CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer

Abstract

In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events.

Fig 1

TLR4 and TLR6 cooperatively mediate the macrophage inflammatory response to oxLDL. OxLDL-induced chemokine gene expression in (a) wild-type, Tlr2^−/−, Tlr4^−/−, Tlr6^−/− and (b) Myd88^−/− and Trif^−/− macrophages. Gene expression was analyzed by QRT-PCR and the fold increase in mRNA levels in oxLDL stimulated cells (50 μg/ml, 12h) compared to unstimulated cells is reported (*p<0.05, compared to wild type). (c) Expression of IL-1β mRNA in wild type, Tlr4^−/−, Tlr6^−/− and Myd88^−/− macrophages stimulated with oxLDL (50 μg/ml, 12h). (d) OxLDL-induced expression of an NF-κB luciferase reporter gene in HEK293 cells expressing the indicated TLRs. (e) Effect of sMD-2 on oxLDL-induced NF-κB luciferase reporter gene expression. All data are mean ± s.d. of triplicate samples and are representative of an experimental n of 3-4.

Fig 2

The co-receptor CD36 is required for TLR4-TLR6-dependent responses. (a) Effect of CD36 and CD14 on oxLDL-induced expression of an NF-κB luciferase reporter gene in TLR4-TLR6 expressing HEK293 cells. (b-c) Chemokine gene expression analyzed by QRT-PCR in (b) wild type and Cd36^−/− macrophages stimulated with oxLDL (50 μg/ml, 6h), and (c) aortas of Apoe^−/− and Cd36^−/−Apoe^−/− mice (n=3) fed a high fat diet for 12 weeks to induce atherosclerotic lesion formation. (d) Reactive oxygen species production in wild type, Tlr4^−/−, Tlr6^−/− and Cd36^−/− macrophages stimulated with oxLDL (50 μg/ml) or zymosan (1 μg/ml) for 45 min. Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of an experimental n of 3. *p≤0.05.

Fig 3

The Alzheimer’s disease peptide β-amyloid activates CD36-TLR4-TLR6 signaling. (a-b) β-amyloid induced expression of an NF-κB luciferase reporter gene in HEK293 cells expressing the indicated receptors. Cells were stimulated with (a) Aβ_1-42 or the (b) control peptide revAβ (10 μM, 5 h) and luciferase activity was measured. (c) Effect of CD36 and CD14 on Aβ_1-42-induced expression of an NF-κB luciferase reporter gene in TLR4-TLR6 expressing HEK293 cells. (d) Comparison of the ability of endogenous and microbial ligands of CD36 to activate an NF-κB luciferase reporter gene in HEK293 cells expressing TLR4-TLR6 and TLR2-TLR6 heterodimers. Cells were stimulated with LTA (1 μg/ml), S. aureus (10:1), Aβ_1-42 (10 μM) or oxLDL (50 μg/ml) and luciferase activity was measured. Data are the mean ± s.d. of triplicate samples and are representative of an experimental n of 3. *p≤0.05.

Fig. 4

CD36-TLR4-TLR6 signaling induces microglial inflammatory responses that promote neurotoxicity. (a-b) Production of nitric oxide in (a) wild type, Tlr2^−/−, Tlr4^−/−, Tlr6^−/−, Cd36^−/− and (b) Md2^−/− microglia stimulated with Aβ_1-42 (10 μM) or LPS (100ng/ml) for 24 hours. (c) Reactive oxygen species production in microglia of the indicated genotype stimulated with Aβ_1-42 (10 μM) for 45 min. Data are the mean ± s.d. of triplicate samples and are representative of an experimental n of 3. *P<0.005. (d) IL-1β and (e) RANTES mRNA measured by QRT-PCR in wild type, Tlr4^−/−, Tlr6^−/−, Myd88^−/− or Trif^−/− macrophages stimulated with Aβ_1-42 (10 μM, 6h). Data are the mean ± s.d. of triplicate samples and are representative of an experimental n of 2-3. *P<0.05. (f) CAD mouse neuronal cells co-cultured with wild type, Tlr2^−/−, Tlr4^−/− and Tlr6^−/− microglial cells stimulated with Aβ_1-42 (10 μM, 72 h) and stained with anti-neuronal class III beta tubulin Ab (green) and DAPI nuclear stain (blue). Quantification of % neuronal survival is shown at right.

Fig 5

CD36-ligand induced TLR4-TLR6 complex formation. (a-b) Immunoblot analysis of CD36-precipitated proteins from THP-1 monocytes treated with oxLDL (50 μg/ml) or Aβ_1-42 (50 μg/ml) for the indicated times. Precipitated proteins were immunoblotted with antibodies to TLR4, TLR6 and CD36. (c) Fluorescence microscopy of HEK293 cells transfected with CD36 and fluorescently tagged TLR4-YFP and TLR6-CFP. Cells were unstimulated (left) or treated with oxLDL (right) for 30 min, and stained with an anti-CD36 Alexa647 conjugated antibody. In the false-colored merged image triple co-localization of TLR4 (blue), TLR6 (red) and CD36 (green) appears as areas of white (arrowheads). Expression of individual receptors is shown in bottom panels. Scale bar 10 μm. (d-e) Inhibition of dynamin-dependent endocytosis blocks CD36-TLR4-TLR6-signaling in macrophages and microglia. QRT-PCR analysis of gene expression in (d) macrophages stimulated with oxLDL (50 μg/ml, 6h) or (e) microglia stimulated with Aβ_1-42 (10 μM, 6h) in the presence and absence of the dynamin inhibitor dynasore (80 μM).

Fig. 6

TLR4-TLR6 activation is triggered by a membrane proximal signaling event initiated by CD36. (a) Effect of wild type or C-terminal domain mutants of CD36 (CD36^Ala, CD36^Y463F, CD36^C464S) on oxLDL-induced expression of an NF-κB luciferase reporter gene in TLR4-TLR6 expressing HEK293 cells. (b) Immunoblot analysis of TLR4-precipitated proteins from HEK293 cells transfected with TLR4-tagged with a fluorescent protein (YFP), TLR6 and wild type CD36 or CD36^Y463F. Cells were untreated or treated with oxLDL (50 μg/ml) for 15 min and TLR4-precipitated proteins were immunoblotted with antibody to TLR6 or TLR4 (α-GFP). (c) CD36 immunoblot of Src- or Lyn-precipitated proteins from empty vector or CD36 transfected HEK293 cells (upper panel). Lyn immunoblot of proteins precipitated by a His-tagged CD36 C-terminal peptide in the presence or absence of soluble peptide competitors corresponding to the terminal 6, 9, or 13 amino acids of CD36 (460-472). (d) Effect of kinase inhibition on oxLDL-induced expression of an NF-κB luciferase reporter gene in CD36-TLR4-TLR6 expressing HEK293 cells. Cells were treated with oxLDL (50 μg/ml, 5 h) in the presence of kinase inhibitors, [Genistein (5 μM); PP1 (10 μM); LY294002 (LY, 5 μM)]; Wortmannin (10 nM) or inactive analogs [Daidzen (5 μM); PP3 (10 μM)] and cellular luciferase levels were measured. (e) Effect of kinase inhibition on co-precipitation of CD36 with TLR4 and TLR6 in THP-1 monocytes. Immunoblot analysis of CD36-precipitated proteins from THP-1 monocytes treated with oxLDL (50 μg/ml) in the absence or presence of kinase inhibitors described in (d). Data in all experiments are representative of three separate experiments. Data in a, d are the mean ± s.d. of triplicate samples (**p≤0.005).