants also failed to drive activation of the NFκB promoter following Pam3Cys-Ser-(Lys)4 (Pam3Cys) stimulation demonstrating the ability of pSer-727 STAT3 to modulate cytokine production. Examination of cytokine production following TLR stimulation showed that siRNA knockdown of STAT3 abolished Interleukin-6 (IL-6) production and diminished Tumour Necrosis Factor (TNF) levels. The findings here suggest that pSer-727 STAT3 co-localises to the mitochondria, where it is proposed to interact with the electron transport chain (ETC) and generate ROS production, this in turn drives production of pro-inflammatory cytokines. Confocal microscopy conducted on TLR-stimulated cells demonstrated rapid nuclear localisation of pSer-727 STAT1, with Tyr-701 STAT1 only translocating to the nucleus at 60 minutes. A range of TLR ligands was able to induce pSer-727 STAT1 nuclear localisation demonstrating this to be a board TLR-induced response. Interestingly, STAT1 S727A mutants failed to drive NFκB and IL-6 activation, but not TNF following Pam3Cys stimulation. STAT1 S727A BMMs however were found to produce less TNF, but not IL-6 or Regulated on Activation Normal T Cell Expressed and Secreted (RANTES). Nuclear translocation of STAT1 was not observed to be different in WT and STAT1 S727A Bone Marrow-Derived Macrophages (BMMs), but may be an issue with being unable to directly detect pSer-727 STAT1. To further assess STAT1s role in driving inflammatory responses Glycoprotein 130 (gp130)Y757F/Y757F (FF) mice with STAT1 knocked out Mice Embryonic Fibroblasts (MEFs) were employed. Following TLR stimulation, TNF production was severely ablated, whilst IL-6 production was not affected. This aligns with the BMM data, suggesting the pSer-727 STAT1 predominately regulates TLR-induce inflammatory responses through regulation of TNF production. My findings propose a new and novel means of regulating inflammatory responses by TLRs. The rapid serine phosphorylation of STAT1 and STAT3 may be used by the TLRs as a means of fine-tuning the innate immune response, with STAT1 acting upon TNF and STAT3 predominately regulating IL-6. Serine phosphorylation of the STAT is traditionally thought to increase transcriptional activity, but this study is the first in demonstrating its ability to modulate pro-inflammatory responses