Oral Presentation 15th Lorne Infection and Immunity 2025

Endocytosis and endosomal signaling are distinct and independent outcomes of TLR4 activation (#34)

Thomas E Schultz 1 , Carmen D Mathmann 1 , Timothy W Muusse 2 , Hyoyoung Kim 2 , James W Wells 1 , Glen C Ulett 3 , Jessica A Hamerman 4 , Andrew J Brooks 1 , Bostjan Kobe 2 , Matthew J Sweet 5 , Katryn J Stacey 2 , Antje Blumenthal 1
  1. Frazer Institute, The University of Queensland, Brisbane, QLD, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
  3. School of Pharmacy and Medical Sciences and Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia
  4. Immunology Program, Benaroya Research Institute, Seattle, WA, USA
  5. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

Toll-like receptor 4 (TLR4) is a significant mediator of cellular activation and antimicrobial responses through recognition of pathogen-derived and endogenous ligands. Its clinical relevance is underscored by associations with beneficial or deleterious outcomes in diverse disease contexts including infection, chronic inflammation, and cancer. TLR4 drives distinct signalling outputs from the cell surface and endosomes, with current thinking in the field proposing that internalisation of cell surface-activated TLR4 is required to enable subsequent signalling from endosomal compartments. Unlike canonical TLR signalling pathways, the identities, hierarchies and interplay of the molecular regulators that govern TLR4 endocytosis are not well characterised. To address this knowledge gap, we utilised an array of genetic and pharmacological tools to probe the molecular regulation of TLR4 endocytosis and endosomal signalling in primary and immortalised macrophages. We demonstrate that, in contrast to the currently accepted model of TLR4 signalling, endosomal TLR4 signalling does not require activation of cell surface TLR4 or endocytosis of the receptor. Moreover, the established requirement for the accessory molecule CD14 in endosomal TLR4 signalling was attributable to previously described functions of CD14 as a binding and internalisation receptor for the TLR4 agonist lipopolysaccharide (LPS), rather than a specific mechanistic contribution to driving TLR4 endocytosis. Our data further show that despite canonical TLR adaptors and signalling cascades being dispensable for TLR4 endocytosis, activity of the TLR4 Toll/Interleukin-1-Receptor (TIR) signalling domain as well as E1/E2 ubiquitination enzyme activity was critical for this process. Collectively, our study identifies that rather than sequential steps in one pathway, pro-inflammatory cell surface signalling, TLR4 endocytosis, and endosomal signalling are independently regulated signalling modalities. This revised understanding of TLR4 signalling might be harnessed for selective context-specific amplification or restriction of TLR4 activity for the development of vaccines and therapeutics.