Objective— Toll-like receptors (TLRs) initiate inflammatory signaling in response to conserved microbial molecules. It has been proposed that dietary saturated fatty acids (SFAs) may also serve as endogenous ligands of TLR2 or TLR4, thereby promoting diseases associated with inflammation and dyslipidemia, including atherosclerosis and insulin resistance.

Methods and Results— We investigated the effects of SFAs on TLR-dependent signaling using a broad range of cell types and readouts. In HEK-293 cells transfected with TLR2, TLR4, or TLR5, SFAs complexed with fatty-acid-free bovine serum albumin (BSA)-stimulated TLR-dependent signaling. However, SFAs alone did not elicit a similar response. Further analysis showed that the effect seen with the complexed SFAs was attributable to LPS and lipopeptide contamination of fatty-acid-free BSA. Additional studies in macrophages, endothelial cells, smooth muscle cells, adipocytes, skeletal muscle cells, and human peripheral blood mononuclear cells confirmed the lack of stimulation of TLR-dependent signaling pathways or expression of TLR-target genes by SFAs.

Conclusions— SFAs do not directly stimulate TLR-dependent signaling, suggesting that alternative mechanisms link dietary fat intake with TLR-associated pathologies. LPS and lipopeptide contamination of the widely used reagent fatty-acid-free BSA explains the previously reported stimulation of TLR2 and TLR4 by SFAs.