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

Area of Science:

• Immunology
• Structural Biology
• Glycolipid Signaling

Background:

• The Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex is a key innate immune sensor, primarily known for responding to bacterial lipopolysaccharide (LPS).
• While endogenous molecules are proposed to activate TLR4-MD-2, structural evidence for direct activation by these ligands is lacking.
• Sulfatides, sulfated glycolipids, have demonstrated varied roles in modulating inflammatory responses.

Purpose of the Study:

• To investigate the direct interaction of sulfatides with the mouse TLR4-MD-2 complex.
• To elucidate the structural basis of sulfatide-mediated TLR4-MD-2 activation.
• To determine the functional consequences of sulfatide binding to TLR4-MD-2 in different species.

Main Methods:

• Biochemical assays to assess TLR4-MD-2 activation by sulfatides.
• Crystallography to determine the structure of mouse TLR4-MD-2 complexed with C16-sulfatide.
• Functional studies in mouse and human macrophages to evaluate cytokine production and LPS antagonism.

Main Results:

• Short-chain fatty acid sulfatides directly activate mouse TLR4-MD-2, triggering MyD88- and TRIF-dependent signaling and cytokine production (TNFα, IFN).
• In contrast, these sulfatides antagonize LPS-induced TLR4-MD-2 activation in human cells.
• Crystal structure reveals sulfatides binding to MD-2's hydrophobic pocket, inducing an active receptor dimer conformation mimicking LPS/lipid A interactions.

Conclusions:

• Sulfatides act as direct agonists for mouse TLR4-MD-2 and antagonists for human TLR4-MD-2, suggesting species-specific roles in inflammation.
• The sulfate group and fatty acid chain length are critical for sulfatide activity, with shorter chains promoting agonism and longer chains potentially favoring antagonism.
• Findings indicate sulfatides may mediate sterile inflammation or modulate bacterial-induced inflammation, and other endogenous lipids may also interact with TLR4-MD-2.