The role of adaptor protein Mal/TIRAP in transducing TLR signalling

Abstract

Toll-like receptor 4 (TLR4) plays an essential role in the innate immune system, protecting the body from gram-negative bacteria. However, while TLR4 activation is essential for host defence, it can also drive harmful inflammatory responses, contributing to conditions such as sepsis. Understanding the precise molecular mechanisms of the TLR4 signalling pathway is important to distinguishing between its protective and damaging responses and developing targeted therapies. Mal/TIRAP is an adaptor protein and the first intracellular component of the TLR4 and TLR2 signalling pathways. In the traditional model of signalling, ligand stimulation dimerises TLR4 and TLR2 which then recruits Mal/TIRAP. Mal/TIRAP links the TLRS to the formation of the supra molecular organising centre, the myddosome. The precise association of Mal/TIRAP with the TLRs and the myddosome is poorly understood. Understanding the timing and the stoichiometry of Mal/TIRAP in complex with TLR4 and TLR2 and the myddosome is critical to understanding the molecular mechanisms of TLR signalling initiation and regulation. Previous studies have been limited due to the non-physiological levels of Mal/TIRAP expression. In this thesis, I investigate the molecular mechanisms of Mal/TIRAP function and the initiation of intracellular TLR signalling using gene editing and microscopy. I developed CRISPR/Cas9 HDR approaches to introduce a HaloTag onto the endogenous Tirap gene in immortalized bone marrow-derived macrophages (iBMMs). The introduction of the HaloTag did not affect the function of the Mal/TIRAP protein which retained its wild-type function (validated by LPS-induced TNF-α and RANTES production). High resolution single molecule imaging techniques were developed and optimised to visualise Mal/TIRAP-Halo molecules in live and fixed cells, allowing the first physiological visualisation of Mal/TIRAP in immune cells. The single molecule imaging revealed critical insights into the association and stoichiometry of Mal/TIRAP in complex with TLR4, TLR2, and the myddosome. In immortalised and primary macrophages, I showed that TLR4 exists as either monomers or preformed dimers, and no ligand induced dimerisation occurs in response to LPS. Mal/TIRAP was pre-associated with TLR4 and TLR2 in both naïve and activated states. The stoichiometry and association of Mal/TIRAP with TLRs remained unchanged upon ligand stimulation, suggesting that signal transduction is mediated by pre-associated TLR-Mal/TIRAP complexes rather than ligand induced recruitment of Mal/TIRAP. These findings challenge the traditional model of ligand induced TLR dimerisation and Mal/TIRAP recruitment, instead suggesting a novel hypothesis in which preformed TLR-Mal/TIRAP complexes undergo small ligand-induced conformational changes to initiate intracellular signalling. This model positions Mal/TIRAP as a streamlining adaptor, Mal/TIRAP streamlines rapid inflammatory responses by selectively associating with a subset of dimerised TLRs. Mal/TIRAP associated with Myddosomes between 10–20 minutes post-stimulation, dissociating completely by 30 minutes while the Myddosomes persisted. A previously unknown cytoplasmic Mal/TIRAP population that appears not to be involved in TLR signalling was also identified, suggesting a potential role for Mal/TIRAP beyond TLR signalling. The insights gained from my study redefine our understanding of TLR signalling through Mal/TIRAP, which will inform how inflammatory pathways may be targeted therapeutically.