Identification of key intermediates for the spatio-temporal regulation of TLR2 and TLR3 signalling

Toll-like receptors (TLRs) are the first line of host defence and one of the most potent triggers of immune responses. They play a pivotal role in a multitude of pathologies including cancer, chronic inflammatory diseases, and infection. Pioneering studies have shown that the localization of TLRs at the cell surface and at the endosomes is not as rigid as previously envisaged, and that cell surface TLRs may travel to the endosome to engage signalling. However, how the endosomal machinery directs TLR trafficking and modulates spatiotemporal signalling remains incompletely mapped, hindering its biological and therapeutic manipulation. In Chapter 4, through use of flow cytometry and advanced microscopy approaches, including LSM Confocal, Airyscan and Total internal reflection fluorescence microscopy in both endogenously expressing TLR cell lines (E.G. RPE1), in addtion to overexpression models (E.G. HEK293-Blue-TLR2), I provide novel insight on a common endocytic route mediated by Clathrin, shared by TLR2 and TLR3 following activation, including identification of a candidate targeting loci. Complimenting trafficking assays with functional readouts, my results in Chapter 5 further identify a common endosomal sorting machinery, the endosomal sorting complexes required for transport (ESCRT) that promotes TLR2 signalling, while terminating TLR3 signalling by inducing its degradation, signifying a signalling divergence from a common machinery. Promisingly, these observations were mirrored in overexpression assays in addition to endogenous and biologically relevant models such as human derived blood macrophages. Furthermore, in Chapter 6, through trafficking and signalling approaches, I go on to reveal a novel relationship between TLRs and the Retromer complex, which may be able to differentially regulate different TLRs and allows us behind the curtain that provides greater molecular details to TLR biology and regulation for targeting. Together, my results shine a light on how TLR trafficking is a novel regulatory intersection for TLR signalling, and identify new interacting partners of TLRs and signalling regulators. These findings will help pave the way for future selective targeting of the molecular machineries regulating TLR-driven immunity, utilising differential TLR regulation in bacterial and viral pathways to help define new targeted therapeutics.