β-Sitosterol modulates TLR4 receptor expression and intracellular MyD88-dependent pathway activation in J774A.1 murine macrophages

• β-Sitosterol (SIT) down regulates TLR4, MyD88 and IRAK1 expression.
• SIT up regulates SOCS3, a negative regulator of TLR4 pathway.
• SIT had no effect on lipid rafts.

Recent evidence has shown that dietary phytosterols (PS) possess anti-inflammatory properties both in vivo and in vitro. Our previous work shows that PS β-Sitosterol (SIT), may function by down-regulating pro-inflammatory transcription factors NF-kB and STAT1 in response to LPS stimulation, possibly through modulation of the TLR4 pathway. The objective in this study was to determine the effects of SIT on TLR4 surface expression and localization into lipid rafts, as well as to investigate its effects on intracellular MyD88 dependent pathway activation. J774A.1 macrophages were pre-treated with cyclodextrin vehicle loaded with cholesterol or SIT, then stimulated with LPS (100 ng/ml) for 30 min. ImageStream cytometry demonstrated that SIT down-regulates TLR4 expression without affecting lipid raft distribution. Western blot demonstrated that SIT down-regulated the adaptor protein MyD88 and the activity of IRAK1 but increased SOCS3 expression. Together, these results provide evidence that SIT may indeed elicit anti-inflammatory properties by down-regulating some components of the TLR4 pathway.