A sphingomyelin-cholesterol complex regulates TLR4 activation in microglia
- 1Centro de investigaciones Biológicas-Margarita Salas (CSIC)
Abstract
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/Trem2R47H 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.
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BibTeX
@misc{antonbarros2026sphingomyelincholesterol,
title = {{A sphingomyelin-cholesterol complex regulates TLR4 activation in microglia}},
author = {C. Anton-Barros and B. Villarejo-Zori and S. Carmona-Lorenzo and J. M. Barbero-Paredes and D. Gonzalez-Diaz and M. Uceda and A. Fernandez-Bernal and M. Portero-Otin and I. Ayuso-Fernandez and C. Galiana-Rosello and F. Garcia-Garcia and E. Navarro and J. Montesinos},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.10.750647},
url = {https://www.biorxiv.org/content/10.64898/2026.09.10.750647v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A sphingomyelin-cholesterol complex regulates TLR4 activation in microglia
AU - C. Anton-Barros
AU - B. Villarejo-Zori
AU - S. Carmona-Lorenzo
AU - J. M. Barbero-Paredes
AU - D. Gonzalez-Diaz
AU - M. Uceda
AU - A. Fernandez-Bernal
AU - M. Portero-Otin
AU - I. Ayuso-Fernandez
AU - C. Galiana-Rosello
AU - F. Garcia-Garcia
AU - E. Navarro
AU - J. Montesinos
PY - 2026
DA - 2026/09/10
PB - bioRxiv
DO - 10.64898/2026.09.10.750647
UR - https://www.biorxiv.org/content/10.64898/2026.09.10.750647v1
AB - 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/Trem2R47H 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.
ER -