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.

许多内源性分子，主要是蛋白质，据称可以激活 Toll 样受体 4 (TLR4)-髓样分化因子-2 (MD-2) 复合物，这是一种源自革兰氏阴性菌的脂多糖 (LPS) 的先天免疫受体。然而，没有结构证据支持内源性配体直接激活 TLR4-MD-2。硫化物 (3- O-sulfogalactosylceramides) 是天然的、丰富的硫酸化糖脂，已通过各种方式显示可引发或抑制炎症反应。我们在这里展示了短脂肪酸 (FA) 链硫苷脂直接激活小鼠 TLR4-MD-2，而不依赖于 CD14，触发 MyD88 和 TRIF 依赖性信号传导，并刺激肿瘤坏死因子 α (TNFα) 和 I 型干扰素 (IFN) 的产生在小鼠巨噬细胞中。与对小鼠受体的激动剂活性相反，测试的硫苷脂拮抗 LPS 在人巨噬细胞样细胞中对 TLR4-MD-2 的激活。硫苷脂的激动和拮抗活性需要硫酸基团的存在，并且与 FA 链长成反比。小鼠 TLR4-MD-2 与 C16-硫苷脂复合物的晶体结构表明，三个 C16-硫苷脂分子与 MD-2 疏水袋结合并诱导受体复合物的活性二聚体构象，类似于 LPS 或脂质 A 诱导的受体复合物。 . 三个 C16-硫苷分子部分模拟了脂质 A 的详细相互作用以实现受体激活。我们的研究结果表明，硫苷脂可能介导无菌炎症或抑制 LPS 刺激的炎症，并且具有多达六个有限长度的脂质链的额外内源性带负电荷的脂质也可能与 TLR4-MD-2 结合并激活或抑制该复合物。三个 C16-硫苷分子部分模拟了脂质 A 的详细相互作用以实现受体激活。我们的研究结果表明，硫苷脂可能介导无菌炎症或抑制 LPS 刺激的炎症，并且具有多达六个有限长度的脂质链的额外内源性带负电荷的脂质也可能与 TLR4-MD-2 结合并激活或抑制该复合物。三个 C16-硫苷分子部分模拟了脂质 A 的详细相互作用以实现受体激活。我们的研究结果表明，硫苷脂可能介导无菌炎症或抑制 LPS 刺激的炎症，并且具有多达六个有限长度的脂质链的额外内源性带负电荷的脂质也可能与 TLR4-MD-2 结合并激活或抑制该复合物。