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Quantitative single-molecule imaging of TLR4 reveals ligand-specific receptor dimerization

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Sci. Signal.  31 Oct 2017:
Vol. 10, Issue 503, eaan1308
DOI: 10.1126/scisignal.aan1308

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Resolving TLR4 signaling

The pattern recognition receptor TLR4 recognizes lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. Ligand binding to TLR4 stimulates two distinct signaling pathways, and different LPS types and their derivatives can bias signaling through either pathway depending on their composition, which has implications for the use of TLR4 agonists as vaccine adjuvants. Krüger et al. used quantitative single-molecule localization microscopy to examine the effects of co-receptors and different LPS chemotypes on the oligomeric state of TLR4 in live cells. In the presence of co-receptors, TLR4 was evenly divided between monomeric and dimeric forms. Agonistic LPS shifted the balance toward dimeric TLR4, which activated an inflammatory signaling pathway, whereas an antagonistic LPS chemotype favored monomeric receptor. This type of analysis should yield a more complete understanding of the factors underlying biased TLR4 signaling.

Abstract

In humans, invading pathogens are recognized by Toll-like receptors (TLRs). Upon recognition of lipopolysaccharide (LPS) derived from the cell wall of Gram-negative bacteria, TLR4 dimerizes and can stimulate two different signaling pathways, the proinflammatory, MyD88-dependent pathway and the antiviral, MyD88-independent pathway. The balance between these two pathways is ligand-dependent, and ligand composition determines whether the invading pathogen activates or evades the host immune response. We investigated the dimerization behavior of TLR4 in intact cells in response to different LPS chemotypes through quantitative single-molecule localization microscopy. Quantitative superresolved data showed that TLR4 was monomeric in the absence of its co-receptors MD2 and CD14 in transfected HEK 293 cells. When TLR4 was present together with MD2 and CD14 but in the absence of LPS, 52% of the receptors were monomeric and 48% were dimeric. LPS from Escherichia coli or Salmonella minnesota caused the formation of dimeric TLR4 complexes, whereas the antagonistic LPS chemotype from Rhodobacter sphaeroides maintained TLR4 in monomeric form at the cell surface. Furthermore, we showed that LPS-dependent dimerization was required for the activation of NF-κB signaling. Together, these data demonstrate ligand-dependent dimerization of TLR4 in the cellular environment, which could pave the way for a molecular understanding of biased signaling downstream of the receptor.

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