Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub

Abstract

Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.

eLife digest

Cells in the immune system have proteins at their surface that detect molecules produced by invading microbes. One of these proteins is Toll-like receptor 4, TLR4 for short. Once TLR4 is activated, the immune cells form MyDDosomes – intricate complexes made of many different proteins. These structures form a signal that mobilizes the cell to fight the infection. In particular, the complexes set up a chain of events that leads to a gene-regulating protein getting access to the cell’s DNA. There, the protein switches on genes which produce other proteins important for inflammation, one of the body’s most important tools to fight an infection.

The activation of TLR4 is thought to be an all-or-nothing mechanism: the receptors are either ‘on’ or ‘off’. However, different microbial molecules recognized by TLR4 trigger different levels of inflammation, ranging from mild to severe. It remained unclear how an all-or-none response from the frontline receptors could lead to a gradual response from the cell.

Here, Latty et al. compare what happens to TLR4, MyDDosomes and the gene-regulating proteins when living immune cells are stimulated by different doses of two microbial molecules. These agents are both recognized by TLR4, but they lead to different levels of inflammation.

The type of microbial molecule, or their concentration, does not change how TLR4 is activated. Two TLR4 proteins can loosely associate with each together to form a dimer. When they bind a microbial molecule, the dimer becomes more stable. This changes the shape of the TLR4 proteins, which in turn triggers the formation of a scaffold of MyDDosomes. More stable TLR4 dimers are formed when the cells is in contact with a microbial molecule that triggers a strong immune reaction, and possibly when its concentration is higher.

Crucially, the different microbial agents and their concentration levels modify how MyDDosomes assemble. By ‘tagging’ each protein in the complex with a fluorescent chemical, Latty et al. can follow its formation as it actually happens. When the cells are stimulated with microbial molecules that provoke a strong inflammation, the MyDDosomes may be bigger, in greater numbers, and form more quickly. In turn, under strong microbial activation, the gene-regulating protein that switches on the immune response genes goes to the DNA faster and in higher numbers. This suggests that the pace of assembly, the size and the number of MyDDosomes control the strength of the immune response.

TLR4 is involved in diseases such as cancer or Alzheimer’s disease, where the body has an incorrect inflammation response. Knowing in greater detail the cellular processes activated by TLR4 could help efforts to find new drug targets for these conditions.