Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico

Edouard Mobarak,Malin Levin,Moutusi Manna,Jan Borén,Tomasz Rog,Mikael Rutberg,Ilpo Vattulainen,Liliana Håversen,Rosie Perkins

doi:10.1038/s41598-018-31926-0

Abstract

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.

Highlights

Toll-like receptors (TLRs) are expressed on the surface of sentinel cells such as macrophages and dendritic cells
Inhibition of GluCer synthase (GCS) with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride (D-PDMP) resulted in significantly lower concentrations of GluCer and higher concentrations of sphingomyelin in lipid domains isolated from plasma membrane of macrophages incubated with LPS or cell medium as control (Fig. 1A)
Atomistic molecular dynamics simulations of the Toll-like receptor 4 (TLR4) dimer complex in membranes showed that LPS induced a tilted orientation of the extracellular domain of TLR4 and that GluCer reduced the LPS-induced tilt

Summary

Introduction

Toll-like receptors (TLRs) are expressed on the surface of sentinel cells such as macrophages and dendritic cells. Several studies have shown that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for proinflammatory TLR4 signaling and that LPS promotes accumulation of TLR4 in these lipid domains[7]. Sphingolipids [including sphingomyelin, ceramides, glucosylceramide (GluCer), lactosylceramide (LacCer) and complex glycosphingolipids] have emerged as key modulators of many cellular signaling functions, including differentiation, proliferation and immune responses[8]. The role of endogenous glycosphingolipids in the LPS/TLR4-induced proinflammatory response of macrophages has not been investigated. The aim of this study was to elucidate how GCS-induced changes in the sphingolipid composition of plasma membrane lipid domains affect the LPS/TLR4-induced proinflammatory response in macrophages. We performed atomistic molecular dynamics simulations to investigate how changes in the sphingolipid composition affect the LPS/TLR4 complex

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Conclusion