Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

Xuewei Zhu; John S. Owen; Martha D. Wilson; Haitao Li; Gary L. Griffiths; Michael J. Thomas; Elizabeth M. Hiltbold; Michael B. Fessler; John S. Parks

doi:10.1194/jlr.M006486

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol[S]

^*Departments of Pathology/Lipid Sciences, Wake Forest University School of Medicine, Winston-Salem, NC
^†Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC
^§Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC
^*Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
^††Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, Research Triangle Park, NC

Abstract

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1^-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1^-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1^-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1^-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1^-M/-M macrophages. Abca1^-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.

Footnotes

↵1 To whom correspondence should be addressed. e-mail: jparks{at}wfubmc.edu
Abbreviations:

Abca1^+/+

wild type mice at the Abca1 locus

Abca1^+/−^M

heterozygous macrophage-specific ABCA1 knockout

Abca1^-M/-M

homozygous macrophage-specific knockout

Abca1^−/−

total Abca1 knockout

ABCG1

ATP-binding cassette transporter G1

BMDM

bone marrow-derived macrophage

CT-B

cholera toxin B

FC

free cholesterol

fPEG-chol

fluorescein ester of polyethylene glycol-derivatized cholesterol

GLC

gas-liquid chromatography

IκBα

inhibitory κB protein α

IL

interleukin

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

MβCD

methyl-β-cyclodextrin

MyD88

myeloid differentiation primary-response protein 88

NF

nuclear factor

NutSP

Nutridoma SP

PI

phosphatidylinositol

PL

phospholipid

PM

peritoneal macrophage

PNS

postnuclear supernatant

TLR

Toll-like receptor

WT

wild type
This work was supported by National Institutes of Health Grants HL-49373, HL-094525, and AT-27820 (J.S.P.), American Heart Association fellowship 09POST2250225 (X.Z.), and the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences Z01 ES102005 (M.B.F.). The fPEG-cholesterol synthesis was supported by the National Institutes of Health Roadmap for Medical Research Initiative through its establishment of the Imaging Probe Development Center, administered by the National Heart, Lung, and Blood Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
↵[S] The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of Data, Results, and six figures.