Toll-like receptor signalling outcome is determined by the stoichiometry of the endogenous TRIFosome

Toll-like receptors (TLRs) drive innate immunity via assembly of macromolecular signal transduction platforms (supramolecular organizing centers (SMOCs)) coordinated by adaptor proteins such as Toll/IL-1R domain-containing adaptor-inducing interferon-β (TRIF), but whether oligomeric TRIFosomes form is unknown. Here using cryoelectron microscopy and biophysical characterisation of full length TRIF in vitro we show that it forms filamentous oligomers which associate with the TRIF signalling partners RIP1 and RIP3 kinases suggesting oligomeric TRIFosomes could form. Endogenous TRIF, however, is predominantly monomeric in the absence of ligand, only forming TRIFosome oligomers in macrophages after stimulation of TLR4 or TLR3 when a large, macromolecular signalling complexes form. TRIFosomes are fully formed at 45 minutes after TLR3 or 60 minutes after TLR4 stimulation commensurate with activation of nuclear factor kappa B (NF-ΚB) in these cells. TLR3/4 activation triggers rapid interferon signalling prior to TRIFosome formation through monomeric TRIF, unexpectedly suggesting that a macromolecular platform of TRIF is not required to drive this signalling pathway. Collectively these data show TRIFosome macromolecular platform formation, for the first time, and, surprisingly, that TLR signalling can be SMOC-independent in addition to being SMOC-dependent.