A sphingomyelin-cholesterol complex regulates TLR4 activation in microglia

Microglial activation in response to lipopolysaccharide (LPS) requires Toll-like receptor 4 (TLR4) redistribution into cholesterol- and sphingolipid-rich membrane domains, yet the lipid determinants of this process remain unclear. Here, we identify cholesterol accessibility as a key driver of TLR4-dependent microglial activation. We show that LPS increases cholesterol uptake and mobilization, whereas inhibition of intracellular cholesterol trafficking or the plasma membrane-to-ER cholesterol transporter Aster attenuates inflammatory responses. Scavenger receptor class B type 1 (SR-B1) mediates LPS-induced cholesterol uptake, and its inhibition suppresses TLR4 signaling, receptor recruitment to detergent-resistant membrane domains, and TLR4 endocytosis without altering bulk cellular cholesterol. Instead, SR-B1 inhibition remodels the sphingolipidome and expands the sphingomyelin-associated cholesterol pool, as detected by OlyA, while LPS promotes remodeling of this pool and increases accessible cholesterol detected by D4H. Manipulation of sphingomyelin-cholesterol interactions alters membrane properties and TLR4 trafficking, while stabilization of these complexes suppresses inflammatory activation. In vivo, LPS induces remodeling of sphingomyelin-associated cholesterol in microglia, whereas Alzheimer’s disease-related ApoE4/Trem2 ^R47H microglia show impaired cholesterol-pool remodeling and enhanced inflammatory responses. Together, these findings establish sphingolipid-dependent cholesterol accessibility as a key regulatory layer controlling TLR4 organization and microglial inflammatory activation, positioning SR-B1 as an upstream regulator of this lipid-signaling axis.