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. 2009 Oct 2;284(40):27384-92.
doi: 10.1074/jbc.M109.044065. Epub 2009 Aug 1.

Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner

Scott W Wong  1 , Myung-Ja Kwon, Augustine M K Choi, Hong-Pyo Kim, Kiichi Nakahira, Daniel H Hwang
Affiliations

Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner

Scott W Wong et al. J Biol Chem. .

Abstract

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.

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Figures

FIGURE 1.
FIGURE 1.
Lauric acid enhances the recruitment of TLR4 and its adaptor molecules, TRIF and MyD88, into the lipid rafts. RAW264.7 cells were stimulated with LPS (100 ng/ml) or lauric acid (100 μm, C12) for 7 min or DHA (20 μm) for 1 h. The cells were lysed and lipid rafts (Fractions 2 and 3) and non-lipid rafts fractions (Fractions 5–8) were separated by sucrose-gradient ultracentrifugation. Each fraction was trichloroacetic acid precipitated and subjected to immunoblotting using anti-TLR4, anti-TRIF, anti-MyD88, or anti-flotillin-1 antibodies.
FIGURE 2.
FIGURE 2.
DHA inhibits LPS- or lauric acid-induced recruitment of TLR4 into the lipid rafts. RAW264.7 cells were treated with LPS (A) or lauric acid (B) for 7 min in the presence or absence of DHA (20 μm). The cells were lysed, and lysates were fractionated using sucrose-gradient ultracentrifugation as described in Fig. 1, lipid rafts collected in Fractions 1–3 and trichloroacetic acid-precipitated. Fractions were immunoblotted with anti-TLR4, TRIF, MyD88, or flotillin-1. C, Ba/F3 cells stably transfected with GFP/FLAG-tagged TLR4 were treated in identical manner as in A and B, but immunoblotted with anti-GFP or anti-flotillin-1. D, RAW264.7 cells were stimulated with LPS or lauric acid for 1 h with or without DHA (20 μm) or DHA alone for 1 h and followed by incubation with fluorescein isothiocyanate-conjugated cholera toxin B on ice for 10 min. LPS-stimulated cells were incubated in DMEM with 10% FBS, whereas, lauric acid-stimulated cells were incubated in serum-poor DMEM. Cells were analyzed for fluorescein isothiocyanate-conjugated cholera toxin B-stained glycosphingolipid 1 (GM1, green), which is enriched in lipid rafts and TLR4 (red) by confocal microscopy.
FIGURE 3.
FIGURE 3.
LPS or lauric acid induces, but DHA inhibits the homodimerization of TLR4. A, Ba/F3 whole cell lysates were immunoprecipitated with an anti-GFP antibody and then immunoblotted with an anti-FLAG antibody. The membrane was stripped and reprobed with anti-GFP. B, dose-dependent response of Ba/F3 cells toward pretreatment with DHA. Cells were stimulated with LPS or C12, and cell lysates were processed as described in Fig. 2A. For the analysis of NF-κB promoter activity, Ba/F3-TLR4 cells transfected with NF-κB- luciferase construct (C) or RAW264.7 cells transfected with NF-κB- luciferase construct (D) were used. Cells were treated with LPS or lauric acid for 8 h in the presence or absence of DHA. Cells were lysed to determine the luciferase activity. The results were expressed as relative luciferase activity (RLA) against the value of the vehicle treatment. Values are means ± S.E. of the mean of at least three independent experiments. a and b were significantly different from the values for the control group without DHA treatment (p < 0.05). For the analysis of mRNA levels of indicated genes, Ba/F3-TLR4 cells (E) or RAW264.7 cells (F) were treated with LPS or lauric acid in the presence or absence of DHA for 6 h. RNAs were prepared, and reverse transcription-PCR was performed as described under “Experimental Procedures.” Hypoxanthine phosphoribosyltransferase (HPRT) was used as a control.
FIGURE 4.
FIGURE 4.
Lauric acid induces but DHA inhibits TLR4 homodimerization and association of TLR4 with MD-2 in lipid rafts. A, Ba/F3 cells stably transfected with GFP/FLAG-tagged TLR4 and FLAG-tagged MD-2 were treated with LPS or lauric acid in the presence or absence of DHA (20 μm). For the immunoprecipitation, lipid raft Fractions 4 and 5 were pooled from the sucrose gradient. One half of the lipid raft fraction was immunoprecipitated with anti-GFP antibodies and then immunoblotted with anti-FLAG antibodies. The membranes were reprobed with anti-GFP antibodies. The other half of the samples was immunoblotted with anti-flotillin-1 antibodies to show the presence of the lipid raft marker. B, samples from pooled non-lipid raft Fractions 10–12 were immunoprecipitated and immunoblotted as described above in A.
FIGURE 5.
FIGURE 5.
Lipid raft inhibitor nystatin inhibits LPS- or lauric acid-induced homodimerization of TLR4, and the activation of NF-κB and TLR4 target gene expression. A, Ba/F3 cells were treated with nystatin prior to treatment with LPS or lauric acid for 30 min. Cell lysates were immunoprecipitated with anti-GFP antibodies and then immunoblotted with anti-FLAG antibodies. B, Ba/F3 cells were treated as in A but were lysed and fractionated by the sucrose gradient. GFP-TLR4 was immunoprecipitated with anti-GFP antibodies from lipid raft fractions and immunoblotted with anti-FLAG antibodies. C, samples from the non-lipid raft fractions were immunoprecipitated and immunoblotted as in B. For the analysis of NF-κB-luciferase activity, Ba/F3-TLR4 cells (D) or RAW264.7 cells (E) transfected with NF-κB-luciferase construct were treated with nystatin for 8 h before the treatment with LPS or lauric acid. Cells were lysed to determine the luciferase activity. a and b were significantly different from the values for the control group without nystatin treatment. For the analysis of mRNA of indicated genes, Ba/F3-TLR4 cells (F) or RAW264.7 cells (G) were treated with nystatin for 6 h before treatment with LPS or lauric acid. RNAs were prepared and reverse transcription-PCR was performed as described under “Experimental Procedures.”
FIGURE 6.
FIGURE 6.
Lauric acid activates, but DHA inhibits NADPH oxidase and ROS generation. A, RAW264.7 cells were preincubated with 10 μm CM-H2DCFDA for 30 min followed by incubation with DHA (20 μm), DPI (2 μm), NAc (10 mm), or vehicle for 30 min. LPS (100 ng/ml) or lauric acid (150 μm) was incubated with the cells for an additional 30 min. Cells were fixed and imaged by confocal microscopy. B, RAW264.7 cells were incubated with 50 μm lauric acid for the indicated time periods, cells were lysed, and gp91[phox] was immunoprecipitated and immunoblotted with anti-gp91[phox] and anti-p47[phox] antibodies. C, similar to B, cells were incubated with indicated amounts of lauric acid for 30 min. D, RAW264.7 cells were pretreated with DHA for 30 min, followed by incubation with 50 μm lauric acid or 100 ng/ml LPS for 30 min (E). F, RAW264.7 cells were pretreated with NAc (10 mm) for 2 h or DPI (2 μm) for 1 h followed by incubation with 50 μm lauric acid.
FIGURE 7.
FIGURE 7.
Inhibition of NADPH oxidase suppresses LPS or lauric acid-induced TLR4 dimerization. A, Ba/F3 cells were pretreated with DPI (2 μm) for 1 h or NAc (10 mm) for 2 h and then stimulated with LPS (100 ng/ml) or (B) 100 μm lauric acid for 30 min. Cells were lysed, and GFP-TLR4 was immunoprecipitated and immunoblotted with anti-FLAG and anti-GFP antibodies. C, RAW264.7 cells were pretreated with 2 μm DPI for 1 h and treated with 100 μm lauric acid for 30 min. Cells were lysed, and lysates were fractionated by sucrose gradient fractionation. Lipid raft fractions (1–3) were collected, trichloroacetic acid-precipitated, and immunoblotted with anti-TLR4 and anti-flotillin-1 antibodies.
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