Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid

Abstract

Saturated fatty acids can activate Toll-like receptor 2 (TLR2) and TLR4 but polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) inhibit the activation. Lipopolysaccharides (LPS) and lipopetides, ligands for TLR4 and TLR2, respectively, are acylated by saturated fatty acids. Removal of these fatty acids results in loss of their ligand activity suggesting that the saturated fatty acyl moieties are required for the receptor activation. X-ray crystallographic studies revealed that these saturated fatty acyl groups of the ligands directly occupy hydrophobic lipid binding domains of the receptors (or co-receptor) and induce the dimerization which is prerequisite for the receptor activation. Saturated fatty acids also induce the dimerization and translocation of TLR4 and TLR2 into lipid rafts in plasma membrane and this process is inhibited by DHA. Whether saturated fatty acids induce the dimerization of the receptors by interacting with these lipid binding domains is not known. Many experimental results suggest that saturated fatty acids promote the formation of lipid rafts and recruitment of TLRs into lipid rafts leading to ligand independent dimerization of the receptors. Such a mode of ligand independent receptor activation defies the conventional concept of ligand induced receptor activation; however, this may enable diverse non-microbial molecules with endogenous and dietary origins to modulate TLR-mediated immune responses. Emerging experimental evidence reveals that TLRs play a key role in bridging diet-induced endocrine and metabolic changes to immune responses.

Keywords: Docosahexaenoic acid; Docosahexaenoic acid (PubChem CID: 445580); Inflammation; Lipopolysaccharide core (CID: 53481794); Palmitic acid (PubChem CID: 985); Pam(3)Csk(4) (CID: 130704); Polyunsaturated fatty acid; Saturated fatty acid; Toll-like receptor.

Fig. 1. Arrangement of Toll-like receptor (TLR) domains

(a) TLRs consist of an extracellular leucine-rich repeat (LRR) domain, a transmembrane (TM) domain, and an intracellular Toll/IL-1R homology (TIR) domain. The extracellular LRR domain contains 20~27 LRR modules. LRRNT and LRRCT modules cover the N and C termini of the LRR modules, respectively. (b) Classification of TLRs: phylogenetic analysis, the structures of the LRR domains, and the chemical properties of the ligands suggest that TLRs can be divided into two major subclasses. (c) Structural boundaries are important for function. Boundaries dividing the N-terminal, central, and C-terminal subdomains are marked by broken lines. Functionally important areas are colored. The A and B patches of the primary TLR4-MD-2 interface are marked. (Kang and Lee, 2011).

Fig. 2. Crystal structures of the TLR4-MD-2-LPS complexes

(a) Structure of the TLR4-MD-2-LPS complex. TLR4, MD-2, and LPS are in gray, cyan, and red, respectively. (b) The dimerization interface between TLR4 and MD-2 is split and rotated by 90°. Hydrophilic residues of TLR4 and MD-2 colored dark green and dark blue, respectively, surround this hydrophobic core and form hydrogen or ionic interactions. The R2 lipid chain of LPS is in red. Other parts of lipid A and core carbohydrates are in pink and orange, respectively. (c) Structures of Lipid A and Eritorian. Abbreviations: (CO), backbone carbonyl oxygen; LPS, lipopolysaccharide; (NH), backbone amide nitrogen; TLR, Toll-like receptor. (Jin and Lee, 2008; Kang and Lee, 2011).

Fig. 3. Crystal structures of TLR2-TLR1/6 heterodimers induced by binding of triacyl and diacyl lipopeptides

(a) Lipid-binding pocket in the TLR2-TLR1-Pam₃CSK₄ complex. The structures of the TLRs are dissected to reveal the shape of the lipid pocket. (b) Lipid-binding pocket in the TLR2-TLR6-Pam₂CSK₄ complex. The putative lipid channel of TLR6 is blocked by phenylalanines F343 and F365. (c) Summary of the lipopeptide patterns recognized by the TLR1-TLR2 and TLR2-TLR6 heterodimers. (Kang and Lee, 2011).

Fig. 4

Unsaturated fatty acids inhibit, but saturated fatty acid potentiates, LPS-induced nuclear factor κB (NFκB) activation and COX-2 expression in RAW 264.7 cells. Cells stably transfected with NFκB(5 ×) (a) or COX-2 promoter (b) reporter gene were pretreated with various concentrations of each fatty acid for 3 h. Cells were then treated with LPS (200 ng/ml). After 8 h, cell lysates were prepared and luciferase activities were determined. Data are expressed as a percentage of LPS treatment alone. Values are mean ± SEM (n=3). RLA, relative luciferase activity. (c): Cells were pretreated with docosahexaenoic acid (DHA) (20 μM) or C12:0 (75 μM) for 3h and further stimulated with LPS (200 ng/ml for DHA; 1 ng/ml for C12:0). After 8 h, cell lysates were analyzed by COX-2 and actin immunoblotting. DHA, docosahexaenoic acid (C22:6n-3); EPA, eicosapentaenoic acid (C20:5n-3); AA, arachidonic acid (C20:4n-6); LA, linoleic acid (C18:2n-6); OA, oleic acid (C18:1n-9); C12:0, lauric acid. (Lee et al., 2003).

Fig. 5

Unsaturated fatty acids inhibit, but saturated fatty acid potentiates, PamCAG-induced NFκB activation and COX-2 expression. RAW 264.7 cells stably transfected with NFκB(5 ×) (a) or COX-2 promoter (b) reporter gene were pretreated with various concentrations of each fatty acid for 3 h. Cells were further stimulated with a synthetic bacterial lipoprotein (PamCAG, 500 ng/ml). After 8 h, luciferase activities were determined. Data are expressed as a percentage of PamCAG treatment alone. (c): RAW 264.7 cells were pretreated with DHA (20 μM) or C12:0 (75 μM) for 3 h and further stimulated with PamCAG (500 ng/ml). After 8 h, cell lysates were analyzed by COX-2 and actin immunoblotting. (d): 293T cells were cotransfected with NFκB-luciferase reporter plasmid and TLR2 expression plasmid, and treated with PamCAG in the presence or absence of DHA or C12:0. Values are mean ± SEM (n=3). RLA, relative luciferase activity. * Significantly different from the respective control (P < 0.05). (Lee et al., 2003).

Fig. 6

Palmitic acid and Pam₃CSK₄ induce but DHA inhibits the recruitment of MyD88 and p47 phox (subunit of NADPH oxidase 2) into lipid raft fractions. (a) To demonstrate the separation of lipid rafts (LR) and non-lipid raft (NLR) fractions of plasma membrane, cells were lysed and fractionated by sucrose-gradient ultracentrifugation. LR fractions (fractions 2 and 3) and NLR fractions (fractions 6–8) were identified by the presence of flotillin-1 (LR marker) and transferrin receptor (NLR marker), respectively. (* due to overwhelming expression only 20% of input lysate from fractions 6–8 was subjected to SDS-PAGE and immunoblotted with anti-TLR2 antibodies). (b) To determine whether palmitic acid (C16:0) or Pam₃CSK₄ induces recruitment of the downstream signaling components of TLR2 into LR fractions, THP-1 cells were serum starved in 1.0% FBS-RPMI-1640 for 12 h, then treated with C16:0 (150 μM) or Pam₃CSK₄ (100 ng/ml) for indicated time periods. Fractions 1–4 were immunoblotted with anti-TLR2, anti-MyD88, anti-p47phox, and anti-flotillin-1 antibodies. (c) Serum starved THP-1 cells were incubated with DHA (10 μM) for 1 h then treated with C16:0 (150 μM) or Pam₃CSK₄ (100 ng/ml) for 5 min. Cell lysate was separated by sucrose-gradient ultracentrifugation and fractions were immunoblotted. (Snodgrass et al., 2013).

Fig. 7. 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-flo-tillin-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. (Wong et al., 2009).

Fig. 8. TLR2/1 dimerization determined by TR-FRET in monocytes

TR-FRET assay was used to detect dimerization of TLR2 with TLR1 using anti-TLR2. Antibodies labeled with europium cryptate as the donor fluorophore and anti-TLR1. Antibodies labeled with d2 as the acceptor fluorophore: an assay to detect the dimerization of native receptors in the context of intact cell membrane, whereas, X-ray crystallography or immunoprecipitation assays to detect dimerization uses truncated or epitope tagged recombinant receptor protein, respectively. (Snodgrass et al., 2013).

Fig. 9. Schematic diagram of the β-arrestin2 and GPR120-mediated anti-inflammatory mechanism

Red colored letters and arrows indicate the DHA-mediated anti-inflammatory effect, and black colored letters and arrows indicate the LPS- and TNF-α-induced inflammatory pathway. (Oh et al., 2010).

Fig. 10. Schematic diagram of the translocation of TLR4 and TLR2 into lipid rafts

Translocation of TLR4 into lipid rafts and homodimerization of TLR4 induced by LPS or saturated fatty acids, and the inhibition of these steps by DHA (a), or heterodimerization of TLR2 with TLR1 or TLR6 in lipid rafts induced by lipopetides or saturated fatty acids, and its inhibition by DHA (b). For the sake of focusing the dimerization event, the horse shoe shape of TLR4 and exact location of MD2 associated with TLR4 as shown in X-ray crystallographic structure (Fig. 2) are not accurately depicted in this figure.