Sulfatides are endogenous ligands for the TLR4–MD-2 complex

Abstract

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)–myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4–MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4–MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4–MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4–MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4–MD-2 and activate or inhibit this complex.

Sphingolipid metabolism during Toll‐like receptor 4 (TLR4)‐mediated macrophage activation

“…Galactosylceramide is a major lipid constituent of the brain, which can be transported to Golgi complex where it can be sialylated to produce GM4 ganglioside or undergo sulfation (generating a sulfatide) to regulate phagocytosis in macrophages (Popovic et al, 2007). Recent evidence shows that sulfatides are endogenous ligands for the TLR4-myeloid differentiation factor 2 (MD-2) complex (Su et al, 2021).…”

A Global Picture of Molecular Changes Associated to LPS Treatment in THP-1 Derived Human Macrophages by Fourier Transform Infrared Microspectroscopy

“…Overall, the fingerprint analysis on one hand supports the importance of the physicochemical variations in lipids triggered by LPS, as discussed above; on the other, it sheds light on a possible involvement of glycan modifications that could impact on the cell mechanical properties, which in turn may contribute to higher phagocytosis activity [ 55 ]. In addition, the observed changes, upon LPS stimulation, in sulfated sugars could be associated with the biosynthesis of sulfated glycosphingolipids or GAGs that are known to be endogenous ligands for the TLR4/MD-2 complex [ 56 ].…”

Mass Spectrometry Imaging Revealed Sulfatides Depletion in Brain Tissues of Rats Exposed in Real Air with High Fine Particulate Matter

“…Surprisingly, only a few sulfatides (STs) were differentially modulated between control and exposure groups in our MSI analysis (Figure 1). Sulfatides are sulfated galactocerebrosides that present mainly in a myelin sheath containing axons 24 and play vital roles in the regulation of cell differentiation, 25 protein trafficking, 26 immune response, 27 and maintaining the normal function of the myelin sheath. 28 Eight ST species detected in negative mode were downregulated in the corpus callosum (enclosed by a white dashed line in Figure 1) of the rat brains in the treatment group.…”